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Molecular Cell, Volume 57
Supplemental Information
Chromatin-wide Profiling of DYRK1A Reveals a Role as a Gene-Specific RNA Polymerase II CTD
Kinase
Chiara Di Vona, Daniela Bezdan, Abul B.M.M.K. Islam, Eulàlia Salichs, Nuria López-Bigas, Stephan
Ossowski, and Susana de la Luna
Figure S1
A
20
5 min
10 min
15
15 min
10
5
0
IP IgG-Control IP anti-DYRK1A
1
GAL4
κB
*
130
130
95
95
72
GAL4
G4E1b-Luc
GAL4
Luciferase
55
36
36
Wb: anti-G4-DBD
8
6
4
2
0
- DYRK1AWT DYRK1AKR
G
Wb: anti-G4-DBD
G4E1b-Luc
140
Relative transcriptional activity
(fold change)
10
8
G4E1b-Luc TATA Less
120
100
6
4
2
0
G4-DBD:
-
K1
YR A WT
K1
AK
R
C
D
K9
W
T
C
D
K9
KM
12
G5HIV1-Luc
D
14
G4-DBD:
F
YR
G5HIV1-Luc
D
16
Relative transcriptional activity
(fold change)
Relative transcriptional activity
(fold change)
E
YR
D K1A
YR
K
D 1B
YR
D K2
YR
D K3
YR
K4
72
Luciferase
TATA
55
G4E1b-Luc-TATAless
-
D
KM
K9
C
D
kDa
Luciferase
TATA
G4-DBD fusion proteins
R
AK
W
T
K9
K1
YR
D
YR
-
D
kDa
D
His S/T
K179R
KR
G5HIV1-Luc
Reporters
NLS2 PEST
AW
KD
K1
NLS1
Sp
G4DBD-DYRK1A
G4-DBD
N
F-
Effectors
G4DBD-DYRK1AWT
D
G4-DBD fusion proteins
T
C
B
C
kinase activity (cpmx103)
25
80
60
40
20
0
G4-DBD:
-
DYRK1AWT DYRK1AKR
Figure S2
A
Percentage
100
20
60
Promoters
61.3%
(2.7e-77)
1.1%
1.8%
>= 1000 bp
B
62.5%
(1.2e-67)
Genome
ChIP
>= 2000 bp
T98G
cells
466
56
HeLa
cells
C
40
RBM39
ASXL1
19
RPS11
25
DYRK1A
(T98G)
6.7
7
6
DYRK1A
(HeLa)
→
chr20:34,330,000-34,323,000
→
chr20:30,945,000-30,943,000
→
chr19:49,999,000-50,003,000
C
-2.0
0.0
+2.0
D
Expression Scale
-3.00
-7.50
**
-10.0
-3.75
***
0.0
0
3.75
7.50
1
TSS
XL
0.4
-4000
AS
4000
2
H3K27ac
K1
DYRK1A target genes
D
Average profile
40
C
0.6
R
0
EN
80
D
-4000
2
Average profile
A
7L
C
B
LU
11
PS
R
Relative mRNA expression
0
DYRK1A expression
CD8_T cells
Leukemia lymphoblastic_MOLT4
CD105_Endothelial
CD71_Early erythroid
Broquial epithelial cells
Thymus
CD19_B cells
CD4_T cells
BDCA4_Dendritic cells
Colorectal adenocarcinoma
X721_B lymphoblasts
Testis_Germ cells
Fetal Thyroid
Uterus
Leukemia promyelocytic_HL60
CD14_Monocytes
CD56_NK cells
Colon
Leukemia chromic myelogenous
Small intestine
Lymp node
CD34_Lymphatic endothelial cells
Thyroid
Pituitary
Fetal lung
Burkitt’s lymphoma_Daudi
Pineal gland_Day
Whole brain
Tonsil
Testis_Intersitial
Prefrontal cortex
Pineal gland_Night
Amigdala
CD33_Myeloid
Whole blood
Fetal liver
Smooth muscle
Fetal brain
Testis_Seminiferous tubule
Ovary
Salivary gland
Trachea
Prostate
Hypothalamus
Pancreas
Burkitt’s lymphoma_Raji
Testis
Olfatory bulb
Occipital lobe
Cerebellum
Caudate nucleus
Skin
Globus pallidus
Medulla oblongata
Retina
Parietal lobe
Adrenal gland
Testis_Leyding cell
Cardiac Myocytes
Placenta
Pancreatic islet
Dorsal root ganglion
Appendix
Bone marrow
Thalamus
Tongue
Adipocyte
Kidney
Uterus_Corpus
Temporal lobe
Liver
Pons
Cerebellum peduncles
Heart
Lung
Spinal Cord
Skeletal muscle
Cingulate cortex
Superior cervial ganglion
Subthalamic nucleus
Adrenal cortex
Atrioventricular node
Trigeminal ganglion
Ciliary ganglion
Figure S3
Random genes
8
H4K20me1
6
4
2
0
0
Z-score DYRK1A targets expression
11.25
4000
1.2
1
***
3.00
2.25
1.50
0.75
0
-0.75
-1.50
-2.25
15.00
Distance (bp)
TSS
shControl
shDYRK1A.2
0.8
**
**
0.2
0
Z-score Scale
+10.0
DYRK1A expression
DYRK1A targets Z-score
Figure S4
A
B
average conservation score
0.5
DYRK1A
random
Nuclear extracts
-
cold competitor
-
t
mu
wt
-
0.4
- Complex II
- Complex I
0.3
0.2
0.1
-200
-100
0
100
- free probe
200
Distance to DYRK1A-peaks mid point (bp)
kDa
E
(m) (r)
K1AYRK1A
R
)
Y
(m nti-D nti-D G (r)
gG
g
HNE IP I IP a IP a IP I
95
2.5
kDa
DYRK1A (m)
2.0
Wb: anti-DYRK1A (m)
% of input
72
D
IgG
(m) (r)
K1AYRK1A(r)
R
)
n
Y
i
t
)
D nti-D i-H3
(m
oma gG G (r ntit
Chr IP I IP Ig IP a IP a IP an
DYRK1A (r)
1.5
1.0
0.5
DYRK1A
95
0.0
RBM39 RPS11
Histone H3
95
DENR
ASXL
G
0.06
IgG
0.05
DYRK1A (m)
0.01
0
6
2
0
1
2
9
1
R
M3 PS1 DEN SXL C7L PL1 RPS _cov
R
A
R
0
RB
LU
r2
ch
TCTCGCGAGA
250
200
150
100
50
0
pGL2
Multimeric
Luc
100
Relative transcriptional activity
(fold change)
0.02
80
60
40
20
0
shControl
95
DYRK1A
80
KAISO
95
YR
D
Ig
G
IP
H
N
E
130
IP
IS
O
(m
)
(r)
(r)
K1
A
kDa
IP
YR
D
IP
KA
A
(r)
K1
Ig
G
IP
YR
D
Ig
G
IP
IP
E
(m
)
(m
)
(m
)
H
N
RPS6 chr20_cov0
Multimeric reporter
Relative transcriptional activity
(fold change)
0.03
kDa
RPL12
TATA
0.04
H
% of input
F
LUC7L
K1
A
C
p120-catenin
DYRK1A
shDYRK1A.1 shDYRK1A.2
Figure S5
B
Not Promoters
C
Promoters
60
number of cells (x104)
80
60
40
20
0.8
0.6
0.4
0.2
0
sh
35
Cyclin D1
95
DYRK1A
54
Tubulin
30
20
10
1
2
3
4
days
Se
Prolierating cells
Serum starved cells
E
DYRK1A mRNA
1.2
Relative expression levels
1.0
kDa
A.1 A.2
l
tro RK1 RK1
Y
D hDY
sh
s
n
Co
ru
sp m s
ec ta
ifi rve
c
d
on
m
om
C
Pr
o
sp lifer
ec at
ifi ing
c
DYRK1A protein
1.2
shDYRK1A.2
40
0
D
shControl
50
0
Relative expression levels
percentage of peaks
100
Proliferating cells
Serum starved cells
1.0
0.8
0.6
0.4
0.2
0
F
Cell cycle profiles
100
G1
S
G2/M
80
*
percentage of cells
