1. science
2. medicine
3. genetics

Epigenetics and targeting mechanisms in
Drosophila melanogaster
Margarida Figueiredo
Department of Molecular Biology
Umeå University 2015, Sweden
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7601-267-3
Cover: immunostaining in polytene chromosomes (picture by Margarida
Figueiredo) with the creative design by Chris Voss.
Electronic version is available at http://umu.diva-portal.org/
Printed by: Arkitetkopia Umeå, Sweden 2015
Nada é, tudo se Outra
Fernando Pessoa
To my parents, Julieta e Carlos, for everything
CONTENTS
LIST OF PUBLICATIONS ............................................................................ iii
ABSTRACT ....................................................................................................... iv
1. INTRODUCTION ........................................................................................ 1
Epigenetics....................................................................................................... 1
Chromatin: the nucleosome ........................................................................... 2
Nucleosome evolution ................................................................................. 3
Histone modifications ..................................................................................... 4
DNA methylation ............................................................................................ 5
Chromatin states ............................................................................................. 6
Long non-coding RNAs ................................................................................... 7
Drosophila melanogaster............................................................................... 8
Mechanisms of targeting............................................................................... 10
2. SEGMENTAL ANEUPLOIDIES ............................................................. 11
Aneuploidy ...................................................................................................... 11
Buffering ........................................................................................................ 12
Summary and discussion of Paper I ............................................................. 14
3. HP1a TARGETING ................................................................................... 16
Chromosome-wide regulation of the 4th ...................................................... 16
The 4th chromosome .................................................................................. 16
POF ..............................................................................................................17
i
HP1a in chromosome 4 gene regulation ................................................... 19
HP1a and heterochromatin formation ........................................................ 20
Su(var)3-9, Setdb1 and G9a ......................................................................... 23
Summary of Paper II .....................................................................................25
4. MSL TARGETING .................................................................................... 29
Sex chromosomes and sex determination ................................................... 29
Sex Chromosome evolution ......................................................................... 30
Dosage compensation .................................................................................... 31
MSL complex ................................................................................................ 32
roX RNAs .................................................................................................. 34
MSL-complex and heterochromatin.............................................................35
Upregulation of the male X .......................................................................... 36
Mechanisms of MSL targeting ...................................................................... 37
Links between X and 4th chromosomes ....................................................... 38
Summary of Paper III ................................................................................... 38
Summary of Paper IV ................................................................................... 43
CONCLUSIONS ..............................................................................................45
ACKNOWLEDGMENTS ............................................................................. 46
REFERENCES ............................................................................................... 48
ii
LIST OF PUBLICATIONS
This thesis is based on the following publications (reproduced with
permission from the publishers):
I
Lina E Lundberg, Margarida L A Figueiredo, Per Stenberg, Jan Larsson
(2012). Buffering and proteolysis are induced by segmental
monosomy in Drosophila melanogaster. Nucleic Acids Res 40: 59265937.
II
Margarida L A Figueiredo, Philge Philip, Per Stenberg, Jan Larsson
(2012). HP1a recruitment to promoters is independent of H3K9
methylation in Drosophila melanogaster. PLoS Genet 8: e1003061.
III
Margarida L A Figueiredo, Maria Kim, Philge Philip, Anders
Allgardsson, Per Stenberg, Jan Larsson (2014). Non-coding roX RNAs
prevent the binding of the MSL-complex to heterochromatic regions.
PLoS Genet 10:e1004865.
IV
Margarida L A Figueiredo, Philge Philip, Jan Larsson (2015). MSL
interaction with non-roX non-coding RNAs. (Manuscript).
iii
ABSTRACT
Regulation of gene expression can occur at different levels, ranging from
single genes to genomic regions and even to entire chromosomes.
Understanding which epigenetic mechanisms are involved in this regulation,
especially how protein regulators are targeted to chromatin, has been the
focus of my thesis.
I show that genes in monosomic regions are buffered, i.e., expressed at a
higher level than the expected 50% of the wild type level. The buffering is
general, it is primarily affected by gene length and is not affected by the
presence of other monosomic regions. Additionally, the expression of
proteolysis genes is induced in aneuploidies.
Gene expression regulation at a chromosome-wide level has so far only been
described for the X chromosome and for the 4th chromosome of Drosophila
melanogaster. The 4th chromosome gene expression is regulated by both POF
and HP1a, which target exons of active genes. Additionally, HP1a targets
promoters. I found that Setdb1 and Su(var)3-9 recruit HP1a to the 4th
chromosome and to pericentromeric regions, respectively, by di- and trimethylation of H3K9. Importantly, HP1a is recruited to promoters of active
genes independently of methylated H3K9. The promoters bound by HP1a are
enriched in HP2, in A/T content and are DNase sensitive. We propose that
HP1a is bound to the H3 histone core at promoters and that the promoter
targeting functions as the nucleation site from which HP1a spreads via H3K9
methylation.
MSL complex targets the male X chromosome and is partially responsible for
dosage compensation, by upregulation of X chromosome gene expression
almost two times. The importance of roX long non-coding RNAs in MSL
recruitment has been one important focus of this thesis. I found that in
absence of roX, MSL targets high affinity sites on the X chromosome.
Additionally, a complete and active MSL complex is bound to six genes of the
4th chromosome. Interestingly, when roX RNAs are not present, MSL targets
genomic regions enriched in Hoppel transposable elements and repeats. We
propose that the heterochromatic targeting represents an ancient function
of the MSL complex and that the roX RNAs evolved to restrict MSL binding to
the X chromosome. I further found that MSL associates with many different
RNAs when roX are absent, including a set of snoRNAs.
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INTRODUCTION
CHAPTER 1
1. INTRODUCTION
It´s truly fascinating to imagine that once in our life time we were nothing
more than a single cell, with an incredibly unique genome. Rounds of cell
divisions, organized cell movements and cell differentiation shaped us. We
are the result of several different populations of cells sharing the same
genome. How the different cell types express their specific sets of genes,
while keeping others silenced, and how this information is passed on to the
daughter cells and remembered through successive rounds of cell division is
called epigenetics.
Epigenetics
Epigenetics is a field of research focused on understanding how changes in
patterns of gene expression, not due to changes in the DNA sequence, are
established and maintained through mitosis and/or meiosis. DNA
methylation and histone modifications within and around specific genes are
known as the main epigenetic marks that are involved in gene expression
regulation and cell memory.
Not only is the epigenetic information heritable through cell divisions but in
some cases also from generation to generation. Transgenerational
epigenetics can occur when stochastic or environmental-induced (for
instance nutrition) epigenetic changes in the germline of the parents are
transmitted to the offspring (CHONG AND WHITELAW 2004; HEARD AND
MARTIENSSEN 2014). Additionally, epigenetic differences between maternally
and paternally inherited alleles can dictate which allele will be expressed, a
phenomenon called genomic imprinting (DELAVAL AND FEIL 2004). Imprinting
causes some genes in the adult individual to have monoallelic expression. As
an example, during gametogenesis or early embryogenesis, DNA
hypermethylation at the maternal allele of an imprinted gene will ensure that
only the paternally inherited allele is active in the adult (DELAVAL AND FEIL
2004). Development can be compromised in imprinting disorders, the
Angelman and Prader-Willi syndromes are well known examples (BUITING et
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INTRODUCTION
al. 1995). The fact that several human diseases, like cancer and disorders of
the immune, endocrine and nervous system, are caused by dysfunctional
epigenetic mechanisms highlights the importance of this field of research
(FALLS et al. 1999).
Chromatin: the nucleosome
The genome of eukaryotic cells is organized in chromosomes, which consist
of linear molecules of DNA associated with histones and other proteins, in a
complex called chromatin. The basic repeated unit of chromatin is the
nucleosome: an octamer of histones around which 147 base pairs of DNA is
wrapped in 1.65 turns (figure 1) (LUGER et al. 1997). Each nucleosome in the
array is separated from each other by a short “naked” DNA segment of 2090 base pairs – linker DNA (SZERLONG AND HANSEN 2011). The eight histones in
each nucleosome are organized in two dimers of H2A-H2B and one tetramer
of H3-H4. Additionally, the linker histone H1 binds between the nucleosome
and the wrapped-DNA, stabilizing the nucleosome, and also interacts with
the linker DNA (LUGER et al. 1997).
Figure 1. Nucleosome assembly. Each histone molecule (excluding linker histone H1) is
formed by the histone core, consisting of three α-helices separated by two loops, and by Nterminal protruding tails. The histone loops are essential for the interaction nucleosome-DNA,
and the α-helices are essential for histone-histone interaction. The tetramer H3-H4 forms a
more stable core, in contrast to the two H2A-H2B dimers (SMITH AND STILLMAN 1991; LUGER et al.
1997). (adapted from (VENKATESH AND WORKMAN 2015)).
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INTRODUCTION
Different structural states of the nucleosome can be affected by the
incorporation of histone variants that replace the canonical histones H3, H2A
and H2B, and the linker histone H1. Histone variants can be incorporated in
nucleosomes throughout the cell cycle, at specific genomic regions and may
have tissue-specific expression patterns. For instance, CenH3 is a histone
variant that replaces H3 specifically at centromeres and plays a role in the
assembly of the kinetochore (BLACK AND CLEVELAND 2011; HENIKOFF AND SMITH
2015). In fact, CenH3 is considered a better marker for centromeres than the
DNA-sequence itself, since CenH3 localizes to neo-centromeres that lack the
α-satellite DNA repeats that are typical for centromeric DNA (HENIKOFF AND
SMITH 2015). Another example is the histone variant H2A.Z, which has been
proposed to have distinct nuclear functions including transcriptional
regulation, cell-cycle control, DNA replication, DNA damage repair,
chromosome segregation and genome integrity (HENIKOFF AND SMITH 2015). In
embryonic stem cells, H2A.Z is preferentially associated with promoters of
developmentally regulated genes that have both active and repressive marks
of transcription (KU et al. 2012).
Nucleosome evolution
Nucleosomes play a very important role in the organization of the genome
by compacting the DNA and allowing the long chromosome molecules to be
packed inside the small eukaryotic cell nucleus (in humans, approximately 2
meters of total DNA are fitted in a nucleus with a diameter of 6 µm).
Several lines of evidence suggest that the nucleosomes evolved in parallel
with the increase in genome size. Histones are highly conserved proteins,
with H2A, H2B, H3 and H4 being present in all eukaryotes. From the three
domains of life, Bacteria is the only one that doesn´t have histones. However,
even in Bacteria the single circular chromosome is tightly associated with
proteins, forming the nucleoid. The nucleoid–associated proteins (NAPs) are
involved in chromosome organization, by allowing the formation of domains
with different supercoils, and modulating gene expression (SANDMAN et al.
1998; WANG et al. 2011). The evolutionary history of histones can be traced
back to the Archaea domain of life, prokaryotes with a single circular
chromosome. In Methanothermus fervidus for instance, the histone-related
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INTRODUCTION
HMfA and HMfB proteins form tetramers that constrain 60 bp of DNA. These
studies on the origin of the nucleosome are in agreement with one suggested
theory on the evolution of the first eukaryotic cells: they were the result of a
fusion between Archaea and eubacterium, the first becoming the nucleus
and the latter the cytoplasm (YUTIN et al. 2008). In the transition from
prokaryotes to eukaryotes, as the genome size increased, the nucleosomes
became essential to maintain and regulate the conformational flexibility of
DNA and its reversible structural changes (MINSKY et al. 1997). As for the
eukaryotes, the evolution from single-cell species to multicellular ones
originated a disproportional increase in genome size relative to the nuclear
volume. Larger genomes had to compact their chromatin more, and a recent
study has shown that eukaryotic genome expansion was accompanied by an
acquisition of arginines in H2A N-terminus and that this directly affects
chromatin compaction (MACADANGDANG et al. 2014).
Histone modifications
There are a large number of identified reversible histone posttranslational
modifications (PTMs), made by highly specific enzymes on different amino
acids of the histone tails and also on the histone core. While the
modifications on histone tails have been the most studied, it has been
suggested that modifications on residues of the histone core lateral surface
can affect more directly the interaction histone – nucleosomal DNA, altering
nucleosome stability (TESSARZ AND KOUZARIDES 2014). Examples of histone
modifications include: acetylation of lysines, methylation of lysines and
arginines, phosphorylation of serines and threonines, ubiquitination of
lysines, sumoylation of lysines, ADP-ribosylation of glutamic acids,
glycosylation and citrullination of arginines (KHORASANIZADEH 2004; TESSARZ
AND KOUZARIDES 2014). In the work presented in this thesis the focus has been
on acetylation and methylation of histone N-terminal tails.
