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RAPID COMMUNICATION
Drosophila Forkhead Homologues Are Expressed in a Lineage-Restricted
Manner in Human Hematopoietic Cells
By Robert Hromas, Jason Moore, Timothy Johnston, Carolynn Socha, and Michael Klemsz
The forkhead gene (FKH) regulates morphogenesis in Drosophila. It is the prototypeof a new family of transcriptional
activators. Partially degenerate oligonucleotides to two
conserved amino acid sequences of this family were used
to prime a polymerase chain reaction (PCR) amplification
of HEL cell cDNA. Two unique clones, designated H3 and
H8, were isolated that contained homologies to FKH. A
third novel clone, 5-3, was isolated by low stringency
screening of a chronic myelogenous leukemia cDNA library
using H8 as a probe. H3 and 5-3 are preferentiallyexpressed
in restricted hematopoietic lineages, while the expression
of H8 was ubiquitous. Southern analysis showed that FKH
5-3 is conserved through yeast, which is rare among tissuespecific transcriptionfactors. The H3 and 5-3 clones provide
evidence that FKH family members are present in a tissuerestricted manner in humans.
0 1993 by The American Society of Hematology.
T
Because most of phenotypic change during development
is regulated at the level of transcription, we sought to ascertain
whether there were human FKH homologues that might play
a role in hematopoiesis. The fact that some of the Drosophila
FKH domain genes were expressed in developing mesoderm
was especially intriguing, because hematopoiesis begins in
the mesoderm. We synthesized partially degenerate oligonucleotides to two conserved regions of the FKH family. They
were used to prime a PCR amplification of HEL cell cDNA.
Three clones were ultimately obtained that contained strong
homologies to the FKH domain. Two of these clones, H3
and 5-3, are expressed in a lineage-restricted manner in blood
cells.
HE FORKHEAD (FKH) gene controls morphogenic
development in Drosophila.’ FKH and a rat hepatic
transcription factor HNF-3A were recently found to be
related.2,3 The highest region of identity was in the
DNA binding domain of HNF-3A. Thus, FKH defined a
new family of transcription factors that regulated development.
Several other FKH family members from rat liver, Drosophila, yeast, and Xenopus have been cloned4-’ (and T.
Davis, University of Washington, personal communication, July 1992). The new Drosophila FKH genes (FDI
through 5, and SLPI and 2) are expressed in developing
gut, mesoderm, or neural tissue. SLP 1 and 2 are essential
for normal Drosophila morphogenesis. The Xenopus FKH
domain gene (XFKH I ) is activin-inducible and selectively
expressed in the blastopore lip, a region that organizes body
axis formation. The prototype of mammalian FKH genes,
HNF-3A, activates transcription of the liver transthyretin
(prealbumin) gene by binding to the promoter sequence
TGACTAAGTCAATAATCAGA (-1 I O to -90 from the
start ~ i t e ) .This
~ , ~sequence is not homologous to any other
transcription factor DNA binding sequence. HNF-3A is
expressed in other tissues besides the liver,4 and thus may
not be the sole reason for the tissue-specificity of transthyretin expression. The other rat liver FKH genes, HNF3B and C, also bind to and activate transcription from the
same sequence.
Recently, two ubiquitous human FKH domain genes,
termed HTLF and ILF, were cloned based on binding to the
HTLV-1 long terminal repeat (LTR).9,10These human FKH
genes are dissimilar to the other FKH genes, and appear to
bind to unrelated sequences.
From the Departments of Hematology/Oncology, Biochemistry and
Molecular Biology, Microbiology, and Immunology, the Walther Oncology Center, the Indiana University Medical Center, Indianapolis.
Submitted January 22, 1993; accepted February 22, 1993.