A
60
40
20
0
Proliferating Serum Starved
Figure S6
A
HA-DYRK1BWT
B
HA-DYRK1BKR
ut
6)
6)
inp
G1 0)
G1 0)
IgG IgG (8W (N2
IgG IgG (8W I (N2
e
e
it
it
II
I
II
II
s
s
tes ou rabb POL POL
tes ou rabb POL POL
sa m
sa m
Ly IP IP
Ly IP IP
IP
IP
IP
IP
kDa
IIo
IIa
230
96
ST
ST
G
G
Wb: anti-HA
D
-
GST:
kDa
130
A
RK1
DY
E
B
RK1
Antibody
DYRK1A
DY
anti-GST
-
130
DYRK1B
DYRK1BWT
43
GADPH
DYRK1BKR
17
H3
+
H5 (Ser2p)
-
+
hyperP-CTD
hypoP-CTD
F
hyperP-CTD
A.1 A.2
l
tro RK1 RK1
Y
D hDY
sh
s
n
Co
Ser5p
kDa
hypoP-CTD
95
sh
230
130
hyperP-CTD
95
hypoP-CTD
130
Ser7p
DYRK1A
95
130
DYRK1B
54
95
IIo
H5 (Ser2p)
IIo
H14 (Ser5p)
IIo
Thr4p
230
130
kDa
N20
RNAPII
230
G
IIo
IIa
230
GST-CTD
(anti-GST)
95
RNAPII
80
Ser2p
hypoP-CTD
IIo
IIa
230
hyperP-CTD
95
C
WT
DYRK1AKR
Wb: anti-RNAPII
(N20)
in
ol leus omat
c
r
Nu Ch
s
yto
kDa
DYRK1A
*
75
C
D
CT
1B
1A
IgG YRK YRK
l
o
r
D
D
tes Cont anti- antisa
IP
IP
Ly IP
*
DYRK1A
Tubulin
B
H
kDa
95
1
G
RK
l Ig
tro ti-DY
n
ut
Co P an
I
inp IP
] DYRK1B
DYRK1A
72
95
IgG
55
DYRK1B
72
J
3.5
IgG
3.0
DYRK1B
2.5
DYRK1A
2.0
1.5
1.0
IgG
DYRK1B
0.03
0.02
0.01
0.5
0.0
0.05
0.04
% of input
% of input
I
RBM39 RPS11 ASXL CDK12 DENR LUC7L RPL12
RPS6
0.00
RBM39 RPS11 ASXL CDK12 DENR LUC7L RPL12 RPS6
Figure S7
kDa
B
A.1 A.2
l
tro RK1 RK1
n
Co hDY hDY
s
sh
s
3.0
% of input
H3
17
17
H3-Ac
17
H3-Ser10p
17
H3-Ser28p
17
H3-Thr45p
H4-Ac
95
DYRK1A
54
Tubulin
normalized values (8WG16)
DENR
A
1.4
B
C
0.6
***
**
***
***
**
0.4
0.2
0.0
A
B
C
RPL12
A
2.5
EIF4
CDK12
→
B
D
DENR
10 kb
C
D
Ser2p shControl
Ser2p shDYRK1A
Ser5p shControl
Ser5p shDYRK1A
E
2.0
1.5
1.0
*
**
**
*** ***
0.5
A
B
C
E
RPS6
A
1.4
*** ***
D
→
D
1.0
*
CDK12
E
2 kb
1.2
0.8
1.0
3.0
0.0
→
ChIP:N20
1.5
0
normalized values (8WG16)
14
2.0
0.5
C
D
IgG
shControl
shDYRK1A.1
shDYRK1A.2
2.5
normalized values (8WG16)
A
1.5 kb
B
C
D
1.2
1.0
0.8
**
***
0.6
***
0.4
***
**
***
0.2
0.0
A
B
C
D
SUPPLEMENTAL FIGURE LEGENDS
Figure S1. Related to Figure 1
Nuclear DYRK1A is an active nuclear kinase present in high molecular weight
complexes that co-fractionates with RNAPII. (A) DYRK1A-immunocomplexes from
HeLa nuclear extracts were used in in vitro kinase (IVK) assays using DYRKtide as the
substrate to determine the DYRK1A kinase activity at the time points indicated.
Immunocomplexes obtained with mouse IgGs were used as a control. DYRK1A
activates transcription in a dose and kinase-dependent manner when tethered to
promoter regions. (B) Schematic representation of the constructs used in the reporter
assays in Figure 1 and panels E-G of this Figure. The effector plasmid G4DBDDYRK1AWT directs the expression of the full-length DYRK1A protein fused to the Gal4
DNA binding domain (DBD) while G4DBD-DYRK1AKR expresses a kinase-inactive
DYRK1A through mutation of the ATP binding site. KD, kinase catalytic domain; NLS,
nuclear localization signal; PEST region; His, stretch of 13 consecutive His residues;
S/T, region enriched in Ser and Thr residues. The target plasmid G5HIV1-Luc contains
5 Gal4 binding sites (Gal4), the HIV-1 promoter, with NF-κB and Sp1 binding sites
driving the expression of the luciferase reporter gene (Montanuy et al., 2008). The
target plasmid G4E1b-Luc contains 5 Gal4 binding sites upstream of the E1b
adenovirus minimal promoter (de la Luna et al., 1999). (C, D) Soluble extracts from
HEK-293 cells expressing the indicated G4-DBD fusion plasmids were separated by
SDS-PAGE and analyzed in Western blots probed with a G4-DBD specific antibody.
The position of the marker proteins (in kDa) is indicated. The Western blots show that
the G4-DBD/DYRK fusion proteins used in the reporter assays shown in Figure 1G and
1H were expressed at similar levels. (E) HEK-293 cells were co-transfected with the
reporter G5HIV1-Luc together with increasing amounts of the expression plasmids
encoding G4-DBD fusions to wild type DYRK1A (5 ng, 10 ng or 20 ng) or the kinase
inactive DYRK1A (10 ng, 20 ng or 40 ng). A plasmid expressing unfused G4DBD (-)
was used to measure the basal activity of the reporter. Luciferase activity was
measured in triplicate plates and the values were corrected for transfection efficiency
as measured by the Renilla activity. The graph represents transcriptional activity as the
fold change compared to the control G4-DBD value, set as 1 (mean ± s.d.), from a
representative experiment of 3 performed. (F) The efficiency of DYRK1A to activate
transcription was compared with that of CDK9 on the G5HIV1-Luc reporter, at similar
protein levels (as assessed in Western blots, in panel D). (G) The efficiency of
DYRK1A to activate transcription was assessed with the G4E1b-Luc reporter or the
G4E1b-Luc TATA-less reporter, which lacks the TATA-box of the minimal E1b
promoter. The graph represents transcriptional activity as the fold change compared to
the control G4-DBD value, set as 1, for each reporter. Note the strong reduction in the
induction of luciferase expression by DYRK1A on the TATA-less reporter when
compared with the G4E1b-luc reporter.