Acetylation of lysines is generally associated with activation of transcription
and it has been proposed that it neutralizes the positive charges of lysines,
making the interaction between the histone and the DNA weaker and
thereby increasing the accessibility of the transcriptional machinery to the
DNA (LUGER et al. 1997; FENLEY et al. 2010; TROPBERGER et al. 2013). Acetylated
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INTRODUCTION
lysines are recognized by proteins with bromodomains, like histone
acetyltransferases (HAT) – which themselves produce this modification, and
by chromatin remodelling proteins (ZENTNER AND HENIKOFF 2013). In
Drosophila melanogaster, acetylation of lysine 16 of histone H4 (H4K16ac) is
highly enriched on the male X chromosome and is believed to contribute to
dosage compensation by increasing the transcriptional output - this is an
important topic of my thesis, discussed in more detail in chapter 4.
Mono-, di- or tri- methylation can occur on lysines, by the actions of histone
lysine methyltranferases (HKMT), and can be associated with activation of
transcription (H3K4me and H3K36me) or with repression (H3K9me and
H3K27me). Methylated lysines can be recognized by proteins with specific
domains, including chromo domains (ZENTNER AND HENIKOFF 2013). As an
example, H3K9me is recognized by heterochromatin protein 1 (HP1), a
chromo domain containing protein, which is associated with the formation
of a highly compacted chromatin structure – this is an important topic of my
thesis that will be discussed in more detail in chapter 3.
The existence of enzymes like histone deacetylases (HDACs) and histone
lysine demethylases (KDMs) makes these posttranslational modifications
reversible and contributes to dynamic changes in chromatin structure.
The effect of histone modifications on chromatin structure can be direct or
indirect by becoming targets for binding of non-histone proteins, “the
epigenetic readers” which can act on chromatin organization. The readers
can bind specifically to the histone modifications (for instance, by their
bromo or chromo domains) and recruit ATP-dependent chromatin
remodellers or histone chaperones (LUGER et al. 2012).
DNA methylation
DNA can be methylated, for example at cytosines (5-mC) of CytosineGuanine dinucleotides (usually denominated CpG) by DNA
methyltransferases (DNMT) and this is an epigenetic mark that has been
extensively studied in mammals. DNA methylation is conserved in fungi and
plants and can occur at other nucleotides and dinucleotides than CpG (JONES
2012). Although CpG is not globally abundant in mammalian genomes, there
are regions enriched in CpG, called CpG islands (SMITH AND MEISSNER 2013).
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INTRODUCTION
CpG islands at promoters of housekeeping genes and developmentally
regulated genes are usually unmethylated, correlating to their active state.
In contrast, DNA methylation is enriched at satellite repeats and
transposable elements and functions to silence these elements (SMITH AND
MEISSNER 2013). One entire X chromosome in each mammalian female cell is
in fact hypermethylated, contributing to its almost complete gene
inactivation, as part of the dosage compensation system in mammals, which
will be briefly discussed in chapter 4. DNA methylation is recognized by
proteins with methyl-CpG binding domains, which act together with histone
modification readers to recruit chromatin remodellers and affect chromatin
structure.
DNA methylation has been reported to be absent in yeast and C. elegans. In
Drosophila melanogaster the presence of DNA methylation has been
controversial. Although still debated, some studies have shown that DNA
methylation can occur at non-CpG locations and at early embryonic stages
(LYKO et al. 2000; KUNERT et al. 2003; WEISSMANN et al. 2003) and in
retrotransposon silencing (PHALKE et al. 2009). However, the existence and
potential importance of DNA methylation in Drosophila remains
controversial and elusive (RADDATZ et al. 2013).
Chromatin states
Chromatin remodelling is a dynamic process and achieving different levels of
chromatin compaction is important for fitting the long DNA molecules inside
the small nucleus, for the condensation of the mitotic chromosomes, and to
regulate gene expression.
The primary structure of chromatin is arranged as an array of nucleosomes
forming the 10 nm thick “beads-on-a-string” structure, seen under the
electron microscope (OUDET et al. 1975; SZERLONG AND HANSEN 2011). The
presence of the linker histone H1 is important in the formation of a higher
order chromatin structure, where each nucleosome in the array can interact
with other nucleosomes and with DNA, resulting in a DNA fiber compaction
into a secondary structure of 30 nm. Although the 30 nm chromatin fiber has
been extensively studied in vitro, its structure has not been resolved yet and
it has been difficult to prove its existence in vivo (ELTSOV et al. 2008; FUSSNER
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INTRODUCTION
et al. 2012). The most compact chromatin form that exists is the 700 nm thick
metaphase chromosome, which is visible under the light microscope and
requires the actions of condensin and topoisomerase II (KHORASANIZADEH
2004).
In an interphase chromosome there are distinguishable chromatin
arrangements: a highly compacted structure called heterochromatin, and a
less compacted structure called euchromatin. Heterochromatin by definition
fails to decondense after telophase in the cell cycle. The majority of genes
reside within euchromatin, since the open structure facilitates the targeting
by RNA polymerase and transcriptional machinery. Heterochromatin is
usually associated with gene silencing, is typically enriched in H3K9me and
HP1, replicates late in S phase and it doesn´t recombine. Constitutive
heterochromatin can be found at regions that are gene poor and highly
enriched in repetitive DNA sequences, satellite repeats and transposons, like
centromeres and telomeres, and is present in all cell types. Some active
genes reside within constitutive heterochromatin and understanding which
mechanisms allow their expression in such a highly compacted repressive
environment is an interesting topic of research, which will be discussed in
chapters 3 and 4. Additionally, depending on the cell type, the expression of
tissue specific genes needs to be switched on or off. These genomic regions
can switch between euchromatic active states and heterochromatic inactive
states. Facultative heterochromatin exists in regions that are
heterochromatic in some cell types, but euchromatic in other cell types. The
inactive X chromosome in the cells of females and imprinted genes are good
examples of facultative heterochromatin (TROJER AND REINBERG 2007).
Long non-coding RNAs
Eukaryotic genomes contain many genes coding for non-coding RNAs
(ncRNAs). ncRNAs are involved in several cellular processes of gene
regulation and chromatin modification, and it has been proposed that they
have evolved with organismal complexity. ncRNAs can be transcribed
antisense to protein-coding genes and from introns of protein-coding genes.
A large fraction of ncRNAs are small RNAs, like for instance small nuclear
RNAs (snRNAs) involved in splicing; small nucleolar RNAS (snoRNAs) involved
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INTRODUCTION
in modifying nucleotides in rRNAs and other RNAs; and micro RNAs (miRNAs)
which are involved in the RNA interference pathway (MORRIS AND MATTICK
2014).
Long non-coding RNAs (lncRNAs) are RNA transcripts typically longer than
200 bp, polyadenylated and without open reading frames. lncRNAs can form
higher order secondary structures and bind proteins or base-pair with
specific RNA or DNA targets, for instance forming RNA-DNA:DNA triplexes.
lncRNAs are involved in regulation of gene expression (repressing it or
activating it), typically by forming ribonucleoprotein complexes that are
recruited in cis (to genes near the lncRNA transcription site) or in trans (to
genes in other genomic loci). The involvement of lncRNAs in dosage
compensation is evident both in mammals and in Drosophila, and it will be a
topic discussed in the chapter 4 of this thesis. Xist lncRNA in mammals acts
in cis, coating the entire female X chromosome from which it is transcribed
and, by recruiting specific proteins, induces formation of heterochromatin
and contributes to X inactivation. The two lncRNAs roX1 and roX2 in
Drosophila can act both in cis and in trans, coating the entire X chromosome
in males, and by recruiting MSL proteins, induce up-regulation of gene
expression from X chromosomal genes. In either case it is still not known how
these lncRNAs can specifically recognize one entire chromosome, especially
the very distant genes (FATICA AND BOZZONI 2014).
Drosophila melanogaster
Also known as the fruit fly, Drosophila melanogaster is one of the best
studied model organisms in genetics. Although phenotypically very different
from us, approximately 75% of known disease-related genes in humans have
homologs in Drosophila (BIER 2005). 143.7 megabases (Mb) of the Drosophila
melanogaster genome has been fully sequenced and annotated, including
some of the heterochromatic sequences in pericentromeric regions (DOS
SANTOS et al. 2015). The epigenetic landscape of Drosophila melanogaster has
also been extensively studied (for which the modENCODE consortium
contributed significantly) and the genome-wide distribution of a large
number of chromatin proteins, as well as histone modification marks, have
been mapped in different tissues and/or cell types (by ChIP-chip and ChIPseq data) (CELNIKER et al. 2009; ST PIERRE et al. 2014). Gene expression profiles
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INTRODUCTION
at different developmental stages are also available (RNA-seq data). One
advantage of Drosophila in genetic studies has been the possibility to easily
create mutants, for which the engineered balancer chromosomes and the Pelement have proven to be very important tools.
The genome of Drosophila melanogaster is constituted by three autosome
pairs (2, 3 and 4) and a pair of sex chromosomes (XX or XY). The 4th and the Y
chromosomes are highly heterochromatic, being enriched in repeated DNA
sequences and transposons. In the study of chromosomes and epigenetics,
Drosophila has a great advantage in having giant chromosomes in some
tissues, called polytene chromosomes. Polytene chromosomes are formed
when DNA replicates without the cell going through division
(endoreplication) and in salivary glands this results in approximately 2000
copies of DNA molecules aligned together, including both homolog
chromosomes (figure 2).
Figure 2. Drosophila melanogaster chromosomes. Polytene chromosomes from a salivary
gland nuclei in comparison with mitotic chromosomes from a brain nuclei (in the rectangle)
from third instar larvae stained with DAPI and visualized with 40x and 100x lenses,
respectively. Left arms (2L, 3L) and right arms (2R, 3R) of chromosomes 2 and 3 are indicated
as well as the X and the 4th. Note that the Y chromosome, along with other very
heterochromatic regions of the genome (pericentromeric regions and part of the 4 th) are not
seen in the salivary gland nuclei since they don´t endoreplicate. The pericentromeric regions
of all polytene chromosomes are joined together in the chromocenter. In the mitotic nucleus,
notice the two dots corresponding to the two replicated 4th homologous chromosomes.
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INTRODUCTION
Mechanisms of targeting
The main focus of my thesis is to understand how proteins are recruited to
chromatin and how it affects regulation of gene expression at a larger scale
rather than on single genes. In chapter 2, I will discuss how gene expression
is regulated at segmental monosomies. In chapters 3 and 4, the main work
of my thesis, I will discuss the two chromosome-wide targeting mechanisms
in Drosophila melanogaster: the mechanisms on the 4th chromosome and on
the X chromosome, respectively. In chapter 3, I will focus on the role of each
HKMT in HP1a recruitment. In chapter 4, I will discuss the role of roX lncRNAs
in the recruitment of MSL-complex and also the search for new RNAs that
interact with MSL.
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SEGMENTAL ANEUPLOIDIES
CHAPTER 2
2. SEGMENTAL ANEUPLOIDIES
Aneuploidy
Aneuploidy has both been defined as an alteration of the normal number of
whole chromosomes in a cell or organism (chromosomal aneuploidy), and as
an alteration of the normal number of a chromosomal region in a cell or
organism (segmental aneuploidy). A diploid organism, for instance, can have
one or three copies of one of the chromosomes (chromosomal monosomy
or trisomy) and one or three copies of a region of a chromosome (segmental
monosomy or trisomy).
Chromosomal aneuploidies can be caused by merotellic attachments, where
a single kinetochore attaches to microtubules that arise from both poles of
the spindle, by spindle assembly checkpoints defects or by chromosome
cohesion defects (GORDON et al. 2012). Segmental aneuploidies can be
caused by replication slippage, non-allelic homologous recombination and
non-homologous end-joining (SCHRIDER AND HAHN 2010).
The presence of an extra copy or the loss of a copy of a chromosome or
segment is usually detrimental for single cells and organisms, because it can
result in an increase or decrease in production of transcripts from those
genes, or it can alter the doses of regulatory sequences, resulting in gene
network deregulation and possibly leading to genomic instability (GORDON et
al. 2012). Studies in yeast and in mouse embryonic fibroblasts have shown
that in single cells, one extra copy of a chromosome causes slow proliferation
(TORRES et al. 2007; WILLIAMS et al. 2008; PAVELKA et al. 2010). In humans,
aneuploidies are the leading cause of spontaneous abortions and birth
defects (COLNAGHI et al. 2011). Chromosomal monosomies are generally
more deleterious than chromosomal trisomies, and only trisomy of
chromosome 21 (Down´s syndrome) is viable in humans. Trisomies 13 and 18
have severe developmental impairments and usually die during the first
months of life (TORRES et al. 2008). In cancers, aneuploidies do not seem to
be deleterious to the cells and may actually contribute to proliferation of the
cancer cells, which is demonstrated by the fact that aneuploidies are present
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SEGMENTAL ANEUPLOIDIES
in most types of cancers (80% of solid tumours and 60% of hematopoietic
tumours) (STANKIEWICZ AND LUPSKI 2010). Extra copies of chromosomes can be
found in healthy organisms in nature, in fact many plants and animals have
cells with one or more extra copies of all their chromosomes: a condition
called polyploidy (genome doubling). For instance human hepatocytes
contain up to eight copies of all chromosomes (octoploidy) (STENBERG AND
LARSSON 2011; GENTRIC et al. 2012).