Address reprint requests to Robert Hromas, MD, Departments of
Medicine and Biochemistry, The Walther Oncology Center, Indiana
UniversityMedical Center, 975 W Walnut St, Indianapolis,IN 462025121.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8111-0044$3.00/0
2854
MATERIALS AND METHODS
Polymerase chain reaction (PCR). Partially degenerate oligonucleotides were synthesized to two highly conserved regions of the
FKH domain, ITMAIQ for the 5’ primer and NMFENG for the 3’
primer. The actual primer sequences were: 5’-ATCACCATGGC(A/C/T)AT(A/C/T)CAG %fold degeneracy; 3’ primer-(C/G/A)CCGTTCTC(G/A)AACAT(G/A)TT
I 2-fold degeneracy. These sequences were chosen based on codon usage at these positions for
drosophila FKH, HNF-3A, B, C, and XFKH-1.
The 3‘ primer was used to prime a cDNA reaction from 1 pg of
poly A+ RNA from the human erythroleukemia cell line, HEL. The
cDNA synthesis was performed in a final volume of 100 pL, containing 1X TAQ DNA Polymerase buffer, 0.5 mmol/L deoxynucleotides, 2 mmol/L dithiothreitol, 1 pL RNasin (Promega Biotec,
Madison, WI), and 5 U of Molony sarcoma virus reverse transcriptase
(Pharmacia, Piscataway, NJ). After 1 hour at 42”C, the 5‘ primer
was added and the reaction denatured for 5 minutes at 94°C. Five
units of TAQ polymerase (Perkin-Elmer/Cetus, Nonvalk, CT) was
added and the reaction amplified for 30 cycles at 94°C for 2 minutes,
48°C for 2 minutes, and 72°C for 2 minutes.
Cloning. The amplified reaction was electrophoresed on a 2%
agarose gel, and an appropriate-sized fragment (237 bp) was excised
(Fig 1). The isolated fragment was ligated into the EcoRV site of
pBluescript KS+ (Stratagene, La Jolla, CA). The inserts were sequenced using Sequenase 2.0 according to the manufacturer’s instructions (USB, Cleveland, OH). Sequencingthese PCR amplification
products showed two novel FKH domains. These were called FKH
H3 and H8.
These two inserts were used to screen for other FKH genes under
low stringency a chronic myelogenous leukemia cDNA X GTI 1 library, as previously described.” One other novel human FKH domain, 5-3, was obtained this way. The Genbank accession numbers
fortheseclonesareasfollows: FKH H3 (L12141), FKHH8 (L12142),
and FKH 5-3 (L12143).
Blood, Vol 81, No 11 (June 1). 1993: pp 2854-2859
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HEMATOPOIETIC FORKHEAD GENES
2855
Expression ana/vsis. Poly A+ RNA was isolated using the Microfasttrack kit (Invitrogen. San Diego. CA). Cell lines were obtained
from the American Type Culture Collection (ATCC, Rockville. MD)
except for HEL cells, which were the generous gift of Dr Thalia Papayannoupolou (University of Washington, Seattle). RNA was denatured by glyoxalation. electrophoresed on a l % agarose gel, and
blotted to a nylon membrane. A normal human tissue Northern blot
and a Southern blot containing digested DNA from multiple species
were purchased from Clontech (Palo Alto, CA). Novel FKH clones
were excised from pBluescript and labeled with '*P by random priming
for hybridization. The blots were hybridized at a stringency of 55°C.
4X SSPE. and 40% fonnamide, and washed at a final stringency of
65°C and 0.1 X SSC. For these hybridization studies, complete cDNAs
obtained from the CML library were used as probes for H8 and 53. For H3, a complete cDNA was cloned from a H e 6 2 hepatoma
cDNA library.
RESULTS
Fig 1. Ethidium bromide agarose gel electrophoresis of PCR
products. The arrow denotes the approximate 237-bp product. This
was isolated and cloned into the EcoRV site of pBluescript KS
Clones were screened for FKH homology by double-strandeddidexy
sequencing. Markers are in the far left lane. HL60 cell cDNA PCR
products are in the lane closest to the markers, while HEL cell cDNA
PCR products are in the far right lane. The intense band below the
indicated FKH band are the degenerate primers.
+.
Three different novel FKH domains
Sequence an&is.
were isolated (Table I). Two were obtained by PCR,H3 and
H8. Although related to the FKH family at the amino acid
level, they were different from any previously described
member. The last FKH clone, designated 5-3. was obtained
by low stringency screening of a CML cDNA X G T 1 I library.