Figure S2. Related to Figure 2
DYRK1A is recruited to promoters. (A) Bar plot showing the relative enrichment of
promoters in the ChIP fragments with respect to the genome background. The light
violet bars represent the percentage of annotated promoters in the genome and the
dark bars the percentage in ChIP fragments. P-values for the significance
(hypergeometric test) of the relative enrichment in ChIP fragments with respect to the
genome background are shown in parentheses. (B) Venn diagram of the overlap
between DYRK1A target chromatin loci of in T98G cells and HeLa cells. (C) DYRK1A
signal maps are shown for representative target genes in T98G cells and HeLa cells.
Figure S3. Related to Figure 3
(A) Profiles of the histone modifications indicated around the TSS for DYRK1A
associated genes (in red) and for random Ensemble genes (in black). (B) DYRK1A
protein levels were down-regulated in HeLa cells by lentiviral delivery of two different
shRNAs targeting DYRK1A and the expression level of several DYRK1A target genes
was determined by RT-qPCR. The data represent the mean and standard deviation of
five independent experiments. (C) Correlation of the expression of DYRK1A and its
targets in human tissues. Data from the GeneAtlas dataset (GEO GSE1133 dataset)
was used. DYRK1A target genes in both HeLa and T98G cells were defined as genes
with DYRK1A peaks between -300 to +1 of the TSS. The graphs show the absolute
(log2) expression values for DYRK1A in different tissues and cell lines, plotted as a
color-coded heatmap, where red indicates stronger expression and green indicates
weaker expression (see "Expression scale"). The correlation analysis is represented as
Z-score values for preferential expression and under-expression of DYRK1A target
genes defined in a different colored heatmap, where red signifies over-expression of
the targets and blue indicates under-expression of the targets. The samples are
ordered according to the Z-score of preferential expression of the DYRK1A targets. (D)
The scatter plot shows the correlation between DYRK1A expression and the Z-score of
the expression of their targets (Pearson correlation coefficient 0.62).
Figure S4. Related to Figure 4
The TCTCGCGAGA-sequence is a regulatory motif in DYRK1A target promoters.
(A) Average conservation of the enriched motif in DYRK1A promoters (red line)
compared to random sequences of equal length (green). On the X axis, 0 indicates the
mid point of the DYRK1A peaks. Minus indicates upstream bases and plus values
indicate bases downstream of the DYRK1A binding site sequence. (B) EMSA was
performed with HeLa nuclear extracts and a 32P-labeled oligonucleotide probe
containing the sequence motif from the RPS11 promoter region. A competition assay
was performed with increasing amounts of the wild type cold probe (wt, 20-fold, 50-fold
and 100-fold) or of a probe of the same size without the consensus sequence (mut, 20fold, 50-fold and 100-fold). The position of the shifted band corresponding to Complex I
and II is indicated, as well as that of the free probe. (C) HeLa nuclear extracts were
immunoprecipitated with the two antibodies against DYRK1A used in the EMSA
experiments (m: mouse monoclonal 7D10 from Abnova; r: rabbit polyclonal from
Abcam). Both the lysates and the immunoprecipitates were analyzed by Western blot
with the anti-DYRK1A monoclonal antibody. IgGs from mouse and IgGs from rabbit
were used as controls in the immunoprecipitation. (D) ChIP-Western from T98G cells
with the two antibodies against DYRK1A used in the ChIP-qPCR assays included in
panels E and F (m: mouse monoclonal 7D10 from Abnova; r: rabbit polyclonal from
Abcam). The chromatin fraction and the immunoprecipitates were analyzed by Western
blot with anti-DYRK1A and anti-histone H3 antibodies as indicated. Note that although
both antibodies immunoprecipitate DYRK1A with similar efficiency, H3 is only detected
in the rabbit anti-DYRK1A immunoprecipitates. (E, F) ChIP-qPCR assays on samples
from T98G cells immunoprecipitated with the two anti-DYRK1A antibodies. ChIP
signals were normalized with inputs. As control, IgG from mouse were used. In F, only
the results with the mouse antibodies are shown to highlight the poor enrichment over
the IgG control. (G) T98G cells were transfected with a pGL2-based plasmid in which
five TCTCGCGAGA sequences (represented as white boxes in the scheme) were
cloned in tandem upstream of the TATA-box of the adenovirus E1B promoter. In the
left panel, the relative transcriptional activity of the multimeric reporter is compared with
that of pGL2-basic. In the right panel, transfections were done in shRNA-control
infected cells or in cells depleted of DYRK1A by lentiviral transduction of two different
shRNAs; values are expressed as the fold change over that of a pGL2-basic reporter in
each of the shRNA-treated cell pools. (H) HeLa nuclear extracts were
immunoprecipitated with two different antibodies to DYRK1A (Abnova mouse
monoclonal and Abcam rabbit polyclonal) or a rabbit polyclonal anti-KAISO antiserum.
Normal mouse IgGs (mIgG) or normal rabbit IgGs (rIgG) were used as controls for
specificity. Both the lysates (10%) and the immunocomplexes were analyzed by
immunoblotting with antibodies against DYRK1A, KAISO and p120-catenin, as
indicated in the Figure.
Figure S5. Related to Figure 5
DYRK1A-peak enrichment T98G cells grown in proliferating or resting
conditions. (A) Distribution of DYRK1A ChIP-Seq target regions as promoters or
regions not associated to promoters in the overlapping peaks between T98G
proliferating cells and T98G serum starved cells (common), in the peaks only detected
in the proliferating ChIP-Seq or only in the T98G serum starved cells ChIP-Seq.
DYRK1A depletion does not affect the cell proliferation. (B) Cumulative cell growth
curve of T98G cells infected with a lentivirus expressing a shControl or shDYRK1A.2.
The graph represents the total number of cells over the period indicated. (C) Total cells
extracts prepared from T98G infected with lentiviruses expressing a shControl or two
different shRNAs to DYRK1A were analyzed by Western blot with antibodies to detect
cyclin D1. It has been shown that DYRK1A depletion in human fibroblast induces
increased cyclin D1 accumulation with one subpopulation of cells arresting proliferation
by co-stabilizing the CDK inhibitor p21 (Chen et al., 2013). No changes in cyclin D1
accumulation were detected in T98G upon DYRK1A depletion, which would be in
agreement with a lack of effect in cell proliferation. (D) Protein levels of DYRK1A were
determined by Western blot in proliferating and serum starved T98G cells. The graph
shows the quantification of two independent experiments. No significant differences
were found. (E) The expression levels of DYRK1A mRNA were determined by RTqPCR in proliferating and serum starved T98G cells. The data represent the mean and
standard deviation of five independent experiments (p-value=0.00187). The reduction
in DYRK1A mRNA levels could be the consequence of the existence of E2Fresponsive elements in one of the DYRK1A promoters (Maenz et al., 2008), which
would be repressed in the serum starved cells. (F) The cell cycle profile of T98G
proliferating and serum starved cells was determined by FACs analysis. The
distribution of the G1, S and G2/M subpopulations is shown. Note that about 90% of
the cells were arrested at G1 phase upon serum withdrawal.