Segmental aneuploidies can result in copy number variations (CNV):
differences in large genomic regions (from 1 kb to several Mb) for different
individuals of the same species, due to duplication or deletion events. In fact,
two individuals from the same species are likely to have dozens to hundreds
of CNVs (SCHRIDER AND HAHN 2010; STANKIEWICZ AND LUPSKI 2010). CNVs along
with single nucleotide polymorphisms (SNPs) and indels (insertion and
deletion of short segments of nucleotides up to 50 bp) account for the
genomic variation between individuals of the same species (SCHRIDER AND
HAHN 2010). CNVs can also be different among human populations, for
instance the gene that encodes a haptoglobin-related protein, used in
defense against trypanosomes, is present in 2 copies in European
populations, in 1-2 copies in Asian populations, but in 4-8 copies in 25% of
African populations (HANDSAKER et al. 2015).
Buffering
An important question regarding chromosomal and segmental aneuploidies
is how the variation in gene dose contribute to the different phenotypes:
how the transcript levels and the protein dose changes. Some chromosomes
can exist in monosomic condition in Drosophila melanogaster healthy
individuals, for instance the X chromosome in males and the 4th
chromosome. It is known that in Drosophila the viability of these two
monosomic conditions depend on the male-specific lethal (MSL)
ribonucleoprotein complex and the Painting of fourth (POF) protein which
are responsible for transcriptional dosage compensation of the X- and 4th
chromosomes, respectively (discussed in chapters 3 and 4 of this thesis). If
we consider the prevalence of CNVs in healthy individuals, we can
hypothesize that there might be systems that compensate for the gene
dosage differences in autosomes – buffering systems. Since duplications and
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SEGMENTAL ANEUPLOIDIES
deletions provide genetic variability and are therefore driving forces of
evolution, incomplete buffering may exist as a way to allow some degree of
genetic variation, while buffering collapse could lead to lethality (STENBERG et
al. 2009).
Several studies in yeast and mammalian cell lines with extra copies of
chromosomes have shown that the transcriptional response increases with
the increase in gene copy number, suggesting that transcriptional dosage
compensatory mechanisms do not exist (TORRES et al. 2007; WILLIAMS et al.
2008; STINGELE et al. 2012). Some of these studies claimed that the respective
protein levels were not increased at the same degree as the mRNA, which
suggests that there might exist a buffering effect at a posttranscriptional
level (TORRES et al. 2007; STINGELE et al. 2012). One of these studies proposed
that in trisomies the increased amount of proteins produced disrupt cellular
physiology interfering with metabolic pathways, and the cell copes with it by
increasing protein degradation (TORRES et al. 2007). In fact, mutations in the
deubiquitylation enzyme UBP6 makes yeast with extra chromosomal copies
grow faster (TORRES et al. 2010). Another study has found that in human
trisomic and tetrasomic cell lines, protein folding is significantly impaired
(DONNELLY et al. 2014).
In contrast, studies using microarray in trisomic fetal cell lines, and in
segmental aneuploidies (deficiencies and duplications) in both Drosophila
and maize have shown that the transcriptional response was not
proportional to the gene dose and thus support the existence of buffering
mechanisms (FITZPATRICK et al. 2002; GUPTA et al. 2006; MAKAREVITCH et al.
2008; STENBERG et al. 2009; MCANALLY AND YAMPOLSKY 2010; ZHANG et al. 2010).
In one particular study in Drosophila it was shown, using microarrays, that
mRNA levels at three deficiencies on chromosome 2L and monosomies for
chromosome 4 were 64% of wild type levels, instead of the expected 50%
(STENBERG et al. 2009). This buffering of RNA levels seen in deficiencies didn´t
occur at the studied duplications, which agrees with the fact that segmental
monosomies are less well tolerated than segmental trisomies. In the cited
study, only genes with expression levels that could be readily measured were
included, since low expressed genes or inactive genes will look like they are
being fully compensated. The buffering effect that was found was suggested
to act in a general mode on all genes included in the deficiency, since the
13
SEGMENTAL ANEUPLOIDIES
buffering effect was normally distributed among the genes, around a mean
of 64% of wild type expression. The authors suggested that the buffering
effect could also be the result of feedback regulation of a few genes which
could result in a local high concentration of transcriptional factors, increasing
the transcription in the entire monosomic region. Additionally, tissue-specific
genes were found to be more buffered than ubiquitously expressed genes
(STENBERG et al. 2009).
Summary and discussion of Paper I
We were interested to study the factors that affect buffering of segmental
aneuploidies, at regional and individual gene levels in more detail.
Additionally, we wanted to investigate the general transcriptional response
to segmental aneuploidies, i.e., how the transcription of genes outside the
monosomic region is affected. In this study we analysed the mRNA
expression levels by microarray on adult female flies from seven lines with
segmental deficiencies (Df). The Df lines we analysed differ in length, total
number of genes and number of expressed genes. We created pairwise
combinations of deficiencies to study the combinatorial effects of buffering.
We confirmed the presence of a weak but significant buffering at the RNA
level and found the mean expression of the genes in each deficiency to be
between 54% and 58% of wild type (WT), instead of the expected 50%, if
there would be no buffering. The fact that the buffering effect obtained was
lower than what previous studies have shown (FITZPATRICK et al. 2002; GUPTA
et al. 2006; MAKAREVITCH et al. 2008; STENBERG et al. 2009) is probably due to
the more stringent cutoff applied on expression levels in this study.
Since cancers have different combinations of aneuploidies and Drosophila
can tolerate monosomy up to approximately 1% of the genome, we
hypothesized that combining deficiencies could decrease the general
buffering (ultimately leading to lethality). When studying the pairwise
combinations of deficiencies, we didn´t find any combinatorial effect on
buffering, i.e., buffering was not enhanced or reduced by combining any two
deficiencies and no deficiency had a directional influence on the buffering of
the other deficiencies.
14
SEGMENTAL ANEUPLOIDIES
We found a weak correlation between the gene expression ratio (Df/WT) and
the total number of deleted genes, i.e., the fewer the deleted genes the
larger the buffering.
When we analysed the buffering at gene level we found that longer genes
are more buffered than shorter genes, independently on both expression
level and whether the genes are ubiquitously expressed or tissue-specific.
These results led us to speculate that buffering mechanisms might involve
transcription elongation. Alternatively, buffering could occur by passive
mechanisms, like the looping out of the monosomic region, since it is
unpaired, to more transcriptional permissive environments.
When analysing the transcriptional changes outside the monosomic regions,
we didn´t find any spreading of the buffering effect from the monosomic
region to the neighbouring diploid regions.
Interestingly, we found that in the individuals carrying monosomic regions,
the transcription of some genes encoding for proteins with peptidase and
proteolysis activities was upregulated. This result agrees with the view that
protein degradation is a general response to aneuploidies, since it´s seen in
both duplications and deficiencies (TORRES et al. 2010; DONNELLY et al. 2014).
It is important to note that the deficiencies used in this study have been kept
in monosomic condition in stocks for years in the lab. It is thus unclear if
general regulatory mechanisms that compensate for gene copy number
imbalances exist naturally or if the DrosDel (RYDER et al. 2007) deficiency lines
analysed in this study had time to select for increases at the transcriptional
level.
15
HP1a TARGETING
CHAPTER 3
3. HP1a TARGETING
Chromosome-wide regulation of the 4th chromosome
Chromosome-wide gene regulation is well known for the X chromosome in
many species, as part of dosage compensation systems. Chapter 2 of this
thesis describes buffering, and it has become evident that buffering systems
at the transcriptional level exist and that they compensate for gene dosage
differences at least for monosomic regions. Less is known about
chromosome-specific systems that regulate gene expression in an autosomespecific manner. So far, the only well described system is the one that targets
and regulates the 4th chromosome of Drosophila melanogaster. The 4th
chromosomes in both males and females are specifically targeted by the
gene regulatory protein Painting of Fourth (POF) – the only known autosomespecific protein (LARSSON et al. 2001). The majority of Drosophila species have
a dot chromosome similar to the 4th (usually referred to as the F element)
(MULLER 1940; ASHBURNER 1989) and the binding of POF to the F element is
conserved in evolution (LARSSON et al. 2004). There are several evolutionary
links between POF and chromosome 4 and the X chromosome dosage
compensation, and this will be a topic of discussion in chapter 4 of this thesis.
The 4th chromosome
The fourth chromosome of Drosophila melanogaster is an atypical autosome
since it is the smallest chromosome, approximately 5 Mb long, and it is highly
heterochromatic (LOCKE AND MCDERMID 1993). The 4th has a pericentromeric
region of 3-4 Mb which is gene-poor and consists of highly condensed
heterochromatin, enriched in A-T and in simple satellite repeats, and this
region is underreplicated in polytene chromosomes (LOCKE AND MCDERMID
1993; RIDDLE AND ELGIN 2006; RIDDLE et al. 2009). The rest of the 4th consists of
a polytenized region of 1.28 Mb where almost all genes reside and which is
also enriched in repetitive DNA, transposable elements and features typical
of heterochromatin, like the presence of HP1a and H3K9me (BARIGOZZI et al.
1966; MIKLOS et al. 1988; EISSENBERG et al. 1992; CZERMIN et al. 2002; SCHOTTA
16
HP1a TARGETING
et al. 2002). Other heterochromatic characteristics of the 4th is that it doesn´t
recombine in meiosis (HOCHMAN 1976), it replicates late in S phase (BARIGOZZI
et al. 1966) and it suppresses the expression of transgenes inserted on the
4th, by the well-studied phenomenon of position-effect variegation – see
below (WALLRATH AND ELGIN 1995; WALLRATH et al. 1996). Although highly
heterochromatic, the 4th chromosome has a gene density similar to the
autosome arms, consisting of at least 92 genes (JOHANSSON et al. 2007a). The
expression of the 4th chromosome genes in this heterochromatic
environment has been proposed to be fine-tuned by both POF and HP1a
which form a balancing system where POF stimulates and HP1a represses
gene expression (JOHANSSON et al. 2007a; JOHANSSON et al. 2007b; JOHANSSON
et al. 2012).
In polytene chromosomes, while HP1a targets the pericentromeric regions
of all chromosomes and the whole 4th chromosome, POF only binds to the
polytenized part of the 4th (figure 3) (LARSSON et al. 2001; JOHANSSON et al.
2007a).
Figure 3. POF targets the 4th (left) while HP1a targets the 4th and the chromocenter (right).
Polytene chromosomes from salivary gland nuclei of wild type third instar larvae,
immunostained with antibodies against POF (left) and HP1a (right). DNA is stained with DAPI
(blue).
POF
Translocation experiments between the 4th chromosome with breakpoints in
the proximal part (closer to the pericentromeric region) or more distal part
(closer to the telomeric region) and other chromosomes suggested that POF
17
HP1a TARGETING
initiates its targeting at the proximal part of the 4th, the nucleation site, and
then spreads in cis to the distal part (LARSSON et al. 2001). These experiments
also showed that POF targets the 4th chromosome with some sequence
specificity since it doesn´t spread in cis to the chromosomes that are
translocated to the 4th. POF was also shown to spread in trans, between the
endogenous 4th and the translocated 4th when these two chromosomes pair
(LARSSON et al. 2001; JOHANSSON et al. 2007a). POF is a 55 kDa protein that
contains an RNA recognition motif and although RNAse treatment doesn´t
affect POF targeting to the 4th (LARSSON et al. 2001; JOHANSSON et al. 2012),
RNA immunoprecipitation followed by tiling arrays (RIP-chip) experiments
have shown that POF has a general affinity for nascent RNAs, and more
specifically to RNAs from the 4th chromosome (JOHANSSON et al. 2012).
The suggestion that POF is responsible for dosage compensation of the genes
on the 4th chromosome came from the fact that homozygous loss-of-function
Pof mutants are lethal when they only have one 4th chromosome, but are
viable and fertile if both 4th chromosomes are present (JOHANSSON et al.
2007a). Whole transcriptome studies by expression microarray experiments
have further shown that homozygous Pof mutants have a significant
reduction in mRNAs encoded from chromosome 4 genes (JOHANSSON et al.