In Table I , these three human FKH domains are compared
with the known FKH genes. The FKH clone 5-3 is the most
0
2
H3
(v
xM
1
Y
1.8-
+
+
5.0-
H8
3.0-
5-3
+
Fig 2. Poly A Northem analysis of the expression pattems of the human FKH genes in several
hematopoietic cell lines. The probe used is indicated
on the left, while the size of the hybridization species
is on the right. H8 is ubiquitously expressed, although it appears to be moderately inducible with
retinoic acid. H3 and 5-3 have restricted patterns
of expression.
ACTIN
+2.1 KB
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2856
HROMAS ET AL
Table 1. Conserved Forkhead DNA Binding Domain
HCM
SLP- 1
SLP-2
FKH
FDI
FD2
FD3
FD4
FD5
HNF-3B
HNF-3A
HNF-3C
XFKH-I
ILF
HTLF
H3
H8
5-3
RRINGELAKKPPYSYATLICLAILQSQEGNVTLSQIYHWIHVHFPYYKQKDASWQNSI
QKMTAGSDTKPPYSYNALIMMAIQDSPEQRLTLNGIYQYLINRFPYFKANKRGWQNSI
PVKDKKGNEKPPYSYNALIMIRQSSEKRLTLNGIYEYI~RFPYFKA~GWQNSI
TYRRSYTHAKPPYSYISLITMAIQNNPTRMLTLSEIYQFIMDLFPRYRQNQQRWQNSI
APHQNKEIVKPPYSYIALIAIQNAADKKVTLNGIYQYIMERFPYYRDNKQGWQNSI
FLHNSHRPEKPPFSYIALIAISSAPNQRLTLSGIYKFIMDKFPYYRENKQGWQNSI
SGSSGPLV"KPPYSYIALITMAILQSPHKKLTLSGICDFIMSRFPYYKDKFPAWQNS1
PSRESYGEQKPPYSYISLTAMAIWSSPEKMLPLSDIYKFITDRFPYY~NTQRWQNSL
PLKMSYGDQKPPYSYISLTAIIHSPQRFVPLSEIYRFIMDQFPFYRKNTQKWQNSL
TYRRSYTHAKPPYSYISLITMAIQQSPNKMLTLSEIYQWIMDLFPPYRQNQQRWQNSI
TFKRSYPHAKPPYSYISLITMAIQQAPSKMLTLSEIYQWIMDLFPYYRQNQQRWQNSI
GYRRPLAHAKPPYSYISLITMAI9QAPGKMLTLSEIYQWIMDLFPYYRENQQRWQNSI
TYRRNYSHAKPPYSYISLITMAIQQAPNKMMTLNEIYQWIVDLFPYYRQNQQRWQNSI
GGDSPKDDSKPPYSYAQLIVQAITMAPDKQLTLNGIYTHITKNYPYYRTADKGWQNSI
QKKKSAT"SKPPYSFSLLIYMAIEHSPNKCLPVKEIYSW1LDHFPYFATAPTG~NSV
GYR~AHAKPPYSYISLITMAIQQAPGK~TLSEIYQWIMDLFPYYRENQ~QNSI
RSR-kSPRHGKPPYSYTALITMAIQNAPDKKITgNGIYQFIMDR_NPFYRDNKQGWQNSI
SKP*KNSIVKPPYSYTALInIQQSPQKKLTLSGICQFISNRFPYYREKFPAWQNSI
- - - - ..KPPYSY--LI-MAI--------TL--IY--I---FPYYR-----WQNSI
...