Figure S6. Related to Figure 6
DYRK1B interacts with the CTD of RNAPII. (A) Soluble cell extracts from HEK-293T
cells expressing HA-DYRK1B wild-type (WT) or a kinase-inactive version (KR) were
immunoprecipitated either with control IgGs (mouse and rabbit, as indicated) or with
the anti-RNAPII antibodies N20 (rabbit) and 8WG16 (mouse). Both the input and the
immunoprecipitates were analyzed with an anti-HA antibody and with the anti-RNAPII
antibody N20. The position of the hypo- and the hyperphosphorylated forms of the
RNAPII (IIa and IIo, respectively) is indicated. The asterisk points to a cross-reacting
band. DYRK1B (wt and kinase-dead) is present in the N20 immunoprecipitates but no
in the 8WG16 immunoprecipitates, similarly to what happens with its paralogue
DYRK1A. (B) The proteins indicated were expressed in HEK-293T cells by transient
transfection of the corresponding plasmids (Flag-tagged in the case of DYRK1A and
HA-tagged in the case of DYRK1B). Soluble cell extracts were incubated with unfused
GST or GST-CTD immobilized on gluthatione-Sepharose beads. The bound proteins
were detected by Western blot with anti-Flag or anti-HA antibodies. Although DYRK1B
is detected as present in the GST control pull-down, a clear increased is observed in
the GST-CTD bound fraction for both the wt and the kinase inactive version. Therefore,
DYRK1B interacts with the non-phosphorylated CTD independently of its kinase
activity. Subcellular distribution of DYRK1B. (C) Panc-1 cells were fractionated into
cytosolic, soluble nuclear and insoluble nuclear (chromatin) fractions. Equivalent
aliquots of each fraction were analyzed by Western blot with antibodies to the indicated
proteins. The purity of the fractions was assessed with the compartment-specific
marker proteins GAPDH and histone H3. DYRK1B is mostly detected in the cytosolic
fraction; however, a small pool of DYRK1B is present in the nuclear soluble and
insoluble (chromatin) fractions. DYRK1B phosphorylates the CTD of RNAPII. (D)
The in vitro kinase assays were performed at equal specific activities of DYRK1A and
DYRK1B obtained from commercial sources (DYRK1A specific activity on DYRKtide
peptide=2,880 nmol/mg; dosage= 6.4, 12.8 ng; DYRK1B specific activity on
DYRKtide=2,210 nmol/mg; dosage=10, 20 ng) as the source of the enzyme and the
fusion protein GST-CTD as the substrate (150 ng). An aliquot of the reaction was
analyzed in immunoblots probed with antibodies to: i) GST, to detect the fusion GSTCTD (note that due to differences in amounts, the GST-DYRK fusions are not
detected); ii) the CTD phosphoresidues indicated; iii) the DYRK proteins. Note that
both kinases phosphorylate Ser2, Ser5 and Ser7 to similar extent. DYRK1A
phosphorylates the RNAPII CTD at Ser2. (E) DYRK1A phosphorylates Ser2 within
the CTD as detected by the H5 antibody. GST-CTD (150 ng) was incubated with GST
(-) or GST-DYRK1A (20 ng) in and IVK assay. An aliquot of the reaction was analyzed
in Western blots probed with antibodies to GST, to detect the fusion GST-CTD, and the
H5 antibody to detect the CTD Ser2 phosphoresidue. Depletion of DYRK1A does not
change global phosphorylation levels of the CTD of RNAPII. (F) Total cells extracts
prepared from T98G infected with lentiviruses expressing a shControl or two different
shRNAs to DYRK1A were analyzed by Western blot with antibodies to detect total
RNAPII (N20) or the H5 and H14 antibodies, which recognize mostly Ser2
phosphorylated (in the context of phosphorylated neighboring Ser5 residues) or Ser5
phosphorylated (Chapman et al. 2007). The position of the hyper- (IIo) and hypo- (IIa)
phosphorylated forms of RNAPII is indicated. The asterisk indicates a cross-reacting
band for the DYRK1A antibody. Assessing the ability of DYRK1B to be recruited to
DYRK1A target sites. (G) Soluble extracts of Panc-1 cells were immunoprecipitated
with rabbit polyclonal antibodies to either DYRK1A or DYRK1B. Both the lysates and
the immunoprecipitates were assayed by Western blot for the presence of the kinases.
Rabbit IgGs were used for control immunoprecipitations. The results show that each
antibody specifically immunoprecipitates the corresponding DYRK protein. (H) ChIPWestern blot on Panc-1 cells with the anti-DYRK1B antibody to assess the ability of
this antibody to immunoprecipitate DYRK1B from formaldehyde-crosslinked cells. (I, J)
ChIP-qPCR assays using samples from Panc-1 cells immunoprecipitated with anti-
DYRK1A and anti-DYRK1B antibodies. As control, rabbit IgGs were used. In G, only
the results with the DYRK1B antibody were shown to highlight the lack of enrichment
over the IgG control.
Figure S7. Related to Figure 7
DYRK1A depletion does not affect globally the acetylation levels of H3 or the
phosphorylation of H3 at Ser10, Ser28 or Thr45. (A) Total cells extracts prepared
from T98G infected with lentiviruses expressing a shControl or two different shRNAs to
DYRK1A were analyzed by Western blot with antibodies to detect histone H3 (H3) or
the following post-translational modifications: acetylated H3, phosphorylated H3 at
residues Ser10, Ser28, Thr45, and acetylated H4. DYRK1A depletion reduces
RNAPII occupancy at target promoters. (B) The presence of RNAPII at DYRK1A
target promoters was assessed by ChIP-qPCR with the N20 antibody in cells infected
with a shControl or two different shDYRK1A. Values are normalized with input. (C, D,
F) DYRK1A depletion leads to a decreased in elongating RNAPII along gene
body. The presence of RNAPII phosphorylated at Ser2 and Ser5 along the indicated
DYRK1A target gene bodies was assessed by ChIP-qPCR with specific antibodies, in
control cells and cells with reduced DYRK1A expression. For each gene, the scheme
shows the basic gene structure and the position of the amplicons. ChIP signals were
normalized with RNAPII (8WG16). Signals in shControl cells were set at 1 for each
amplicon, and the values from the shDYRK1A cells were expressed relative to these
signals. The graphs show the average of two independent experiments with the bars
indicating standard deviations.
Table S1. Related to Figure 2.
DYRK1A ChIP-Seq results on T98G and HeLa cells.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Plasmids
The plasmids to express HA- or GST-tagged, wild type or kinase inactive DYRK1A (in
which the ATP binding Lys179 was replaced by Arg), have been described previously
(Alvarez et al., 2007; Alvarez et al., 2003). To express N-terminal fusions of the
different human DYRKs (Accession Nº for DYRK1A: NP_569120; Acc. Nº for DYRK1B:
NP_004705; Acc. Nº for DYRK2: NP_006473; Acc. Nº for DYRK3: NP_003573; Acc.
Nº for DRYK4: NP_003836) to the yeast Gal4 DNA binding domain (DBD; amino acids
1-147 of the yeast transcription factor), EcoRI/XbaI fragments containing the
corresponding open reading frames were ligated in-frame into the EcoRI/XbaI sites of
pCG4-DBD (de la Luna et al., 1999). The DYRK1B open reading frame was cloned into
pCDNA3-HA to express a HA-tagged version of the kinase. To generate a DYRK1B
kinase inactive mutant, we substituted the codon for Lys140 in the ATP binding site by
an Arg codon, and the loss of activity was confirmed in in vitro kinase assays.
As reporter plasmids we used, pG5HIV-Luc (Montanuy et al., 2008), kindly
provided by C. Suñe (Instituto de Parasitología y Biomedicina López Neyra, Granada,
Spain), and pG4E1b-Luc (de la Luna et al., 1999). The pG4E1b-TATAless-Luc reporter
plasmid was generated with the QuickChange Site-Directed Mutagenesis Kit
(Invitrogen) on the pG4-E1b-Luc using the TATA-less primer (Supplemental Tables).
The reporter plasmid in which luciferase expression is under the control of the
promoter of the RPS11 gene was generated by PCR amplification of the corresponding
genomic regions with primers RPS11-f and RPS11-r (Supplemental Tables), which
was subcloned into pGL2-basic (Promega). The two consensus sequences within the
promoter region were deleted with the QuickChange Site-Directed Mutagenesis Kit and
primers RPS11-mut1 and RPS11-mut2 (Supplemental Tables). For the multimeric
reporter, sense and antisense oligonucleotides with 5 repetitions of the TCTCGCGAGA
sequence followed by the E1B adenovirus TATA box (AGGGTATATAATG) were
annealed and cloned into pGL2-basic.