2007a; LUNDBERG et al. 2013). An additional study has shown that POF
stimulates the expression of 4th genes independently on chromosome 4 copy
numbers (STENBERG et al. 2009). In the cited study, when fold changes in
mRNA expression of haplo-4 flies, compared to wild type, were calculated
based on expression microarray results, it was shown that the genes on the
4th in haplo-4 flies are buffered to 64% of wild type levels, instead of the
expected 50% if there would be no buffering - a similar level of buffering to
what is seen in segmental monosomies (see chapter 2). Chromosome 4 genes
in triplo-4th flies were also buffered to 139% of wild type, instead of the
expected 150% if there would be no buffering, contrary to what was seen for
segmental trisomies (see chapter 2). Importantly, the study showed that the
genes more strongly buffered in haplo-4 flies are the ones that have the
largest decrease of expression in Pof mutants, compared to wild type,
showing that the POF protein in this case is responsible for 4th chromosome
dosage compensation (STENBERG et al. 2009).
18
HP1a TARGETING
It remains elusive by which mechanism POF stimulates transcription output
from the 4th chromosome. It has been shown that genes located in
pericentromeric regions and chromosome 4 genes (genes enriched in HP1a)
show an increased ratio between whole cell transcripts and nuclear
transcripts, compared to other genes. It has been suggested that one
potential role of HP1a and POF might be increasing the export of transcripts
from the nucleus by taking advantage of the positioning close to the nuclear
envelope (JOHANSSON et al. 2012). In fact, highly heterochromatic regions of
the genome are in close proximity to the nuclear envelope and
transcriptionally active genes may interact with the nuclear pore complex
(LANCTÔT et al. 2007; CAPELSON et al. 2010). Whether genes located in
heterochromatic regions take advantage of their location close to the nuclear
periphery when activated remains an important question to answer.
HP1a in chromosome 4 gene regulation
The fact that chromosome 4 gene regulation is not affected only by POF but
also by HP1a was first showed by the general increase in expression from 4th
genes caused by RNAi knockdown of HP1a (LIU et al. 2005; JOHANSSON et al.
2007a). There is in fact a negative correlation between fold change in gene
expression from the 4th caused by Pof homozygous mutations and by HP1a
RNAi (JOHANSSON et al. 2007a). An expression microarray study in first instar
larvae has also shown that HP1a transheterozygous mutants display an
increase in gene expression level from the 4th chromosome (LUNDBERG et al.
2013). These results argue for the existence of a balancing system that
regulates gene expression on the 4th chromosome: POF stimulates while
HP1a represses expression (JOHANSSON et al. 2007a). POF and HP1a colocalize
on the 4th chromosome and their targeting on polytenes has been shown to
be interdependent, since in Pof homozygous mutants there is less HP1a
targeted to the 4th and in HP1a transheterozygous mutants no POF is seen
on the 4th chromosome (JOHANSSON et al. 2007a). A recent study has
challenged this view on POF and HP1a interdependence, showing that in
HP1a transheterozygous mutants, POF ChIP-chip enrichment values on the
4th are unaffected (RIDDLE et al. 2012). The discrepancy between polytene
stainings and ChIP-chip experiments when it comes to POF binding in HP1a
mutants remains to be understood. In a sensitised translocation system, POF
19
HP1a TARGETING
spreading on the 4th chromosome depends on heterochromatin. In this
system, increasing the amount of heterochromatin, by decreased
temperature or removal of the highly heterochromatic Y chromosome, also
led to an increase in POF targeting (JOHANSSON et al. 2007a). Additionally,
experiments using chromatin immunoprecipitation followed by tilling arrays
(ChIP-chip), in both S2 cell lines and salivary gland tissue, have shown at a
high resolution that POF and HP1a bind to the same regions on the fourth
chromosome, with a bias towards exons of actively transcribed genes, but
interestingly, HP1a binds additionally to promoters (JOHANSSON et al. 2007b).
The enrichment of HP1a in promoters of active genes is intriguing and is the
main focus of paper II – see below.
HP1a and heterochromatin formation
HP1a was first identified in Drosophila melanogaster as a non-histone
chromosomal protein associated with heterochromatin that when mutated
acts as a suppressor of variegation - Su(var) (SINCLAIR et al. 1983; JAMES AND
ELGIN 1986; EISSENBERG et al. 1990). Position-effect variegation is a
phenomenon by which an active gene becomes silent when inserted inside
or in the vicinity of a heterochromatic region, probably due to the spreading
of heterochromatin (MULLER AND ALTENBURG 1930; EISSENBERG AND REUTER
2009). This happens in some cells but not in others, which leads to a mosaic
(or variegated) expression of the gene in the adult. The hp1a gene, also called
Su(var)2-5, when mutated suppresses the silencing of a transgene inserted
in a heterochromatic environment. In other words, it suppresses positioneffect variegation (SINCLAIR et al. 1983). Several other mutations have been
identified as suppressors of variegation, like mutations in Su(var)3-9 and
Su(var)3-7 (SINCLAIR et al. 1983; EISSENBERG AND REUTER 2009). Su(var)3-7,
Su(var)3-9 and HP1a are in different ways involved in heterochromatin
assembly and function and they colocalize in the chromocenter, at some
telomeres and euchromatic sites. They might be loaded on the chromatin in
an hierarchical manner: Su(var)3-7 recruits Su(var)3-9 and Su(var)3-9 recruits
HP1a (CLEARD et al. 1997; DELATTRE et al. 2000; SCHOTTA et al. 2002; DELATTRE
et al. 2004; SPIERER et al. 2005). HP1a is an essential protein and it is well
known for being involved in heterochromatin formation, being enriched at
pericentromeric and telomeric regions and binding both di- and
20
HP1a TARGETING
trimethylated H3K9 (JAMES AND ELGIN 1986; LU et al. 2000; LACHNER et al.
2001).
HP1 is a conserved protein family found in many eukaryotic organisms
besides Drosophila, for instance in yeast, plants and humans. There are
several hp1 genes in each species coding for HP1 proteins with different
targeting specificities and probably also different functions (VERMAAK AND
MALIK 2009). For instance, in several Drosophila species, four conserved hp1
genes have been identified: hp1a, hp1b, hp1c, hp1d (LEVINE et al. 2012). In
Drosophila melanogaster it has been shown that: HP1a is bound to
pericentromeric and H3K9me-enriched regions; HP1b targets both
euchromatin and heterochromatin; HP1c localizes primarily to euchromatin;
HP1d targets heterochromatic compartments distinct from HP1a and
H3K9me, and is expressed specifically in the female germline; and HP1e
doesn´t seem to localize to chromatin and its expression is male germline
specific (SMOTHERS AND HENIKOFF 2001; VERMAAK et al. 2005; FONT-BURGADA et
al. 2008; VERMAAK AND MALIK 2009).
Most HP1 studies have been focused on the mammalian HP1α and on the
Drosophila melanogaster HP1a proteins. These studies have shown that HP1
proteins are constituted by a conserved chromo-domain separated from a
conserved chromo-shadow domain by a less conserved hinge region
(LOMBERK et al. 2006). HP1a chromo-domain is involved in the interaction
with H3K9me2,3 (BANNISTER et al. 2001; LACHNER et al. 2001; NAKAYAMA et al.
2001; JACOBS AND KHORASANIZADEH 2002), whereas the chromo-shadow
domain is required for protein-protein interaction, specifically for the HP1a
homodimerization and its interaction with several distinct proteins
(SMOTHERS AND HENIKOFF 2000). In fact, HP1a (and/or HP1α) has been shown
to interact with proteins involved in varied cellular processes not related to
heterochromatin formation, like for instance: transcriptional regulation,
replication, DNA repair, nuclear architecture and chromosomal maintenance
(LOMBERK et al. 2006). HP1a recruitment might depend on RNA, since the
hinge region of HP1a interacts with RNA and is involved in heterochromatin
targeting (SMOTHERS AND HENIKOFF 2001; MUCHARDT et al. 2002). Additionally,
it has been shown that the hinge region can be post-translationally modified
by phosphorylation, which affects HP1a targeting (BADUGU et al. 2005).
21
HP1a TARGETING
The current model for heterochromatin formation postulates that two
chromo-domains of a HP1a homodimer bind to H3K9me2,3 in the tails of two
adjacent nucleosomes, crosslinking the nucleosomes and restricting the
access to transcriptional machinery due to the formation of a more compact
chromatin structure (VERMAAK AND MALIK 2009). Additionally, HP1a interacts
directly with the histone methyltransferase Su(var)3-9 (see below), through
its chromo-shadow domain, which leads to the deposition of more H3K9me
and allows the spreading of H3K9me2,3 as well as HP1a, in a positive
feedback loop that facilitates the spreading of heterochromatin in cis from a
nucleation site (SCHOTTA et al. 2002; VERMAAK AND MALIK 2009).
HP1a targeting to chromatin is still not fully understood, and it might be a
combination of interaction with H3K9me2,3 marks, interaction with
chromatin-associated proteins and a DNA sequence specific recognition (DE
WIT et al. 2005). It has been shown that HP1a binding and silencing occurs
preferentially at regions enriched in DNA repeats, for example, HP1a targets
P-element tandem repeats (FANTI et al. 1998; DE WIT et al. 2005; DE WIT et al.
2007). Intriguingly, besides targeting the highly repetitive DNA from
pericentromeric regions and the 4th chromosome, HP1a also binds to a
euchromatic region of the 2L chromosome (2L:31) in polytene chromosomes
from third instar larvae and it was claimed to silence those genes (HWANG et
al. 2001).
Additionally to being mainly associated with gene silencing, HP1a has been
shown to bind active genes in both euchromatin and heterochromatin and in
these cases HP1a is suggested to be required for their proper expression
(WAKIMOTO AND HEARN 1990; LU et al. 2000; PIACENTINI et al. 2003; DE WIT et al.
2007; RIDDLE et al. 2011). An expression microarray study in Drosophila first
instar larvae has shown that besides HP1a repressing effect on chromosome
4 active genes, HP1a has a stimulating effect on pericentromeric active genes
(LUNDBERG et al. 2013).
The dependence of HP1a targeting on H3K9me has been challenged in some
studies. A study showed that HP1a lacking its chromo-domain is still able to
target heterochromatin (SMOTHERS AND HENIKOFF 2001). Additionally, it has
been suggested that HP1a can be targeted to some chromatin sites by
interacting with Piwi proteins (which bind to their DNA targets by interacting
with Piwi- interacting RNAs – piRNAs) in a manner independent on H3K9me
22
HP1a TARGETING
(HUANG et al. 2013). HP1a has been proposed to bind DNA directly, as it
seems to be the case for telomeric HP1a which doesn´t require H3K9me or
the action of Su(var)3-9 for its binding (ZHAO et al. 2000; PERRINI et al. 2004).
Interestingly, it has been shown that HP1 can bind nucleosomes
independently on the H3 tail and that HP1 in vitro can bind with high affinity
to the H3 histone-fold (ZHAO et al. 2000; NIELSEN et al. 2001; DIALYNAS et al.
2006; LAVIGNE et al. 2009). A recent study in C. elegans has shown that the
genome-wide targeting of protein HP1 Like 2 (HPL-2), is not affected in HKMT
mutant embryos that lack H3K9me (GARRIGUES et al. 2015).
Su(var)3-9, Setdb1 and G9a
In Drosophila melanogaster there are three conserved histone lysine
methyltransferases (HKMTs) responsible for methylating H3K9: Su(var)3-9,
Setdb1 and G9a. Su(var)3-9, discovered in Drosophila, localizes mainly to the
pericentromeric regions but also to some extent to the 4th chromosome, in
polytene chromosome stainings (SCHOTTA et al. 2002; LUNDBERG et al. 2013).
Su(var)3-9 di- and tri-methylates H3K9 in pericentromeric regions, through
its SET domain (REA et al. 2000; SCHOTTA et al. 2002). Additionally, this HKMT
has a chromo-domain possibly involved in interaction with H3K9me, and
interacts with HP1a through its N-terminal domain (REA et al. 2000; SCHOTTA
et al. 2002). Some studies have suggested that Su(var)3-9 can interact with
histone deacetylase enzymes and that deacetylation of H3K9 precedes its
methylation, since Su(var)3-9 only methylates H3K9 when this residue is not
acetylated (CZERMIN et al. 2001; LACHNER et al. 2001). The physical interaction
between Su(var)3-9 and linker histone H1 also seems to play a role in
Su(var)3-9 recruitment to chromatin and H3K9 methylation (LU et al. 2013).
Su(var)3-9 and HP1a binding to chromatin was shown to be interdependent.