HCM
SLP-1
SLP-2
FKH
FD 1
FD2
FD3
FD4
FD5
HNF-36
HNF-3A
HNF-3C
XFKH-1
ILF
HTLF
H3
H8
5-3
RHNLSLNDAFIKTEKSCDGKGHFWEVRPGAETKFFKGENi*RGYEFVKDSLQD
RHNLSLNKCFTKIPRSYDDPGKGNYWILDPSAEEVFIGGSTGKLRRRTTAASR
RHNLSLNKCFVKVPRHYDDPGKGNYWILDPSAEEVFIGETTGKLRRKNPGASR
RHSLSFNDCFVKIPRTPVKPGKGSFWTLHPDSGNMFENG**CYLRRQKRFKDE
RHNLSLNECFVKVARDDKKPGKGSYWTLDPDSYNMFDNG*"SFLRRRRRFKKK
RHNLSLNDCFVKIPRANDSAGKGSYWMLDSSASDMFEQG**NYRRRRTRRQRH
RHNLSLNDCFIKVPREPGNPGKGNFWTLDPLAEDMFDNG**SFLRRRKRYKRA
RHNLSFNDCF1KVPRRPDRPGKGAYWALHPQAFDNFENG"j;SLLRRRKRFKLH
RHNLSFNDCFIKVPRNVTKAGKGSYWTLHPMAFDMFENG**SLLRRRKRFRVK
RHSLSFNDCFLKVPRAPDKPGKGSFWTLHPDSGNMFENGCYLRRQKRFKCE
RHSLSFNACFVKVARSPDKPGKGSYWTLHPDSGNMFENG*KCYLRRQKRFKCE
RHSLSFNDCFVKVARSPDKPGKGSYWALHPSSGNMFENGJinCYLRRQKRFKLE
RHSLSFNDCF1KVPRSPEKPGKGSYWTLHPESGNMFENG';"CYLRRQKRFKCE
RHNLSLNRYFIKVPRSQEEPGKGSFWRIDPASESKL1EQ""AFRKRRPPGVPC
RHNLSLNKCFQKVERSHGK+GKGSLWCVDPEYKPNLI"Q**ALKKQPFSSASS
RHSLSFNDCFVKVARSPDKPGKGSYWALHPSSGNMFENG""CYLRRQKRFKLE
RHNLSLNECFVKVPR_DKKPGKGSYRTLDPDSYNMFENG""SFLRRRRRFKKK
RHNLSLNDCFgKIPREPBKGNYWTLDPQS_DEMFDNG*%3FLRRRKRFK&H
RHNLS-N-CF-K..R...-.GKG--W.L-p...--F--G-...LRR--R---.
Comparison of the amino acid sequence of the entire conserved DNA binding domains of every known FKH gene with the H3, H8, and 5-3 human
FKH genes. A consensus FKH DNA binding domain is listed at the bottom. An amino acid was considered consensus if it was present at 14/18 sites.
The asterisk (*) indicates that there are gaps in the conserved amino acid sequences. The inverted bar (A)indicates that there is an insertion of 8
amino acids here that are not in any other FKH domain. The plus (+) indicates that there is a 2-amino acid insertion in the HTLF not in any other FKH
gene. These three human genes are underlined where they are different from all other previously described FKH domains.
divergent at the amino acid level from the other known FKH
genes of the clones described here.
The FKH domain H3 was different at five amino acids
from the FKH of rat HNF-3C. It also is similar but not identical to HNF-3C in the flanking regions around the FKH
domain. Thus, it is possible that it is the human homologue
to HNF-3C. However, we did sequence another PCR fragment that had only two amino acid differences from the rat
HNF-3C FKH domain, indicating that H3 may not be the
human homologue to HNF-3C.
Based on amino acid identity in the DNA binding domain,
all known FKH genes can be separated into four groups (Table 2). The human FKH genes H3 and H8 are most closely
related to the rat and Xenopus FKH genes. Human FKH 53 is more homologous to H3 and H8 than it is to ILF and
HTLF. ILF and HTLF define their own homology group, as
they are the most distant FKH members to the yeast and
drosophila FKH genes.
Expression analysis. The tissue expression patterns of
the three novel FKH genes were investigated using northern
analysis. Figure 2 shows the three human FKH clones used
sequentially as probes on a Northern blot containing several
hematopoietic cell lines. H3 was found to be expressed only
in the human erythroleukemia line HEL. H3 was not found
in HL60 promyelocytic leukemia cells, K562 CML cells,
Jurkat T cells, and HeLa cervical carcinoma cells. H8 was
expressed as two transcripts in every blood cell line examined.