For the pull-down assays, pGEX-CTD and pMyc-Cyclin T1 (Taube et al., 2002)
were kindly provided by M. Peterlin (University of California, San Francisco, USA). For
the expression of cyclin L2, the open reading frame of human cyclin L2 (Acc. Nº
NP_112199) was cloned in-frame into pCDNA-3 with a Flag-tag at the N-terminus to
generate pFlag-Cyclin L2.
Cell culture and DNA transfection
The HeLa, T98G, Panc-1, HEK-293T and HEK-293 cell lines were supplied by the
American Type Culture Collection (http://www.atcc.org). All the cell lines were cultured
at 37ºC and in 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen)
with 10% fetal bovine serum (FBS; Invitrogen) and supplemented with antibiotics (100
u/ml penicillin and 100 µg/ml streptomycin; Invitrogen). The cells were transfected by
the calcium phosphate precipitation method and processed 24-48 h after transfection.
For serum starvation conditions, T98G cells were grown in DMEM without FBS for 48
h. For harmine treatment, T98G cells were treated with 10 µM harmine or with vehicle
(0.02% DMSO) for 16 h.
Lentivirus preparation and infection
HEK-293 cells were transfected with pVSV-G (Stewart et al., 2003) and pCMV∆R8.91
(Zufferey et al., 1997), together with the pLKO.1-puro non-targeting vector (Sigma
Mission clone SHC001) or pLKO.1-shRNA DYRK1A (Sigma Mission clones
TRCN0000022999, TRCN0000199464 and TRCN0000199188), and the supernatant
was harvested after 72 h. Viral particles were concentrated by centrifugation through a
4% sucrose cushion at 87,500 x g for 90 min at 4ºC. The cells were infected by adding
the virus in DMEM with 5 µg/ml hexadimethrine bromide (Polybrene, Sigma). The
medium was removed 24 h after infection and the cells processed 48 h later to quantify
mRNA expression or for ChIP analysis. For selection, 1 µg/ml puromycin was added
for 3 days and the cells were cultured for a further 24 h in the absence of puromycin.
For the rescue experiments, the open reading frames of DYRK1Awt and the kinaseinactive version were cloned into the pWPXL lentiviral vector (Didier Trono's group;
Addgene plasmid 12257). The expression of DYRK1A from these lentivectors is
resistant to the effect of the DYRK1A shRNA expressed by the Mission clone
TRCN0000199188 that targets the DYRK1A 3'-UTR.
FACS analysis of cell cycle parameters
Cells harvested in PBS-5 mM EDTA were fixed and permeabilized overnight with 100%
ethanol. The cells were then resuspended in phosphate-buffered saline (PBS)
containing 0.5 µg/ml Ribonuclease A (Sigma), 50 µg/ml propidium iodide (Sigma) and
3.8 mM sodium citrate, and analyzed by FACS flow cytometry with the Cell Quest
software (Becton Dickinson).
Cell lysate preparation, glycerol gradient sedimentation and size exclusion
chromatography
To prepare the total cell lysates, 1x106 cells were resuspended in 150 µl 2x sample
loading buffer (100 mM Tris-HCl [pH 6.8], 200 mM dithiothreitol [DTT], 4% [w/v] sodium
dodecyl sulfate [SDS], 20% [v/v] glycerol, 0.2% [w/v] bromophenol blue) and heated for
10 min at 98ºC. Soluble extracts were prepared by incubating cells for 15 min at 4ºC in
lysis buffer A (50 mM HEPES [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1% [v/v] Nonidet P40 [NP-40], protease inhibitor cocktail [PIC; Roche Diagnostics] and phosphatase
inhibitors [2 mM sodium orthovanadate, 30 mM sodium pyrophosphate, and 25 mM
sodium fluoride]), followed by centrifugation at 13,000xg for 30 min at 4ºC.
Subcellular fractionation of cells was performed using NE-PER (Nuclear and
Cytoplasmic Extraction Reagent; Pierce) according to the manufacturer’s protocol.
Protein quantification was performed with the commercial BCA Protein Assay Kit
(Pierce). For the subnuclear fractionation, 3 mg of HeLa nuclear extracts (HNE; CIL
Biotech) was applied to a 10 ml 10–40% glycerol gradient in 20 mM HEPES [pH 7.9],
150 mM KCl, 0.2 mM EDTA, 0.1 % NP-40 and PIC. After centrifugation at 33,000 rpm
in a SW41 Ti rotor (Beckman) for 20 h at 4°C, 24 fractions (500 µl each) were collected
from the top of the gradient and a 30 µl aliquot of each fraction was analyzed in
Western blots. Molecular mass standards (Gel Filtration High Molecular Weight
Calibration Kit, Amersham) were used to identify the fractionation profiles within the
gradient and the marker proteins were detected by silver staining of SDS-PAGE gels
using standard protocols.
Western blotting
Cell lysates were resolved on SDS-polyacrylamide gels and the proteins transferred
onto Hybond-ECL nitrocellulose membranes (Amersham Biosciences). Membranes
were blocked for 30 min at room temperature (RT) with 10% (w/v) skimmed milk diluted
in TBS (10 mM Tris-HCl [pH 7.4] and 100 mM NaCl)-0.1% Tween-20 (TBS-T), and
then probed for 1 h at RT with primary antibodies diluted in 5% skimmed milk in TBS-T.
After several washes, the membranes were incubated at RT for 45 min with
horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) diluted in 5%
skimmed milk in TBS-T. Antibody binding was detected by chemiluminiscence
(SuperSignal West Pico Chemiluminescent Substrate, Pierce) on a LAS-3000 image
analyzer (Fuji PhotoFilm). Primary antibodies are listed in the Supplemental Tables for
Extended Experimental Procedures.
Immunoprecipitation
Soluble cell extracts were incubated overnight at 4°C with protein G-Magnetic beads
(Invitrogen) pre-bound with a mouse monoclonal anti-DYRK1A antibody. Mouse IgGs
or rabbit IgGs (Santa Cruz) were used as controls. The beads were washed four times
with lysis buffer A, adding 0.1% NP-40 to the three initial washes, and they were finally
resuspended in sample loading buffer. Samples were resolved by SDS-PAGE and
analyzed by immunoblotting or used for in vitro kinase assays. The antibodies used in
the immunoprecipitation experiments are listed in the Supplemental Tables for
Extended Experimental Procedures.
GST-fusion protein expression in bacteria and pull-down assays
GST-fusion constructs were transformed into E. coli BL21(DE3)pLysS (Stratagene)
and protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactoside for 3 h
at 37ºC for the GST-CTD fusion protein and for 8 h at 20ºC for GST-DYRK1A (WT or
KR). Cells were lysed in lysis buffer B (10 mM Tris-HCl [pH 8], 100 mM NaCl, 1 mM
EDTA, 0.5% NP-40 and a protease inhibitor cocktail), and the bacterial lysates were
incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for 45 min at
RT and then washed four times with lysis buffer B.
For pull-down assays, prey proteins were transcribed and translated in vitro in the
presence of [35S]-methionine using the TnT T7 Coupled Reticulocyte Lysate System
(Promega), following manufacturer’s instructions. Equivalent amounts of synthesized
proteins were incubated with unfused GST or GST-CTD immobilized on glutathioneSepharose beads that had been previously equilibrated in binding buffer (20 mM
HEPES-KOH, 200 mM KCl, 0.1% Triton-X-100, 0.05% NP-40, 5 mM EDTA, 0.3% BSA
and 5 mM DTT). After 3 h rolling at 4ºC, the beads were washed extensively with cold
binding buffer containing 500 mM KCl, and the bound proteins were resolved on SDSPAGE gels that were then dried and exposed to a film.