In HP1a homozygous mutants, Su(var)3-9 relocalizes to ectopic places on
chromosome arms besides its normal sites, where it causes H3K9me (SCHOTTA
et al. 2002). In addition, on polytene chromosomes from Su(var)3-9
homozygous mutants, HP1a and H3K9me are lost from the chromocenter but
remain on the 4th chromosome (SCHOTTA et al. 2002; EBERT et al. 2004;
FIGUEIREDO et al. 2012). It has also been shown that, in Kc cells, the binding of
HP1a to most genes on the 4th chromosome is independent on Su(var)3-9
(GREIL et al. 2003).
23
HP1a TARGETING
In fact, from immunostaining experiments on polytene chromosomes, it was
shown that Setdb1 is the enzyme responsible for H3K9 methylation and HP1a
recruitment on the 4th chromosome, not acting in the chromocenter (SEUM
et al. 2007b; TZENG et al. 2007). In polytene chromosomes from Setdb1
homozygous mutants, POF, HP1a and H3K9me2,3, enrichments are
decreased on the 4th chromosome (TZENG et al. 2007). ChIP-chip analysis of
modENCODE data also showed that in Setdb1 mutants, POF recruitment is
impaired (RIDDLE et al. 2012). These results suggest that Setdb1 is required
for POF binding to the 4th, and in fact POF and Setdb1 were shown to interact
in vivo (TZENG et al. 2007). Whereas Su(var)3-9 and G9a are not required for
normal fly development, Setdb1 is required for normal oogenesis and was
claimed to be essential for survival (STABELL et al. 2006a; CLOUGH et al. 2007;
SEUM et al. 2007b; YOON et al. 2008).
G9a has H3K9 methylation activity in Drosophila melanogaster and like
Setdb1, was discovered as a homolog to the human protein. Contrary to
Su(var)3-9 and to Setdb1, which localize primarily to the chromocenter and
to the 4th, respectively, G9a localizes to euchromatic bands in polytene
chromosome arms and it´s role in global H3K9 methylation is still elusive (MIS
et al. 2006; STABELL et al. 2006b; SEUM et al. 2007a).
The three HKMTs appear to exert their methylation activities in distinct
regions of the genome, and accordingly: Su(var)3-9 is a strong suppressor of
variegation of transgenes inserted in pericentric heterochromatin, while
Setdb1 is a strong suppressor of variegation of transgenes inserted on the 4th
and G9a appears to be a weak suppressor of variegation of genes inserted in
pericentric heterochromatin (BROWER-TOLAND et al. 2009). Interestingly,
although Su(var)3-9 doesn´t seem to affect H3K9me or HP1a recruitment to
the 4th, a study showed that both Setdb1 and Su(var)3-9 mutants display
decreased gene expression from the 4th (LUNDBERG et al. 2013). In this study,
it was suggested that Setdb1 and Su(var)3-9 have some degree of
redundancy, since Setdb1 and Su(var)3-9 affect the expression of the same
sets of genes.
24
HP1a TARGETING
Summary of Paper II
The roles of each HKMT in methylating H3K9 and in recruiting HP1a genomewide in Drosophila melanogaster are still elusive and previous studies were
based on results from immunostaining experiments on polytene
chromosomes. In this project we were interested in the division of labour
between HKMTs at a high resolution, and we therefore performed ChIP-chip
experiments in salivary gland tissue from third instar larvae, to compare with
polytene chromosome stainings. We mapped H3K9me2, H3K9me3 and HP1a
genome-wide in wild type and in mutants for Setdb1 (Setdb110.1), for
Su(var)3-9 (Su(var)3-9evo/06) and for G9a (G9aRG5). Additionally we also
mapped HP1a in Pof mutants (PofD119).
Our mapping of HP1a and H3K9me2,3 in wild type showed that these factors
are mainly enriched on chromosome 4 and in pericentromeric regions of all
chromosomes. This is in agreement with previous results from others who
performed ChIP-chip and DamID (DNA adenine methyltransferase fused to a
chromatin protein of interest) in Drosophila cell lines, embryos and fly heads,
which indicates the stability of HP1a and H3K9me2,3 throughout
development (DE WIT et al. 2005; DE WIT et al. 2007; JOHANSSON et al. 2007b;
FILION et al. 2010; KHARCHENKO et al. 2011; RIDDLE et al. 2011; YIN et al. 2011).
In one of these studies, the authors performed DamID experiments for 53
chromatin associated proteins, in embryonic cell lines, and they found that
the chromatin in Drosophila can be divided in five types (black, blue, green,
red and yellow), depending on their unique combination of proteins (FILION
et al. 2010). The chromatin type defined as green is enriched in HP1a,
Su(var)3-9 and H3K9me2, and corresponds to pericentromeric
heterochromatin and to the 4th chromosome.
Our ChIP-chip and immunostaining performed in salivary gland polytene
chromosomes in HKMT mutants confirm previous results from other groups,
and show that: Su(var)3-9 is responsible for H3K9me2,3 and HP1a
recruitment in pericentric regions of all chromosomes; Setdb1 produces
H3K9me2,3 and recruits HP1a to the 4th chromosome and to the 2L:31
region; and G9a doesn´t affect H3K9me or HP1a recruitment genome-wide
(SEUM et al. 2007b; BROWER-TOLAND et al. 2009). Interestingly, since region
2L:31 in wild type polytene chromosomes has been shown to ocassionaly
bind POF, it seems like Setdb1 methylates H3K9 at POF-bound regions
25
HP1a TARGETING
(LUNDBERG et al. 2013). It is intriguing that Su(var)3-9 doesn´t methylate H3K9
on chromosome 4 nor recruits HP1a, since it was shown that Su(var)3-9 is
enriched on the 4th chromosome (SCHOTTA et al. 2002; EBERT et al. 2004;
LUNDBERG et al. 2013).
Interestingly, our results show that HP1a recruitment to promoters of active
genes, on the 4th chromosome, 2L:31 region and pericentromeric regions,
doesn´t depend on methylated H3K9. In Setdb1 mutants, H3K9me2,3 is lost
from promoters of active genes at all the regions mentioned above, but HP1a
remains enriched (figure 4). We suggest that the initial H3K9me independent
binding of HP1a at promoters of active genes constitutes a nucleation event,
and that HP1a will then spread to the gene bodies by recognition of H3K9me
marks.
Figure 4. HP1a at promoters of active genes on the 4th chromosome binds independently on
H3K9me, on Setdb1 and on POF. Left: ChIP-chip profiles, depicted for a region on the 4th
chromosome, of HP1a, H3K9me2 and H3K9me3 binding on salivary glands from third instar
larvae of wild type and mutants for Pof and Setdb1. Y-axes show ChIP enrichment in log2 ratios
and x-axes show chromosomal position in Mb. Right: metagene profile representing the
average HP1a targeting on 4th chromosome active genes, based on ChIP-chip profiles of HP1a
in wild type, and mutants for Su(var)3-9, Pof and Setdb1. Y-axes show ChIP enrichment in log2
ratios and the x-ratios represent the different gene features: IG – intergenic; P – promoter;
E1-5 – exon bins; IN – intron.
26
HP1a TARGETING
The most prominent H3K9me-independent HP1a promoter peaks are seen
on the 4th chromosome active genes in Setdb1 mutants. In these promoters,
HP1a targeting is decreased, which might be due to the fact that the lack of
Setdb1 causes less H3K9me on gene bodies and consequently less HP1a
being recruited. Since HP1a is essential for survival, contrary to Su(var)-3-9,
G9a, and Setdb1 (Setdb1 mutants could be recovered although less viable
than wild type), it might be that HP1a at promoters is essential for viability.
Otherwise, the essential role of HP1a might relate to its activity in several
distinct cell functions. It is intriguing that Setdb1 produces H3K9me2,3 at
promoters of pericentromeric regions, since it has been reported that this
enzyme does not act in these regions (SEUM et al. 2007b; TZENG et al. 2007;
LUNDBERG et al. 2013).
Our results on Pof mutants are intriguing since they showed that in the
absence of POF, HP1a is lost from bodies of active genes on the 4th, as it was
previously shown (JOHANSSON et al. 2007b), but it remains at promoters
(figure 4). The metagene profile from chromosome 4 active genes for HP1a
targeting is very similar between Setdb1 and Pof mutants. It was shown
before that Setdb1 colocalizes with POF and HP1a on the 4th chromosome,
and that in Sedtb1 mutants, POF and HP1a enrichments are decreased on the
4th chromosome (TZENG et al. 2007; LUNDBERG et al. 2013). Our results suggest
that the HP1a spreading from its nucleation site at promoters depends on
the presence of both Setdb1 and POF at gene bodies. The dependence on
POF and Setdb1 for HP1a targeting seems to be specific to active genes. In
fact, we found a region on the 4th chromosome with two unexpressed genes
in which the high enrichment in HP1a doesn´t depend on Setdb1 nor on Pof.
Since HP1a can bind RNA, we propose that POF interaction with nascent
transcripts from the 4th stabilizes the interaction between HP1a and H3K9me.
We further studied the H3K9me independent HP1a-bound promoters for the
presence of a sequence motif or for the specific enrichment in chromatinassociated protein factors and/or histone marks. Using CompleteMOTIFS
platform for motif search, we found that the H3K9me independent HP1a
promoters, in comparison to promoter regions of 2L (excluding
pericentromeric and 2L:31 regions), are enriched in A/T. This is in agreement
with previous results showing that HP1 is enriched at A/T rich regions (GREIL
et al. 2003).
27
HP1a TARGETING
After analysing the mapping of 51 chromatin factors in S2 cells, from
modENCODE, we found that the H3K9me independent binding of HP1a at
promoters correlates very well with the binding of HP2 (heterochromatin
protein 2). HP2 protein has been shown to colocalize with HP1a on polytenes,
to be a suppressor of variegation and to interact with HP1a, and was
proposed to drive HP1a dimerization (SHAFFER et al. 2002; MENDEZ et al.
2011). Since mutations that affect HP1a dimerization abolish the interactions
between HP1 and non-modified H3, we suggest that HP2 interaction with
HP1 is required for HP1 dimer to bind to the core of H3 (LAVIGNE et al. 2009).
Our results support the model that HP1a high affinity binding to H3 histone
fold happens at promoters of active genes, and is a nucleation site for HP1a
chromatin targeting (DIALYNAS et al. 2006). This model suggests that HP1
binds with high affinity to the H3 histone fold when H3 becomes exposed, in
regions of high transcription or replication, and subsequently HP1a can
spread by binding with low affinity to H3K9me sites. It has been shown
previously that HP1a binds with higher affinity to the fold region of H3 than
to the methylated lysine 9 on the N-tail (NIELSEN et al. 2001).
By analysing DNase hypersensitivity data from embryos (THOMAS et al. 2011),
we found that on average, promoters at the 4th chromosome are more DNase
sensitive than the 2L promoters, even though the 4th chromosome promoters
are enriched in HP1a. In fact, it was previously suggested that HP1a opens
the chromatin structure at some promoters of active genes on the 4 th
(CRYDERMAN et al. 2011).
The role of HP1a at promoters of active genes is still elusive. A study showed
that for 4th chromosome genes from Setdb1 mutants, which have HP1a at
promoters but not at gene bodies, there is an increased transcriptional
output from these genes, compared to wild type (LUNDBERG et al. 2013). It
was suggested that the role of HP1a at promoters is activating transcription
while at gene-bodies it represses transcription. In support of this, a study has
proposed that HP1a at promoters of active genes on the 4th promoters can
open the chromatin structure by recruiting chromatin-remodelling
complexes (CRYDERMAN et al. 2011).
28
MSL TARGETING
CHAPTER 4
4. MSL TARGETING
Sex chromosomes and sex determination
Among species with sexual differentiation, i.e. distinct males and females,
there is a large variety of sex determination mechanisms. In some animals
sex is determined by the environment, for instance by the temperature of
incubation of eggs. In other animals there are genetic mechanisms of sex
determination, for example the existence of heteromorphic sexchromosomes. Both in mammals and in Drosophila, males are the
heterogametic sex and have one X and one Y chromosome, in contrast to
females which have two X chromosomes and are thus the homogametic sex.
The X and the Y chromosomes are morphologically and genetically different
and are believed to have evolved from a homologous pair of autosomes.
In mammals, sex determination is conferred by the gene SRY (sex
determining region Y) on the Y chromosome, which is the master trigger of
male embryonic differentiation (LAHN et al. 2001). In Drosophila
melanogaster, the primary determinant for sex is the ratio X-chromosomes:
Autosomes (X:A), or more specifically, the number of X-chromosomes
relative to the number of precellular nuclear divisions (CLINE AND MEYER 1996;
MARIN AND BAKER 1998; ERICKSON AND QUINTERO 2007). Females will develop
from XX:AA embryos with an X:A ratio of 1.0, and males will develop from
XY:AA embryos with an X:A ratio of 0.5. When comparing flies, with two pairs
of autosomes, and humans: XXY flies are viable and fertile females whereas
XXY humans are sterile males with Klinefelter syndrome; XO flies are sterile
males, whereas humans XO are sterile females with Turner syndrome.