H8's expression was moderately induced in promyelocytic
HL60 leukemia cells when they are stimulated with retinoic
acid (
mol/L for 3 days). FKH 5-3 was expressed in the
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HEMATOPOIETIC FORKHEAD GENES
2857
Table 2. Homology Groups Within the Forkhead Family
Group II
Group I
Yeast HCM
Group 111
Drosophila FKH
Xenopus XFKHl
Drosophila FD1-5
Drosophila SLP-1.2
Rat HNF-3A.B.C
Human, H3, H8, 5-3
Group IV
Human ILF,
HTLF
v
d
d
The known FKH genes can be divided into homology groups based
on their relationshipto each other in the DNA binding domain. The human
FKH genes ILF and HTLF are the most distant of the mammalian FKH
genes to the yeast and drosophila FKH genes. FKH 5-3 has a homologue
in yeast, but is unrelated to the known yeast FKH gene HCM.
CML cell line K562 and in the Jurkat T-cell leukemia line,
but was not found in HL60, HEL, or HeLa cells.
The expression of the human FKH genes in normal human
tissue was analyzed (Fig 3). FKH gene H8 was found to be
9
5-3
1
ACTIN
+
Fig 4. Poly A
Northem analysis of 5-3. 5-3 is expressed only
in the pluripotent N-TeraZ/Dl cells. I t s expression is markedly in-
H3
H8
5-3
ACTIN
+
Fig 3. Poly A Northem analysis of the expression patterns of
the human FKH genes in normal human tissues. H 8 is expressed
in every tissue, although the smaller transcript is much fainter on
this blot than in the one containing the blood cell lines. H 3 has a
restricted pattern of expression, while 5-3 could not be found in
any of the tissues examined.
duced when these cells are stimulated t o differentiate with retinoic
acid.
ubiquitously expressed in all tissues. As above, there appeared
to be a faint 3-kb transcript and a highly expressed 5-kb transcript. Human FKH clone H3 was expressed in normal liver
and pancreas as a single I .8-kb message. H3 transcripts were
not found in heart, brain, placenta, lung, muscle, or kidney.
We also found H3 to be expressed in malignant liver tissue
(the HepG2 hepatoma cell line), but not in KB squamous
cell carcinoma (data not shown).
FKH 5-3 was not expressed in any of the tissues in this
blot. These and subsequent blots were reprobed with actin
to assess the integrity and quantity of the RNA.
Figure 4 shows a Northern analysis of FKH 5-3 expression
in several additional cell lines. Significantly, 5-3 is only expressed in the pluripotent teratocarcinoma cell line N-teraZ/
DI. The expression is induced when the N-TeraZ/Dl cells
are stimulated with retinoic acid
mol/L for 7 days).
FKH 5-3 was not seen in normal human marrow, JY and
Eskol B-cell lines, or in U937 monocytic leukemia.
Species homologies. Southern analysis was next performed using EcoRI-digested DNA from multiple species (Fig
5). H3, H8, and 5-3 all had hybndizable DNA fragments in
the human, monkey, rat, mouse, dog, cow, rabbit, and
chicken genome. FKH genes H3 and H8 were not present
in yeast; however, FKH 5-3 did have a single cross-hybridizing
fragment present in yeast.
H3 and H8 appear to represent single copy genes in all of
these species. 5-3 hybridized to multiple digestion fragments,
suggesting that it may be cross-hybridizing to closely related
FKH genes. This cross-hybridization may indicate that 5-3
has its own subfamily. 5-3 does not hybridize to H3 or H8,
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2858
HROMAS ET AL
H3
H8
'0
scripts that are novel to the FKH family. These are designated
H3, H8, and 5-3.
Sequence analysis of these clones found that H3 and H8
were more closely related to the drosophila FKH genes than
5-3.5-3 probably represents a subgroup of FKH genes based
on its divergence from the drosophila FKH genes (especially
in the NMFENG region) and its cross-hybridization to multiple fragments on Southern analysis. This suggests that there
are additional FKH genes closely related to 5-3.