In vitro kinase assays
For in vitro kinase (IVK) assays, we used GST-DYRK1A expressed in bacteria as a
purified enzyme source (Alvarez et al., 2007). Both the substrate (GST-CTD) and the
enzyme were eluted from the glutathione beads with 10 mM reduced glutathione
(Sigma) in 50 mM Tris-HCl pH 8 and dialyzed against a buffer containing 50 mM
HEPES pH 7.4, 150 mM NaCl and 2 mM EDTA. For the IVK assays, we used 100-150
ng of GST-CTD and 5, 10 or 20 ng of GST-DYRK1AWT or GST-DYRK1AKR. The
proteins were incubated for 20 min at 30oC in 20 µl of phosphorylation buffer (50 mM
HEPES [pH 7.4], 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT and 50 µM ATP]. For the
comparative IVK assays of DYRK1A and DYRK1B, we used commercial proteins (Life
Technologies). The IVK assays were performed at the same specific activity for each
kinase as determined by the supplier on DYRKtide (DYRK1A: 2,880 nmol/mg;
DYRK1B: 2,210 nmol/mg).
For the IVKs, in which DYRK1A was purified by immunoprecipitation from cells,
the immunocomplexes were incubated for 20 min at 30oC in 20 µl of phosphorylation
buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT and 50 µM
ATP [γ32P-ATP, 2.5x10-3 µCi/pmol] in the presence of the peptide substrate DYRKtide
(Himpel et al., 2000) and 32P incorporation was determined as described (Alvarez et al.,
2007). Harmine was used as a DYRK1A inhibitor (Bain et al., 2007) and in this case
the immunoprecipitates were preincubated for 15 min with 10 µM harmine or with the
vehicle alone (DMSO) prior to carrying out the IVK reaction in the presence of the
inhibitor.
Electrophoretic mobility-shift assay (EMSA)
A [32P]-labeled double stranded oligonucleotide (0.03 pmol) spanning either the wt or
the mutant DYRK1A motif (for details see Supplemental Experimental Procedures) was
incubated with 5-10 µg of nuclear cell extract and 30 ng of poly-dI-dC (Roche
Diagnostics) at 20°C for 20 min in 250 mM Tris-HCl [pH7.5], 250 mM NaCl, 5 mM
MgCl2, 5 mM DTT, 1 mM EDTA and 50% glycerol. For the supershift assay, 2 µg of
either mouse IgG (Santa Cruz), mouse monoclonal (Abnova H00001859) or rabbit
polyclonal (Abcam ab69811) anti-DYRK1A antibodies were pre-incubated with the
extracts for 15 min on ice before the addition of the DNA probe. Binding reactions were
analyzed on non-denaturing 4% acrylamide gels.
Reverse transcription (RT) and quantitative PCR (qPCR)
Total RNA was isolated with the RNeasy extraction Kit (Qiagen), treated with DNase I
(Ambion, 2 U/µl) and quantified with Nanodrop. Superscript II Reverse Transcriptase
(RT: Invitrogen) was used to synthesize cDNA from the total RNA (500 ng) as
recommended by the manufacturer’s instructions. RT-qPCR reactions were performed
with SYBR Green (Fermentas) in 384 well plates using the Roche LC-480 cycler
(Roche Applied Science). The template was denatured at 95º for 5 min, and subjected
to 50 cycles of 15 s 95º, 20 s 60º, 20 s 72º. Each sample was assayed in triplicate. The
Ct (threshold cycle) was calculated for each sample using the Relative Quantification
2nd Derivative Maximum method with the Lightcycler 480 1.2 software (Roche). No
PCR products were observed in the absence of template and all the primer sets gave
narrow single melting point curves that were checked at the end of each run. For RTqPCR, analysis was performed using a 1/10 dilution of cDNA as the template. A "noRT" control was included in each experiment to ensure that the PCR products were not
amplified from contaminating DNA, and all results were normalized to GAPDH, β-actin
or EF1α expression.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed from approximately 107 HeLa or T98G cells per
experiment. Briefly, formaldehyde was added to the culture medium to a final
concentration of 1% for 10 min at RT and the crosslinking was then quenched with
0.125 M glycine for 5 min. Crosslinked cells were washed twice with TBS, resuspended
in 1 ml of Buffer I (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40 plus PIC) and
incubated on ice for 10 min. Cells were centrifuged at 800 x g for 5 min, and the cell
pellet was resuspended in 1.2 ml of Buffer II (1% SDS, 10 mM EDTA, 50 mM Tris-HCl
[pH 8.0], 1 mM phenylmethylsulfonyl fluoride and PIC) and incubated for a further 10
min on ice. Samples were centrifuged and the cell pellet was resuspended in 1.2 ml of
IP Buffer (100 mM Tris-HCl [pH 8.6], 0.3% SDS, 1.7% Triton X-100 and 5 mM EDTA)
prior to sonicating the chromatin to an average size of 200-500 bp (Bioruptor,
Diagenode). Extracts (600 µg of total protein) were diluted in 1 ml of IP Buffer and
immunoprecipitated overnight at 4°C with the corresponding antibodies (listed in the
Supplemental Tables for Extended Experimental Procedures) or control IgG
antibodies. Immune complexes were recovered by incubating for 4 h at 4°C with
protein G-Sepharose beads (40 µl), and the beads were then washed with three
successive 1 ml washes in Low salt Buffer III (50 mM HEPES [pH 7.5], 140 mM NaCl,
1% Triton X-100 plus PIC), and one wash with High salt Buffer IV (50 mM HEPES [pH
7.5], 500 mM NaCl, 1% Triton X-100 plus PIC). DNA was eluted twice from the beads
for 1 h at 65ºC in Elution Buffer (1% SDS, 100 mM NaHCO3) with constant agitation
(1,000 rpm). The crosslinking was reverted by further incubating the DNA at 65ºC
overnight at 1,000 rpm. The eluted material was phenol/chloroform extracted and
ethanol-precipitated, and the ChIP DNA was resuspended in 50 µl of H2O for further
analysis. For ChIP-qPCR, a 1/10 dilution of ChIP DNA was used as the template for
the PCR reaction, and a 1/1000 dilution of input DNA was used as standard for
normalization.
For all antibodies, ChIP signals were normalized with inputs. In the case of
antibodies detected post-translational modifications, the data was represented as the
ratio to the non-modified protein: H3-T45p/H3, H3-Ac/H3, Ser2p/8WG16,
Ser5p/8WG16. For standarization, values in T98G cells infected with a control
lentivirus (shControl) were set at 1 and values from the cells infected with a lentivirus
expressing a shRNA against DYRK1A (shDYRK1A), or other treatments, expressed
relative to these signals.
Statistical analysis
All results are expressed as the mean ± SD of three independent experiments unless
otherwise stated. Statistical analyses were performed with a two-tailed unpaired
Student's t-test to determine statistical significance between the groups. P-values are
represented in all Figures as follows: *, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤
0.001.
Computational analysis
Sequencing and read mapping
ChIP-Seq libraries were sequenced to a length of 36bp on an Illumina GAIIx
sequencer, and an average of 25,752,900 reads were obtained per library. The quality
of the sequenced reads was controlled using FastQC (Trinklein et al., 2003). The reads
were aligned to the hg19 human reference genome using bwa (Li and Durbin, 2009),
allowing up to 3 mismatches and no gaps (whereby >97% of the reads were aligned).