In female flies, the X-linked gene sex-lethal (sxl) is expressed in its active
form, and is responsible for somatic sex determination, dosage
compensation and oogenesis. The components of the X:A ratio signal are
proposed to be numerators, denominators and maternal genes, and will act
by activating or repressing the sxl RNA (SCHUTT AND NOTHIGER 2000). According
to the model, numerator genes are located on the X chromosome and are
responsible for activation of sxl. If numerator genes are duplicated in XY:AA
29
MSL TARGETING
embryos, it leads to activation of sxl causing male lethality; when numerator
are deleted in XX embryos, it prevents activation of sxl leading to female
lethality. SXL is a splicing factor and an RNA binding protein, which is
activated early in XX embryos and initiates its own positive feedback
regulation. SXL also regulates downstream genes essential for female
differentiation: it activates transformer (tra), represses translation of malespecific lethal 2 (msl2) transcript and activates genes involved in oogenesis
(SCHUTT AND NOTHIGER 2000). The TRA protein is a splicing factor involved in
the regulation of double sex (dsx) transcript, which is a transcription factor
that in the spliced form, in females, represses male-specific genes and
activates female-specific genes, and in the unspliced form, in males, does the
opposite. MSL2 is part of the dosage compensation system, which will only
function later in development, and it is active only in males – see below.
Sex chromosome evolution
The prevalent theory for the evolution of sex chromosomes postulates that
the acquisition of a male-determining gene on a chromosome from an
homologous pair (for instance, the sry in humans) gives rise to the proto-Y
chromosome, which, since it will only be transmitted through males, will
likely accumulate male advantageous mutations. The proto-Y will diverge
from the proto-X and to avoid intersex there will be a selection for
suppression of recombination, for which inversions contribute. The loss of
recombination will in turn lead to the accumulation of more deleterious
mutations, mobile elements, and loss of genes, which promotes progressive
proto-Y degeneration.
Despite the fact that the formation of sex chromosomes occurred
independently several times in evolution, the Y chromosomes display
similarities among species, often (but not always) being small, gene-poor and
rich in repetitive sequences (LAHN et al. 2001). The view that X and Y in
Drosophila evolved from a pair of autosomes has been challenged since these
two chromosomes share very little homology, contrary to mammals that
have many homologous regions through which the X and the Y recombine
(CARVALHO 2002). The Y chromosome in Drosophila is constituted by
repetitive sequences and fewer than 20 protein coding genes mainly involved
30
MSL TARGETING
in spermatogenesis, and apparently there is no homology between them and
genes on the X chromosome (CARVALHO 2002; CARVALHO et al. 2009). The only
homologous region between the sequenced portions of Drosophila X and Y
chromosomes is the rDNA locus, which helps in their pairing in meiosis
(CARVALHO 2002; TSAI AND MCKEE 2011). Interestingly, the Y chromosome
genes in Drosophila have paralogs on autosomes, which led to the hypothesis
that the Y chromosome might have evolved through the accumulation of
male-related genes from autosomes on a B-chromosome (B chromosomes
are additional dispensable chromosomes that are present in a fraction of
individuals) (CARVALHO 2002; CARVALHO et al. 2009).
Dosage compensation
Y chromosome degeneration leads to a gene dose problem since genes on
the male X chromosome are in only one copy, when compared to autosomal
genes and to X chromosomal genes in females, which are in two copies.
Different animals have evolved distinct mechanisms of dosage compensation
which balance the relative expression levels of X-linked genes between sexes
and in accordance to the autosomes. It has been proposed that an increased
expression of the monosomic X-linked genes in males was the first event in
dosage compensation (OHNO 1967; LARSSON AND MELLER 2006). In fact, studies
in mammals have shown that expression from X genes is on average twice
that from autosomal genes, although this is still in debate (GUPTA et al. 2006;
NGUYEN AND DISTECHE 2006; YILDIRIM et al. 2012; FAUCILLION AND LARSSON 2015).
A recent study has also showed that both in male and female human cell
lines, the average half-lives for transcripts from the X chromosome is longer
than for transcripts from autosomes and the ribosome density is higher on
X-linked transcripts (FAUCILLION AND LARSSON 2015). The increase in X-linked
gene expression in females would create the need to compensate for the
presence of two X chromosomes, which in mammals is achieved by silencing
one of the X chromosomes in each female cell (LARSSON AND MELLER 2006). In
Drosophila, the dose problem is solved by a ribonucleoprotein complex – the
MSL (male-specific lethal) complex, which is only formed in males, due to the
male specific production of one protein of the complex: MSL2 (BASHAW AND
BAKER 1995). The MSL complex specifically recognizes the male X
31
MSL TARGETING
chromosome and contributes to the 2-fold upregulation of almost all its
genes (HAMADA et al. 2005; DENG AND MELLER 2006; ZHANG et al. 2010).
MSL complex
The MSL complex is formed by the association of five proteins and two long
non-coding RNAs. The five genes encoding for the proteins of the complex
were discovered in screens for mutations with male-specific lethal
phenotypes (BELOTE 1983), and their names are suggestive for that: male
specific lethal 1 (msl1), male specific lethal 2 (msl2), male specific lethal 3
(msl3), maleless (mle) and male absent on first (mof). The two redundant long
non-coding RNAs are RNA on the X 1 (roX1), and RNA on the X 2 (roX2), and
their genes are located on the X chromosome, more than 7 Mb apart.
Contrary to the protein components of the complex, roX1 or roX2 alone are
not essential for survival of males, but if both roX RNAs are removed than the
male flies die (MELLER AND RATTNER 2002). Immunostainings on polytene
chromosomes for each of the proteins of the complex and RNA in situ
hybridizations for each roX show that they specifically target the X
chromosome in males with similar distributions (seen for MSL3 in Figure 5).
Figure 5. MSL complex targets the male X chromosome. MSL3 immunostaining on polytene
chromosomes from salivary glands from male third instar larvae. All the other components of
the complex colocalize with MSL3.
The protein components of the MSL complex are conserved and have
orthologues in other species including humans, while the roX RNAs have low
degree of conservation, even in Drosophila related species (PARK et al. 2003;
32
MSL TARGETING
SMITH et al. 2005). The conservation of MSL-complex proteins suggests that
they might have had a function outside dosage compensation before they
were recruited to the monosomic male X (STENBERG AND LARSSON 2011).
MSL1 and MSL2 are the core components of the complex, being required for
its assembly. In contrast, in msl3, mof or mle mutants, and in roX1 roX2
double mutants, partial MSL complexes form at some locations on the male
X chromosome, although the majority of MSL targeting to the X chromosome
is lost (PALMER et al. 1994; LYMAN et al. 1997; KAGEYAMA et al. 2001; MELLER
AND RATTNER 2002). It has been shown that in wild type females, MLE, MSL3
and MOF are produced, contrary to MSL1 and MSL2 (PALMER et al. 1994). If
in females MSL2 is expressed ectopically or is induced by mutation of Sxl, the
MSL complex is formed and targets both X chromosomes (KELLEY et al. 1995;
LYMAN et al. 1997). MSL2 is the limiting component for formation of the MSL
complex, since its translation is repressed in males by SXL and, as it has been
proposed, the msl2 transcript associates with free MSL complexes in the
nucleus and regulates the production of complexes by a feedback
mechanism (JOHANSSON et al. 2011). It has also been shown that MSL2
positively regulates MSL1 translation or protein stability (PALMER et al. 1994).
Besides the major role of MSL2 in the complex formation, MSL2 requires
MSL1, since in msl1 homozygous mutant males no MSL2 staining is seen on
the X chromosome and MSL2 expression is decreased (LYMAN et al. 1997).
MSL2 and MSL1 have also been shown to interact and form a heteromer
which binds DNA (COPPS et al. 1998; FAUTH et al. 2010).
MSL3 protein contains a chromo-domain and has been shown to bind
nucleosomes marked with H3K36me3 in vitro, a mark of active chromatin,
and this targeting is suggested to be important for MSL recruitment to the
male X chromosome (LARSCHAN et al. 2007).
MOF is a histone lysine acetyltransferase responsible for acetylation of
H4K16 at a high level on the male X chromosome (AKHTAR AND BECKER 2000;
MORALES et al. 2004). MOF has functions outside the MSL complex, and ChIPchip and ChIP-seq experiments in Drosophila cell lines have shown that MOF
is enriched in promoters of autosomal genes (KIND et al. 2008; CONRAD et al.
2012). MOF at promoters of autosomal genes is part of the Non-Specific
Lethal (NSL) complex, formed by six other proteins, and it seems to promote
gene expression (PRESTEL et al. 2010; RAJA et al. 2010). Interestingly, there is
33
MSL TARGETING
a high degree of conservation between the Drosophila MOF and the
mammalian MOF, which also produces H4K16ac and is involved in
embryogenesis and oncogenesis (TAIPALE et al. 2005; GUPTA et al. 2008).
Additionally, in humans, MOF has been suggested to be involved in
upregulation of the X chromosome (DENG et al. 2013).
MLE is a DNA/RNA helicase and has been shown to be essential for
incorporating roX1 and roX2 in the MSL complex, by binding to stem-loop
structures on the roXs in an ATP-dependent manner (PARK et al. 2007; PARK
et al. 2008; ILIK et al. 2013; MAENNER et al. 2013).
roX RNAs
The role of roX redundant long non-coding RNAs in MSL complex targeting to
the male X chromosome is still very intriguing. Although roX1 and roX2 have
little sequence homology, they share conserved domains that are important
for their interaction with MSL proteins (PARK et al. 2007; PARK et al. 2008; ILIK
et al. 2013). The redundancy of roX1 and roX2 is interesting and it has been
suggested that only one roX (roX1 or roX2) is present in each MSL complex
(ILIK et al. 2013). It has been proposed that roX RNAs are incorporated in the
MSL complex co-transcriptionally since they are unstable when not bound to
the MSL complex (MELLER et al. 2000). MSL2 regulates roX transcription
(RATTNER AND MELLER 2004) and it has been shown that roX1 transcription is
regulated by an autoregulatory loop (LIM AND KELLEY 2012).
Interestingly, roX RNAs ectopically expressed from autosomes in roX1 roX2
double mutant males, can recruit MSL, which spreads together with roX RNA
in cis around the transgene, and can also recognize the X chromosome in
trans and recruit MSL to the X chromosome (KELLEY et al. 1999; MELLER et al.
2000; PARK et al. 2002; ALEKSEYENKO et al. 2008; STRAUB et al. 2013). It has
been suggested that the role of roX RNAs is to mediate the spreading of the
MSL complex on the whole X chromosome (KELLEY et al. 1999). MSL spreading
ectopically to the genes surrounding the transgene seems to increase the
expression of those genes (KELLEY AND KURODA 2003; VENKEN et al. 2010).
The importance of roX RNAs for correct MSL targeting to the X chromosome
was further shown by the fact that in roX1 roX2 double mutants, MSL2 and
H4K16ac are decreased on the X chromosome and target new genomic
34
MSL TARGETING
regions like the chromocenter, the 4th chromosome and a few other
autosomal sites (MELLER AND RATTNER 2002; DENG AND MELLER 2006; JOHANSSON
et al. 2011). This is an important topic of study in paper III.
The role of roX RNAs in MSL targeting shows parallels with the mammalian
dosage compensation system. In mammal females, the Xist long non-coding
RNA is expressed from the Xic locus on one of the X chromosomes, coats the
entire chromosome in cis, and recruits several proteins and chromatin
modifiers that, for instance by producing DNA methylation, lead to a
complete compaction of the chromatin structure and silencing of the
majority of genes on that chromosome (HEARD AND DISTECHE 2006).
MSL-complex and heterochromatin
The male X chromosome seems to require heterochromatin associated
proteins to keep its normal shape. Using DamID technique it has been shown
that HP1a is enriched on the male X chromosome compared to an autosome
and compared to the female X chromosome (DE WIT et al. 2005).
Interestingly, in HP1a and in Su(var)3-7 mutants, the X chromosome loses
compaction, shows a bloated phenotype, and this phenotype depends on a
functional MSL complex (SPIERER et al. 2005). Another link between the MSL
complex and heterochromatin is provided by JIL-1, an essential protein
kinase involved in Histone 3 Serine 10 (H3S10) phosphorylation, which was
shown to colocalize and physically interact with the MSL complex (JIN et al.