Northern analysis of the expression of the human FKH
genes showed that there were both tissue-specific and ubiquitous patterns. H8 was expressed ubiquitously in both hematopoietic and nonhematopoietic lineages. H3 was expressed in normal liver, pancreas, and HEL erythroleukemia
cells.
FKH 5-3 is a candidate for a transcriptional regulator active
during early hematopoietic development. It is expressed in
differentiating N-TeraZ/DI cells. a model system for in vitro
human pluripotent development.'* It is also expressed in
K562 and Jurkat cells, but it is not expressed in any of the
other tissues studied, including total normal human marrow.
On the basis of expression, it provides an intriguing connection between a pluripotent cell and two leukemias, one of
CML origin (K562) and the other of T-cell origin (Jurkat).
In addition, 5-3 expression goes up as the N-TeraZ/DI cells
are stimulated to differentiate. Further studies of the expression and effect of transduction of 5-3 into murine ES cells
as they differentiate into embryoid bodies with blood islands
are in progress.
There are two possible reasons that FKH 5-3 is not expressed in normal marrow. First, the concentration of 5-3
expressing progenitors may be too low to be assaved. Second.
5-3 may only be expressed in transformed marrow cells. To
address this question, we are examining 5-3 expression during
the differentiation of interleukin-3-dependent progenitor cell
lines (32D and FDCP mix A4).
Southern analysis found that 5-3 is highly conserved. The
human gene can cross-hybridize to a yeast species, despite
high-stringency washes. 5-3 is not related closely to the yeast
FKH gene HCM. HCM was not found to be essential for
yeast survival (T. Davis, personal communication). It will be
interesting to investigate whether the highly conserved 5-3
will be essential for yeast survival. Knock-out experiments
in yeast could lend insight into the general role of 5-3. It will
also be significant if human 5-3 can rescue yeast with deficient
5-3.
The exact function ofthese human FKH genes is unknown.
If structural similarity to the Drosophila and rat FKH genes
can provide insight into functional similarity, then the human
FKH genes may be transcriptional activators that regulate
phenotypic change during lineage development. Drosophila
FKH and SLP I and 2 are known morphogenetic regulators.
Rat HNF-3A, B, and C are known transcription factors. Yeast
HCM is a suppressor of a calmodulin mutation (T. Davis,
personal communication), although it probably also works
by trans-activating other genes.
Identifying consensus DNA binding sequences by mobility
shift selection and PCR amplification, and then searching
these consensus sequences against the database may turn up
some of the genes that human FKH H3, H8, and 5-3 might
r
.
-*K3*q*;
I '
5-3
Fig 5. Southem analysis of the H3, H8, and 5-3 human FKH
genes. For H3 and H8, there are hybridizable homologues in every
species examined, except yeast. 5-3 has a cross-hybridizing band
in yeast DNA. The 5-3 blot was exposed longer to intensify the
yeast band.
which are more closely related to drosophila FKH genes (data
not shown).
The cross-hybridization of FKH 5-3 to a yeast DNA fragment is of interest. The one known yeast FKH gene, HCM,
is not homologous to 5-3 at either the nucleotide or amino
acid level. The conservation of a homologous gene in yeast
is extraordinarily rare for tissue-specific mammalian transcription activators.
DISCUSSION
This study shows that drosophila FKH transcription factor
family members are expressed in a lineage-specific manner
in human blood cells. Using PCR and low-stringency cDNA
library screening we have isolated three human FKH tran-
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HEMATOPOIETIC FORKHEAD GENES
regulate. Another approach will be to force expression of these
genes in defined cell culture systems and assay for phenotypic
changes.
In summary, these data show that human members of the
FKH family of transcription activators can be expressed in
restricted hematopoietic lineages.
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From www.bloodjournal.org by guest on January 12, 2015. For personal use only.
1993 81: 2854-2859
Drosophila forkhead homologues are expressed in a lineage-restricted
manner in human hematopoietic cells
R Hromas, J Moore, T Johnston, C Socha and M Klemsz
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