The resulting SAM files were sorted by position and transformed to BAM using
samtools (Li et al., 2009). Picard (http://picard.sourceforge.net) was used to mark
clonal duplicate reads in the BAM files (e.g. ~20% duplicate reads marked in TG98G)
and ‘samtools flagstat’ to obtain quality information about the mapping.
Peak identification and summary of the ChIP-Seq data
The ChIP-Seq peak caller ‘shore peak’ (Moyroud et al., 2011; Pose et al., 2013) from
the open source package SHORE (Ossowski et al., 2010)
(http://sourceforge.net/projects/shore/) was used for the calling and visualization of
peaks. BAM files were converted to the native SHORE format map.list using ‘shore
convert’, and the peaks were predicted by the peak caller ‘shore peak’, using only
uniquely aligned reads and allowing for up to 3 duplicate reads at a position. Peaks
were accepted when: i) fold-change (FC, DYRK1A IgG versus control IgG) was ≥ 3; ii)
the false discovery rate (FDR) was ≤ 0.05; iii) peak size was at least 80 bp; and iv) the
x-shift (distance between forward and reverse peak) was at least 10 bp. The
parameters of peak calling were:
shore peak –i ChIP.map.list –c Control.map.list -S 10000 -V 10 -Q 0.5 -J 80 -B 3 -F 0 -
n 3 --min-foldchange=3 -H 1,1 --rplot=500 –r hg19.index.shore
T98G ChIP-Seq: 966 peaks were detected with a FDR≤10-2 (598 peaks with a
FDR<10-3). However, only 604 peaks also showed a significant x-shift (x-shift≥80) and
normalized FC scores (454 peaks for a FDR<10-3). Visual inspection indicated that the
top 500 peaks mostly present an S-shaped coverage pattern.
MACS (Zhang et al., 2008) was used, in no-model mode, assuming the fragment
sizes estimated by SHORE, to produce wiggle files for visual inspection in IGV. MACS
returned 713 peaks with a FDR<5%. More than 90% of the top 500 SHORE peaks
were also detected with MACS. Visual inspection of the overlapping peaks as well as
removal of peaks with a small x-shift (<80 bp) or forward-reverse coverage imbalance
resulted in a final set of 539 high quality peaks.
Serum-starved T98G ChIP-Seq: SHORE identified 477 peaks with a FDR≤0.05 (247
for a FDR≤10-2). Correction for x-shift resulted in 341 peaks (221 for a FDR≤10-2), of
which 233 (199 for a FDR≤10-2), were included within the top 500 MACS results. Visual
inspection and quality filtering by x-shift and fwd-rev coverage distribution resulted in a
final selection of 337 high quality peaks.
HeLa ChIP-Seq: Peak detection in HeLa ChIPs revealed substantially fewer peaks
than the T98G ChIPs. To reduce false positives, we used SHORE’s ability to analyze
two independent biological replicates together. We selected 73 peak predictions with pvalue < 0.05 (p<10-2: 64 peaks), of which 68 were found in the top 100 MACS results.
77 SHORE peaks showed a good x-shift (≥80), of which 67 (87%) were found in the
top 100 MACS results.
Peaks resulting from the three ChIP-Seq experiments were loaded into a MySQL
database, and SQL commands were used to overlap the three peak sets using a 10%
minimal overlap between peaks as threshold (Supplemental Table S1).
Peak annotation
An in-house gene and promoter annotation pipeline (ChIPanno) was used to annotate
potential target genes and promoters. ChIPanno uses gene, transcript, promoter and
transcription start site (TSS) annotation data from Ensembl, NCBI RefSeq,
SwitchGear-TSS (Landolin et al., 2010; Trinklein et al., 2003)
(http://www.switchgeargenomics.com/) and the UCSC genome browser database
(Meyer et al., 2013), all obtained via the UCSC Table Browser (Karolchik et al., 2004).
TSS from SwitchGear-TSS were filtered by setting confScore > 20 to decrease false
positive TSS annotations (Landolin et al., 2010). We further used the promoter
definition provided by the annotation tool HOMER (Heinz et al., 2010) and a custom set
of promoters defined as SwitchGear-TSS 1 kb upstream to 100 bp downstream (called
SW-TSS-1.1k from here on). All information was uploaded into the MySQL database to
facilitate further motif, peak and functional enrichment analyses.
Peak distribution at various genomic locations and peak density heatmaps
The distribution of various genomic features in binding sites was assessed using CEAS
software (Shin et al., 2009), and with in-house Python and Perl scripts. The distribution
of the peaks within a -1 kb to +1 kb window centered on the TSS of each RefSeq
transcript was calculated in 25 bp bins using scripts contained in the HOMER package
(Heinz et al., 2010) and plotted using R.
Expression of target genes according to public microarray data analysis
Normalized mRNA expression (Affymetrix Human U133A/GNF1H Gene Atlas
microarray chip) in 73 normal human tissues was downloaded from the BioGPS
database (Wu et al., 2009). When more than one probe was present for the same
gene, they were averaged. Based on the ‘‘gene-normalized’’ expression of the
DYRK1A targets in the GeneAtlas data, we carried out Sample Level Enrichment
Analysis (Gundem and Lopez-Bigas, 2012) using Gitools (www.gitools.org; (PerezLlamas and Lopez-Bigas, 2011)), which compares the median expression value of the
genes in each module (DRKY1A targets) with a distribution of random modules of the
same size drawn from the expression values for the sample. The result is a z-score,
which is a measure of the difference between the observed median expression value
compared with the expected value. Positive and negative z-scores indicate significantly
higher or lower expression level of genes in the module under this condition,
respectively. Similarly, DYRK1A-target expression was analyzed in brain samples from
six Down syndrome individuals and compared to controls (GEO dataset GSE5390;
(Lockstone et al., 2007), as well as in teratozoospermic samples with respect to the
average level in samples from 14 normospermic individuals (GEO dataset GSE6872;
(Platts et al., 2007).
Conservation score
We extracted sequences 50 bp upstream and 50 bp downstream of each peak's midpoint, and calculated the average conservation score of this region based on UCSC
PhastCons conservation score data (Siepel et al., 2005) in placental mammals (hg19,
phastCons46way for human). The calculation was done using the scripts from
ChIPseeqer (Giannopoulou and Elemento, 2011). To plot the conservation score, the
score was calculated for 250 bp upstream and downstream regions from the peak’s
summit. We compared the conservation score of random peaks within 5 kb region of
each DYRK1A peak and plotted an average conservation score of 10 bp window using
R.
De novo motif search
MEME was used for de novo identification of motifs in peak regions and associated
promoters (Bailey et al., 2009) (http://meme.nbcr.net/downloads/meme_4.6.0.tar.gz).
Motifs were considered if they were found in at least 5 sites (-minsites 5), and MEME
was requested to retrieve the 10 motifs with lowest p-value (-nmotif 10) and to search
within a motif length range of 6-20 bp (-minw 6 -maxw 20). In separate executions,
MEME was performed for a fixed motif length of 10 bp (-minw 10 -maxw 10) and 11 bp
(-minw 11 -maxw 11) to optimize the prediction for the DYRK1A associated motif
selected in the initial motif search.
Gene ontology analysis and other analysis
For Gene ontology analysis, the online software DAVID (Huang da et al., 2009) was
used. The Venn diagrams were generated with the web application BioVenn (Hulsen et
al., 2008).