2000; WANG et al. 2013; LINDEHELL et al. 2015). JIL-1 targets all chromosomes
in females but is highly enriched on the male X chromosome dependent on
a functional MSL complex (JIN et al. 1999; JIN et al. 2000). JIL-1 is actually
increased on the female X chromosome when MSL2 is expressed ectopically
(JIN et al. 2000). JIL-1 on the male X chromosome was proposed to be
important for a normal transcription, since genome-wide mapping of JIL-1
showed that its binding correlates with marks of active chromatin
(H3K36me3 and H4K16ac) (REGNARD et al. 2011), and RNAi mediated
knockdown of JIL-1 caused decreased transcription from the male X
chromosome (REGNARD et al. 2011). It has also been shown that in JIL-1 null
mutants, HP1a and H3K9me2 enrichments are increased on the male and
female X chromosome(s) (ZHANG et al. 2006). Additionally, Su(var)3-7 loses
its chromocenter specific binding and is ectopically targeted to chromosome
35
MSL TARGETING
arms in JIL-1 null mutants (DENG et al. 2010). Decreasing the dose of Su(var)37 or Su(var)3-9 rescues the lethality of JIL-1 null mutants (DENG et al. 2007;
DENG et al. 2010). Some studies suggested that Su(var)3-7 acts downstream
of Su(var)3-9 and H3K9me in heterochromatic formation and gene silencing,
and that JIL-1 counteracts this, repressing the spreading of heterochromatin,
possibly helping to keep an open chromatin structure on the male X
chromosome (DENG et al. 2010; REGNARD et al. 2011).
Upregulation of the male X
It is still unclear how dosage compensation upregulates gene expression
from the male X chromosome approximately two times. H4K16ac has been
proposed to open the chromatin but it´s still elusive how this affects
transcription (AKHTAR AND BECKER 2000). It has been suggested that the MSL
complex acts by facilitating transcription elongation by reducing the pausing
of RNA polymerase II (RNA Pol II) (LUCCHESI 1998). Supporting this view, it was
shown by ChIP-chip experiments that MSL complex targeting is linked to
active transcription since MSL complex and H4K16ac are enriched in active
gene bodies with a 3´ end bias (ALEKSEYENKO et al. 2006; GILFILLAN et al. 2006;
ALEKSEYENKO et al. 2008; KIND et al. 2008; PRESTEL et al. 2010). In contrast to
the transcription elongation theory, it has been claimed that MSL acts by
increased transcription initiation, through increased recruitment of RNA Pol
II to promoters of X linked genes, in comparison to autosomes and female X
chromosomal genes (CONRAD et al. 2012), but this is still controversial
(FERRARI et al. 2013; STRAUB AND BECKER 2013; VAQUERIZAS et al. 2013).
Several lines of evidence suggest that MSL is not alone responsible for the
full dosage compensation of X chromosomal genes. Only 75% of the actively
transcribed genes on the male X are bound by MSL (CONRAD AND AKHTAR
2012). Gene expression studies suggest that MSL only accounts for ~1.4-fold
increase in transcriptional output from the X chromosome, and it has been
suggested that the rest of the compensation, up to the final 2-fold is, at least
partly, accomplished by general buffering effects that act on all monosomic
regions, discussed in chapter 2 (STENBERG et al. 2009; ZHANG et al. 2010;
LUNDBERG et al. 2012). In fact it was shown that 15% of the expressed genes
on the male X chromosome are dosage compensated but unbound by MSL
and have reduced levels of H4K16ac (PHILIP AND STENBERG 2013). It has been
36
MSL TARGETING
hypothesized that, during the evolution of sex chromosomes, the MSL
complex targeting was a secondary adaptation, while buffering mechanisms
were the initial response to the monosomic state of the X chromosome in
males (STENBERG AND LARSSON 2011).
It´s important to keep in mind that the MSL complex and other compensatory
mechanisms can cause dosage compensation not only by regulating
transcription, but also by regulating for instance RNA stability, translation or
protein stability (FAUCILLION AND LARSSON 2015).
Mechanisms of MSL targeting
It´s still elusive how the MSL complex specifically recognizes and targets the
male X chromosome, but the prevailing model postulates that it is recruited
in a sequence-dependent manner to approximately 250 High Affinity Sites
(HAS), defined by the enrichment of a GA-rich DNA sequence motif, and then
spreads to neighbouring genes that are being actively transcribed (LARSCHAN
et al. 2007; ALEKSEYENKO et al. 2008; STRAUB et al. 2008; GELBART AND KURODA
2009; CONRAD AND AKHTAR 2012; STRAUB et al. 2013). HAS, initially called
chromatin entry sites, were defined as the regions on the male X that still
bind MSL1 or MSL2, when one of the other proteins of the complex is
removed (in msl3, mle or mof mutants) (LYMAN et al. 1997; KELLEY et al. 1999).
Another characteristic of HAS is that they can recruit MSL when inserted on
an autosome in males (OH et al. 2004; ALEKSEYENKO et al. 2008). roX1 and roX2
genes are themselves two of the strongest HAS on the X chromosome (KELLEY
et al. 1999; ALEKSEYENKO et al. 2008; STRAUB et al. 2013). Other features
besides the DNA sequence will likely contribute to HAS recognition by the
MSL complex, since the GA-rich motif enriched at HAS is found elsewhere in
the genome, and by itself it cannot predict HAS (ALEKSEYENKO et al. 2008). In
fact one feature typical for HAS is that they have reduced nucleosome
density (ALEKSEYENKO et al. 2008; STRAUB et al. 2008; STRAUB et al. 2013).
The spreading beyond HAS requires an MSL complex constituted by all five
proteins and at least one roX RNA (LYMAN et al. 1997; MELLER AND RATTNER
2002). Besides the preference of MSL to target actively transcribed genes,
and regions enriched in H3K36me3, other features might affect the binding
of the complex (ALEKSEYENKO et al. 2006; STRAUB et al. 2008).
37
MSL TARGETING
Links between X and 4th chromosomes
Several lines of evidence suggest an evolutionary relationship between the X
and the 4th chromosomes. The X and the 4th are the only chromosomes that
have a chromosome-wide gene regulatory mechanism that compensate for
dose differences. In several species related to Drosophila melanogaster, POF
targets the F element (equivalent to the 4th chromosome) and the MSL
complex targets the X chromosome, like in Drosophila simulans, Drosophila
pseudoobscura and Drosophila virilis. However, this is not the case for all
Drosophila species. For instance, in Drosophila ananassae and Drosophila
malerkotliana, both POF and the MSL complex target the male X
chromosome, while the F element is targeted only by POF and in both males
and females. Interestingly, in Drosophila busckii, the F element is located at
the base of the X and the Y chromosomes, and POF targets the entire male X
chromosome which is also enriched in H4K16ac (including the F element
part) (LARSSON et al. 2001; LARSSON et al. 2004; STENBERG AND LARSSON 2011).
These facts led to the suggestion that the 4th and the X chromosomes have
the same origin and that POF originated as part of an ancient dosagecompensation mechanism (STENBERG AND LARSSON 2011).
Summary of Paper III
To study the role of roX RNAs in the recruitment of MSL complex to
chromatin, we performed immunostaining experiments and ChIP-seq
experiments on polytene chromosomes from third instar larvae to map
MSL1, MSL2 and MOF, in wild type or double mutants for roX1 and roX2. Our
ChIP-seq results correlate very well with the immunostainings and show that
in roX1 roX2 double mutants, MSL is restricted to the HAS on the X
chromosome, and that MSL in addition targets ectopic places: the
chromocenter and specifically three regions on the 4th chromosome (Figure
6).
38
MSL TARGETING
Figure 6. MSL complex targets three bands on the 4th chromosome in the absence of roX.
ChIP-seq enrichment profiles showing that MOF and MSL1 are enriched on three regions of
the 4th chromosome (top) that correspond to the three bands of MSL2 immunostaining seen
in the polytene 4th chromosome (down).
Our results clearly show that the role of the protein components of the MSL
complex is distinct from the role of roX RNAs. While both in roX1 roX2 and in
msl3 mutants the complex loses most of its binding from the male X, in roX1
roX2 mutants the complex is relocated to ectopic sites. In order to test if the
pericentromeric targeting of the MSL complex was restricted to polytene
chromosomes, we analysed the colocalization between the 1.686
pericentromeric repeat (enriched in pericentromeric regions of
chromosomes 2 and 3) and MSL by combined DNA in situ hybridization and
immunostaining in brain cells from third instar larvae. We found a high
degree of co-localization between the pericentromeric probe and the MSL
complex in roX mutants but not in wild type or mof mutants, confirming the
results from polytenes. We speculate that in interphase chromosomes of
roX1 roX2 mutants, the pericentromeric regions are in close proximity to the
X chromosome, in a region where local MSL concentration is high. One study
has previously shown that HAS are in closer proximity to each other in males
than in females (GRIMAUD AND BECKER 2009). We didn´t find colocalization
between MSL and the 1.686 repeat on mitotic chromosomes. Additionally,
MSL targeting on mitotic chromosomes was restricted to the euchromatic
part of the X chromosome, in both wild type and roX mutants. The high
39
MSL TARGETING
compaction of the heterochromatic regions of the mitotic chromosomes
might repress the MSL targeting to heterochromatin, although this still
remains elusive.
Importantly, we show that all five proteins of the complex colocalize at the
ectopic places on the chromocenter and on the 4th chromosome in roX1 roX2
mutants and that H4K16ac also increases in these regions. These results
show that the MSL complex doesn´t require roX RNAs for complex assembly
and activity. When we looked at the mRNA levels from the 4th genes
specifically targeted by MSL in roX mutants, by quantitative PCR in third
instar larvae, we found that their mean expression levels are not significantly
different from wild type. Our results suggest that the presence of H4K16ac is
not enough to stimulate transcription, at least from the 4th chromosome. It´s
important to keep in mind that on the 4th chromosome, gene expression is
regulated by both POF and HP1a which might counteract the effect caused
by H4K16ac. MSL targeting on the 4th didn´t affect HP1a or POF
immunostaining pattern (data not shown).
We found that JIL-1 is also targeted to the chromocenter and 4th
chromosome in roX1 roX2 mutants. JIL-1 seems to be part of the MSL
complex or to be recruited to chromatin by the MSL complex. Intriguingly, if
MSL1 and MSL2 are overexpressed in males, MSL2 targets also the
chromocenter and the 4th chromosome (DEMAKOVA et al. 2003). The
chromocenter and the 4th chromosome are among the most
heterochromatic regions of the genome. Our results suggest that the levels
of roX RNAs relatively to the levels of MSL protein components dictate where
the MSL complex will bind. MSL might have an intrinsic affinity to
heterochromatin, and when there is excess of MSL protein components
relatively to the roX RNAs, as in roX1 roX2 mutants or when MSL1 and MSL2
are overexpressed, the complex is recruited to heterochromatin.
It has been proposed that heterochromatin functions as a sink to limit the
quantities of silencing factors (EISSENBERG AND REUTER 2009). It was shown that
increasing the amount of Y chromosomes (or the 4th or the heterochromatic
portion of the X) suppresses PEV, possibly because the amount of
heterochromatin compaction elsewhere in the genome decreases. Since it
was shown that in roX double mutants the flies that escape the lethality are
more likely to be roX1 roX2/0 than roX1 roX2/Y, it might be the case that
40
MSL TARGETING
increasing compaction of heterochromatin in the genome (i.e. in X/0 males)
makes the MSL complex leave the chromocenter and the 4th and target the
X. To test the MSL dependence on heterochromatin, we performed MSL
immunostainings on polytene chromosomes from null mutants for the
heterochromatin proteins: Su(var)3-7, Su(var)3-9, HP1a and HP2 (data not
shown). It was reported that in mutants lacking Su(var)3-7, MSL targeted the
4th and the chromocenter (SPIERER et al. 2008); however, we couldn´t
reproduce these results. In the heterochromatin mutants tested we couldn´t
see any distinct difference compared to wild type: MSL always targeted
preferentially the male X chromosome. We also found HP1a and Su(var)3-7
targeting on polytenes not to be affected by the lack of roX RNAs (data not
shown). We couldn´t determine if HP1a was required for MSL targeting to
the 4th chromosome, since roX1 roX2; Setdb1 triple mutants died before
reaching third instar larval stage.
Since the 4th and the X chromosomes, as well as POF and MSL, share
evolutionary links, it might be that the 4th specific genes targeted by MSL
were on the X chromosome in Drosophila melanogaster related species, but
we haven´t found any evidence for this possibility.
By analysing our ChIP-seq data we found that all the enrichment peaks for
MSL on the X chromosome in roX mutants corresponded to the previously
mapped HAS (ALEKSEYENKO et al. 2008; STRAUB et al. 2008; STRAUB et al. 2013).
Out of 263 reported HAS, we found 208 to be independent on roX RNAs.