Antibodies used in this study
Target
Host
Source
Assay
DYRK1A
Mouse
Abnova, #H000001859
Western blot
Immunoprecipitation
ChIP
DYRK1A
Rabbit
Abcam, #ab69811
Immunoprecipitation
ChIP
DYRK1A
Rabbit
Sigma, #D1694
Western Blot
DYRK1B
Rabbit
Abcam, #ab113968
Western blot
Immunoprecipitation
ChIP
Histone H3
Rabbit
Abcam, #ab1791
Western blot
ChIP
H3-Ser10p
Rabbit
Millipore, #04-817
Western blot
H3-Ser28p
Rabbit
Millipore, #17-10269
Western blot
H3-Thr45p
Rabbit
Abcam, #ab26127
Western blot
ChIP
H3-Ac
Rabbit
Millipore, #06-599
Western blot
ChIP
Histone H4-Ac
Rabbit
Millipore, #06-866
Western blot
RPB1 (RNAPII)
Rabbit
N20, Santa Cruz Biotechnology, #sc-899
Western blot
Immunoprecipitation
ChIP
RNAPII-Ser2p
Rabbit
Abcam, #ab5095
Western blot
ChIP
RNAPII-Ser5p
Rabbit
Abcam, #ab5131
Western blot
ChIP
RNAPII-IIa
Mouse
8WG16, Covance, #MMS-126R
Western blot
Immunoprecipitation
ChIP
RNAPII-Ser2p
Mouse
H5, Covance, #MMS-129R
Western blot
RNAPII-Ser5p
Mouse
H14, Covance, #MMS-134R
Western blot
RNAPII-Tyr1p
Rat
Active Motif, #61383
Western blot
RNAPII-Thr4p
Rat
Active Motif, #61361
Western blot
RNAPII-Ser7p
Rat
Active Motif, #61087
Western blot
Gal4-DBD
Rabbit
Santa Cruz Biotechnology, #sc-577
Western blot
Lamin B1
Mouse
Santa Cruz Biotechnology, #sc-20682
Western blot
KAISO
Rabbit
Santa Cruz Biotechnology, #sc-23871
Western blot
Immunoprecipitation
CDK9
Rabbit
Santa Cruz Biotechnology, #sc-484
Western blot
Cyclin T1
Rabbit
Santa Cruz Biotechnology, #sc-8127
Western blot
p120-catenin
Mouse
BD-Bioscience, #610133
Western blot
GAPDH
Mouse
Millipore, #374
Western blot
α-tubulin
Mouse
Sigma, #T6199
Western blot
Vinculin
Mouse
Sigma, #V9131
Western blot
Flag
Mouse
M2, Sigma, #F1804
Western blot
Immunoprecipitation
HA
Mouse
Covance, #MMS-101R
Western blot
Immunoprecipitation
Cyclin D1
Mouse
Cell Signaling, #2926
Western blot
GST
Mouse
Santa Cruz Biotechnology, #sc-138
Western blot
Primers used for mutagenesis and cloning
Name
Sequence
TATA-less
GGCTCGCCTCTGAGACTAGACGCTAGATTC
RPS11-f
CCCGGGAACAAAGATGGCGACGCCGC
RPS11-r
AGATCTCTTCCCGGCCGCCTGAAAA
RPS11-mut1
CAGGATGGACTCCGTACGACATACGGGCGGGCTGAAGGC
RPS11-mut2
GGCGTTGTGGGGCCTAAGACCACCGTCTAGCACTTCCCGC
Primers used for ChIP-qPCR
Region (gene)
Forward primer
Reverse primer
RPS11 (-2)
ACGCCCGGCTAATTTTTGT
TGAGGTCAGGGGTTCAACA
RPS11 (-1)
GCCACACATGCTCCTAGCAC
GCTCTGTTCGTTCAGCCTTC
RPS11 (0)
GAAGGCTGAACGAACAGAGC
CGTACGGAGTCCATCCTGTT
RPS11 (+1)
GGTTTAAGTGCAGCCTGTCAA
ACGTAAGGAGAAACGCAGCA
RPS11 (+2)
GAGGCTCAAGCGTTTTAGGA
CCGTTTCCACAGAATTACCC
RBM39
AATTTGAGCGGCCGAAGTAT
GAATGGGGGATGGGAATATC
RPS11
GCTGAAGGCTGGTCACATCT
GGGCACTGTGAAGGACTGAC
ASXL1
AGCATCGCCTCCCAGAAT
CACCGACCTCAGCTAGGAAC
CDK12-A
TGATAAGCAGGGGAATGAGG
CTCCCTCACACAGACCCAGT
CDK12-B
GGAATGCCCAATTCAGAGAG
CTTCGATACCAAGCGGTGAC
CDK12-C
TGGTTTGGTGCACTTTTCTG
ATCCCGATGCAGGAAATTCT
CDK12-D
ATCCTGCCTTCAGCAGAACA
GCTCTTGGGTTTTCATCAGC
CDK12-E
AAAACTCTATCGGGGGCCTA
GAACTGGATGGAAGGATGGA
DENR-A
ACGCTCCGCAATTTTTCTC
CTCCCGCGAGACAATGAG
DENR-B
GTTTGTGAAATGGCTGCTGA
GGACTCGAAGTGGGTAATCG
DENR-C
TGGAAGGGGTCAAATAAAACA
TGCAAGGCCACATACTCTTG
DENR-D
CGAAGATCTTGGAGAAGTAAAGAA
TTTAAAAGGCCTCTCTCCCTTT
LUC7L2
CCGGGAGGGAATGTAATGTA
CTCCTCCCGCCCCTTTAC
CDC5L
CTCTGCCACTCGGTGACG
CGCATGTCCAAAACAGAATG
SMEK2
GGGAGTACTTCGGCGAGAC
GGGAGATCGCGAGAACCT
RPL12
GCGGACAAGCCAGATATAGG
CTGCCCACAACAAACATGG
RBM15
GAAAAAGGGGGTCGAAAGAG
AAACCCGTCCTTTAAACCACTA
RPS6-A
CATCTTGAAGCAGCTGAACG
CTCACTTCCGCTATCCCGTA
RPS6-B
CATGAAGCAGGGTGTCTTGA
ACAATGCAACCACGAACTGA
RPS6-C
CTAGGACCAAAGCACCCAAG
GCATATTCTGCAGCCTCTTCTT
RPS6-D
CGAAGAGACGCAGACTTTCC
TCAGCAATGAAAAGTCAACAGA
chr19_cov0
ATCTTGGTTCACCGCAACCT
AATTAGCTGGGTGTGGTGGT
chr20_cov0
GCTTGGCCAACAAGGTAAAA
CTCTCTGCAACCTCCACCTC
Primers used for RT-qPCR
Gene
Forward primer
Reverse primer
RPS11
GCTTCAAGACACCCAAGGAG
TGACAATGGTCCTCTGCATC
LUC7L2
TCGTCAACGAATCAAATTCAG
ATCCGCTCTTAAAGCCAGGT
ASXL1
TGCAGGTCATAGAGGCAGAA
GAGCGTGAAAAGGCTGATTC
CDC5L
AGCTGCCCAAAGAGACAATG
TGGCTTCAGAAAGCATCTCA
DENR
CGATTACCCACTTCGAGTCC
CAGCTTCTTGTTTGGGTGAA
RPS6
GCTAGCAGAATCCGCAAACT
CTGCAGGACACGTGGAGTAA
CDK12
TCCCACCCTTATTACCTGGA
AGCTCTGGAGGGAGAGGAAG
GADPH
ACCCAGAAGACTGTGGATGG
TTCAGCTCAGGGATGACCTT
EF1α
AGGTGATTATCCTGAACCATCC
GAACGGCGATCAATCTTTTCC
DNA-oligonucleotide probes used in EMSAs
Name
DYRK1A-WT
DYRK1A-MUT
Forward probe
Reverse probe
GGCCTAAGACTCTCGCGAGACACC
GTCTAG
GGCCTAAGACAGGTGTACAACACCG
TCTAG
CCGGATTCTGAGAGCGCTCTGTGGC
AGATC
CCGGATTCTGTCCACATGTTGTGGC
AGATC
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