Additionally, by performing motif analysis using MEME software tool, we
show that the GA rich motif found in the roX independent HAS, is identical to
the one previously described for wild type (ALEKSEYENKO et al. 2008), and that
this motif is conserved, since it was also enriched in the MSL1 peaks from
Drosophila simulans (ChIP-seq for MSL1). Since partial MSL complexes target
HAS in absence of MSL3, MLE or MOF, and as we found now, in absence of
roX1 and roX2 RNAs, it seems that MSL1 and MSL2 are the only components
of the complex required for HAS targeting. It´s still elusive which
characteristics of HAS determine this targeting.
We were very intrigued by the link between MSL and heterochromatin and
this led us to study the properties of the heterochromatic regions targeted
by MSL in roX mutants. By doing motif search (using MEME) on the
heterochromatic MSL-bound regions of chromosomes 2, 3 and 4 in roX
41
MSL TARGETING
mutants (which mainly include pericentromeric sequences), we found a
motif containing repeats from the Hoppel (1360) transposable element.
Hoppel are non-autonomous DNA transposons highly enriched in
pericentromeric heterochromatin and on chromosome 4 (COELHO et al.
1998). When we mapped the motif containing Hoppel repeats along the
chromosome arms we confirmed it to be enriched towards the centromeres.
We performed in situ DNA hybridization with a probe containing the motif
combined with MSL immunostaining and found them to colocalize on the
three bands on the 4th in roX mutants. The affinity for Hoppel was further
confirmed when we aligned all the ChIP-seq reads (including highly repetitive
sequences) to sequences from repeats included in the Repbase Update
database. We found that in roX mutants MSL is enriched at PROTOP_B,
PROTOP_A and NTS (Non-transcribed Spacer). Interestingly, PROTOP is a
family of autonomous DNA transposons that have been suggested to be
ancestors of Hoppel and P-element. Since both Hoppel and the other
transposable elements are usually in repeated copies in the genome, we
wondered if MSL has affinity to repeats in general.
In roX mutants MSL also binds to some autosomal sites, and we asked if these
were also enriched in repeats. Indeed, we found that the percentage of
repeat masked sequences (from UCSC) is higher around autosomal genes
targeted by MSL in roX mutants, than around autosomal genes not targeted
by MSL. To test if MSL has affinity to any sequence when repeated in tandem,
we analysed flies carrying 7 or 2 copies in tandem of the P[lacW] transgene.
Very interestingly, we found that in a roX mutant background, MSL is
recruited to the 7 tandem repeat transgene but not to the 2 tandem repeat
transgene. In contrast, in a wild type background, no MSL targeting was seen
to these repeat transgenes. These results show that sequences in tandem are
enough to recruit MSL when roX RNAs are not present. It´s still unclear if the
sequence plays a role in the targeting, since P[lacW] contains a mini-white
gene (X-linked).
We propose a model in which the heterochromatic targeting of the MSL
complex represents an ancient targeting of the complex, before specializing
in dosage compensation. MSL at heterochromatin could for instance play an
important role in allowing transcription of genes in repressive environment.
In this model, roX are evolutionary younger than the MSL and they act in
42
MSL TARGETING
restricting the MSL targeting to the X chromosome, which in a monosomic
state required mechanisms to upregulate its expression.
Summary of Paper IV
Although the absence of roX1 and roX2 causes male lethality, there are some
escapers (MELLER AND RATTNER 2002; DENG et al. 2005; MENON AND MELLER
2009). roX1 roX2 mutant male escapers might survive due to the
incorporation of additional roX RNAs in the MSL complex which could restore
dosage compensation. In wild type individuals, such RNAs have not been
identified (OH et al. 2003; ILIK AND AKHTAR 2009; JOHANSSON et al. 2011). It is
known that MLE requires RNA to target chromatin, since RNase treatment
causes MLE dissociation from wild type polytene chromosomes (RICHTER et
al. 1996). Interestingly, when roX RNAs are absent, MLE, together with the
other proteins of the MSL complex, targets heterochromatic regions of the
genome (FIGUEIREDO et al. 2014). Therefore, we hypothesized that in roX
mutants MSL is recruited to chromatin by recognition of non-roX RNA
molecules, possibly via MLE.
In order to test if there are other RNA species that associate with the MSL
complex and target it to heterochromatin in roX mutants, and if there are
additional roX RNAs, we performed RNA immunoprecipitation followed by
deep sequencing (RIP-seq) with an antibody against MSL2, in wild type and
in roX1 roX2 mutant male larvae. As expected, we found MSL2 to be enriched
in roX1 and roX2 RNAs in wild type. There is no evidence for the existence of
additional roX RNAs, confirming previous results. We found several RNAs,
transcribed from genes on the X and on other chromosomes, to be MSL2
bound in roX mutants and the majority corresponds to snoRNAs. We next
calculated the MSL2 RIP/input average ratio for each gene of D.
melanogaster genome and obtained a list with the top MSL2-bound RNAs
enriched in wild type and the top MSL2-bound RNAs enriched in roX mutants.
From this list we selected some RNAs for further analysis, depending on
concordance between the RIP/input average values and the RIP/input
enrichment profiles seen in integrated genome browser (IGB). We found
Sgs3 transcript to be enriched in MSL2 in wild type, and the transcripts from
Lsp1ß, 7SLRNA:CR32864, snoRNA:SC35-a and snoRNA:Psi28S-2562 to be
enriched in MSL2 in roX mutants. We tested the targeting of these RNAs in
43
MSL TARGETING
polytene chromosomes and in brain cells nuclei, by RNA in situ hybridization
(FISH) with antisense RNA probes against each RNA, combined with MSL2
immunostaining. None of the RNAs tested targeted chromatin in polytene
chromosomes or colocalized with MSL2 in brain cells nuclei. snoRNA:Psi28S2562 and snoRNA:SC35-a target the nucleolus in polytene chromosomes and
in brain cells nuclei, 7SLRNA:CR864 targets the cytoplasm, and both Sgs3 and
Lsp1ß showed a diffused pattern in the nucleus.
In order to select only the RNAs that associate with chromatin, we analysed
the MSL2-bound RNAs in roX mutants that were previously shown to be
chromatin associated (caRNAs) in embryos (SCHUBERT et al. 2012). From the
list of 28 caRNAs, we selected the ones which showed consistency between
the MSL2 RIP/input average ratio values and the MSL2 RIP/input enrichment
profiles seen in IGB. We tested each selected RNA by RNA-FISH combined
with MSL2 immunostaining and none them was seen targeting chromatin in
polytenes or colocalizing with MSL2 in brain cells nuclei. The majority of the
selected caRNAs bound by MSL2 are snoRNAs, which was seen for Rps7,
Uhg4, CG10576, Uhg2, Rps8 and CG13900 that target the nucleolus in both
polytene chromosomes and brain cells nuclei. Rps16 transcript showed a
dispersed pattern in the nucleus.
Our results indicate that MSL binding to heterochromatin probably doesn´t
occur via recognition of specific RNAs. Alternatively, the MSL complex might
incorporate several RNAs promiscuously and this binding can be lower than
the limit of detection by in situ hybridization and immunostaining.
Additionally, we can´t exclude the possibility that other non-tested RNA
molecules might target MSL to chromatin. Since the majority of RNAs bound
by MSL2 are snoRNAs, we can speculate that snoRNAs have similar stem-loop
structures to the roX RNAs. Also, since we found before that in roX mutants
MSL has affinity for non-transcribed spacer sequences (NTS) present in
ribosomal genes, one possibility is that the higher local concentration of MSL
in the rDNA locus, and in the nucleolus, promotes MSL binding to snoRNAs.
44
CONCLUSIONS
CONCLUSIONS
Paper I




Genes in monosomic condition are buffered.
The buffering is general and is not affected by other monosomic
regions.
Gene length is the primary determinant for buffering: longer genes
are more strongly buffered than shorter genes.
Genes involved in proteolysis are upregulated in individuals carrying
segmental monosomies.
Paper II




Setdb1 is responsible for H3K9me dependent recruitment of HP1a to
chromosome 4 and to region 2L:31.
Su(var)3-9 is responsible for H3K9me dependent recruitment of
HP1a to pericentromeric regions.
HP1a targets promoters of active genes in a manner that is
independent of H3K9me and of POF.
HP1a promoter peaks are enriched in AT content, in HP2 and are
DNase sensitive.
Paper III

MSL recruitment to HAS is independent on roX RNAs.

A complete and active MSL complex forms at six genes in the 4th
chromosome, independently from roX RNAs.

In the absence of roX RNAs, MSL has affinity to Hoppel transposable
elements and to repeats in general.
Paper IV

In the absence of roX, MSL associates with several RNAs, especially
with snoRNAs.

The RNAs associated with MSL don´t seem to target chromatin.
45
ACKNOWLEDGMENTS
ACKNOWLEDGMENTS
I am glad I chose to do my PhD studies in the cold far-north Umeå, there has
been a lot of exciting research and so much fun outside the lab. The beautiful
white cold winters and the fresh green summers will be missed, along with
the walks in the forests, the cycling, the skiing and the swimming in the lakes.
Many people were important during these last five years.
I want to thank you Jan, if you wouldn´t have accepted me as a master
student in your lab, I would have missed the chance to experience all of this.
Workwise I have to thank you so much for wanting to share your great
scientific knowledge with me during these PhD years. I hope I have learned
something from you, like how to do good and honest research, how to always
do my best, how to be patient and last but not least, how to be fair and kind
to people. The scientific world would be a better place with more people like
you. Also, thank you for giving me freedom.
I also want to thank the people in Jan Larsson´s and Per Stenberg´s groups
that I have been closely working and socializing with. Philge, thank you for
your great bioinformatics knowledge, for the help throughout the years and
for the kindness. I was really lucky that you were sitting in the office next
door and that I could always come to discuss the projects. Masha, thank you
for sharing your scientific knowledge with me, for all the help in the lab and
in the project we had together, for being always available to discuss science
and other subjects too. Thank you Per for all your ideas and comments during
group meetings and project discussions, they were really important. Without
Philge, Masha, Per and Jan, this thesis wouldn´t be possible. Anna-Mia, thank
you for being such a good colleague, for all the encouraging words! Your
smile and positive energy are contagious! Marie-Line, thank you for sharing
your excitement about science, but also for all the lunches, outdoor
activities, game nights and trips. Lina, thank you so much for all the great
times at work, for our long conversations, travels and so much more! It was
never the same here without you. I am so happy that we are friends and that
we always keep in touch! To Anders and to Henrik: thank you for the good
mood! I want to thank everyone in Yuri Schwartz´s group and in Per
Stenberg´s group for the interesting journal clubs, scientific discussions and
gatherings! Good luck to EpiCoN!
46
ACKNOWLEDGMENTS
I also want to thank everyone in the fly floor for the good environment!
Thank you Erik for the good times at work and outside work: everything gets
funnier when you are around! Thank you for your friendship! Thank you
Fredrik for the entertaining and loud conversations: it was fun teaching with
you! Thank you Hande for the good times teaching together and outside
work too. I want to thank everyone in the friday fika group for the delicious
cakes, specially to the fika king John! Also, to the people that I hanged out
with in the beer corner, it was fun! Sveta, thank you so much for all the great
times inside and outside the department and especially for all our
adventurous travels! So many good memories! I am so happy that we are
friends!
I also want to thank the friends outside work that I spent my time with in
Umeå, you made the dark cold winters warmer and brighter! A special thanks
to the band members for the fun and for teaching me! I loved that one music
day per week! Thank you Johanna, you are the best flatmate! Thank you Kathi
for all the fun we had together! Come back to Portugal soon! Melanie, thank
you so much for your friendship! We had so many good times together and
Umeå was never the same without you. Your words really motivate me, in
science and in life. Fariba, thank you so much for your friendship, for caring
for me, for all the good times we had together and for the motivation words!
I hope we will live close to each other in the future!
Aos amigos de Portugal, obrigada por estarem longe mas sempre tão perto!
Bárbara, obrigada por estares sempre aí para mim! A tua amizade é
inigualável. Tânia, Pequita, Anahí, Filipa, Clara, Lúcia, gosto muito de voces!
Ir a Portugal é tão bom por saber que nos vamos encontrar! Obrigada Sílvia,
Joana, Melissa e Diana pela amizade.
À minha família, aos tios, primos e irmão, que sempre se preocuparam e de
quem eu gosto muito, muito obrigada! O maior agradecimento de todos vai
para os meus pais, Julieta e Carlos, não há palavras para descrever todo o
vosso amor, apoio e motivacão desde sempre! Esta tese não seria possivel
sem voces! desculpem estar longe, mas trago-vos sempre comigo!!
Chris, your love is all I ever dreamed of. Thank you for all the fun, caring and
support in this last step of my PhD. I hope the days come easy and the
moments pass slow, and each road leads us where we want to go.
47
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