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Department of Pharmacology, Cornell University Medical College, New York, New York 10021
Abstract
Using a PCR-based cDNA subtractive hybridization method (L. Diatchenko
et al., Proc. Natl. Acad. Sci. USA, 93:
6025–6030, 1996), we cloned a cDNA fragment of a novel gene
that is highly expressed in F9-10; F9-10 is an F9 teratocarcinoma stem
cell line that expresses high levels of exogenous Hoxa-1 mRNA and
protein in comparison to F9 wild-type stem cells, which do not express
endogenous Hoxa-1 mRNA in the absence of retinoic acid (RA). Rapid
amplification of cDNA ends was used to clone the full-length cDNA of
this gene, designated HA1R-62 (Hoxa1 regulated-62). We have shown that
HA1R-62 is also a RA-responsive gene and that it is expressed (mRNA
size, 
4.3 kb) in adult mouse thymus, lung, kidney, and ovary as well
as in 12.5-day mouse embryos. DNA sequence analysis and in
vitro translation experiments have shown that HA1R-62 encodes a
protein with a molecular mass of approximately 26 kDa.
Elucidation of the function of the HA1R-62 gene product will provide
new insights into the functions of RA and homeobox genes.
Introduction
RA,3
a member of the vitamin A family of signaling molecules called
retinoids, influences the proliferation and cellular differentiation of
a wide variety of cell types and plays an important role in vertebrate
development (1
, 2)
. Most of the effects of RA are elicited
through its ability to regulate gene expression via the action of RARs.
The RARs (RAR
, RARß, RAR
, and their isoforms) are activated by
both all-trans-RA and 9-cis-RA, whereas the RXRs
(RXR, RXRß, RXR, and their isoforms) are activated only by
9-cis-RA (3)
. These receptors bind
preferentially as RAR/RXR heterodimers to RA-responsive elements
located in the regulatory regions of target genes. The many diverse
effects of RA are associated with the transcriptional regulation of
target genes, which include transcription factors, extracellular matrix
proteins, enzymes, growth factor receptors, and so forth (4
, 5)
.
F9 murine teratocarcinoma stem cells (F9 cells) resemble the pluripotent inner cell mass cells or ES cells of mouse blastocysts and thus provide an important model system in which to study critical early events in mouse development. F9 cells differentiate into primitive endoderm in response to RA (2 , 6) . The homeobox gene Hoxa-1 is transcriptionally regulated by RA and encodes a transcription factor that has been shown to play important roles in cell differentiation and embryogenesis (7, 8, 9, 10, 11, 12, 13) .
The actions of homeobox genes in embryogenesis and tumorigenesis are complicated. It follows that the identification of the gene targets of this particular homeobox gene, Hoxa-1, is crucial to the elucidation of the molecular mechanisms involved in cellular differentiation. To dissect these actions and identify the key components, it is advantageous to use a model system. By using both differential hybridization and a cDNA subtractive hybridization approach, we were able to identify genes that were differentially expressed in F9-10, a murine F9 teratocarcinoma stem cell line (14) that expresses high levels of exogenous Hoxa-1 as compared to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of RA. The F9-10 cell line was produced in this laboratory by stable transfection of F9 wild-type cells with a cDNA containing the complete coding sequence of the Hoxa-1 protein under the control of mouse metallothionein promoter. On ZnCl2 treatment, Hoxa-1 is ectopically overexpressed in the absence of RA, as assessed by both Northern and Western blot analyses (14) .
Our data have shown that the expression of a wide variety of genes is affected by the level of Hoxa-1 protein, and it is likely that these genes are either direct or indirect target genes of Hoxa-1.4 Here we report the molecular cloning and characterization of one of the putative Hoxa-1 downstream target genes, HA1R-62.
Results
The Identification of Genes That Are Differentially Expressed
in the F9–10 Cell Line versus the F9 Wild-Type Cell
Line.
We sought to isolate genes that were differentially expressed in
the F9 Hoxa-1-overexpressing cell lines versus wild-type F9
cells, which do not express Hoxa-1 mRNA in the absence of RA. The F9-10
cell line expresses exogenous Hoxa-1 mRNA and protein after zinc
addition at about the same level as that of the endogenous Hoxa-1 mRNA
in F9 wild-type cells treated with RA (14)
. Differential
hybridization and a subtractive hybridization method were used to
compare gene expression in F9 wild-type cells and F9-10 cells after
both cell lines were cultured in the presence of
ZnCl2 for 24 h to induce the expression of
exogenous Hoxa-1 cDNA in the F9-10 cell line. Using both methods, 169
candidate clones were picked and then assayed by Northern blot analysis
of RNA from F9 wild-type cells cultured for 24 h in the presence
of 100 µM ZnCl2
versus RNA from F9-10 cells cultured for 24 h in the
presence of 100 µM ZnCl2.
Twenty-six of the clones were differentially expressed in F9-10 cells
as compared to F9 wild-type cells.4
The
characteristics and expression pattern of one of the putative Hoxa-1
target genes, HA1R-62 (Clone-62), are reported here.
Time Course of HA1R-62 (Clone-62) mRNA Induction by RA.
Wild-type F9 cells were examined for expression of both Hoxa-1 and
HA1R-62 mRNAs. As shown by Northern blot analysis (Fig. 1)
, HA1R-62 mRNA was significantly increased in F9 wild-type cells
24 h after the addition of 1.0 µM RA. The level of
HA1R-62 mRNA was further increased 48 and 72 h after RA
treatment. The same blot was hybridized to the Hoxa-1 cDNA probe
to show the induction of endogenous Hoxa-1 mRNA by RA in F9 wild-type
cells (7)
.
|
. The difference between the
endogenous Hoxa-1 mRNA size in F9 wild-type cells (2.2 kb) and the
exogenous Hoxa-1 mRNA in F9-10 cells (1.5 kb) results from the fact
that the exogenous Hoxa-1 mRNA is encoded by a cDNA missing a portion
of its 3' untranslated region (14)
. In F9-10 cells, even
without ZnCl2 treatment, some exogenous Hoxa-1
mRNA expression is seen (Lane 7); this results from the
leakiness of the metallothionein promoter. As shown in Fig. 1, A and B
, the level of HA1R-62 mRNA was increased
in the Hoxa-1-overexpressing F9-10 cells after
ZnCl2 treatment. In contrast, F9 wild-type cells
treated with 100 µM ZnCl2
do not exhibit expression of the HA1R-62 gene, indicating that HA1R-62
mRNA expression is not caused by ZnCl2 treatment
(Fig. 1C)
. These data indicate that there is a correlation
between expression of the Hoxa-1 protein and HA1R-62 mRNA expression in
both wild-type F9 cells after RA addition and in the F9-10 line after
ZnCl2 addition.
Expression of HA1R-62 in HW-F9-pTA-N-Hoxa1-8 Cells.
Additional Hoxa-1 stably transfected F9 cell lines were created more
recently in this laboratory using the Tet-off gene expression system,
and in one of these cell lines, HW-F9-pTA-N-Hoxa1-8, exogenous Hoxa-1
mRNA expression was activated by the removal of tetracycline from the
medium (Fig. 2
, Lane 4). It is also the case that in this
tetracycline-regulated expression vector, the exogenous Hoxa-1 mRNA
(1.5 kb) is not full length, although the Hoxa-1 protein encoded by
this vector is full length (14)
. This HW-F9-pTA-N-Hoxa1-8
cell line was used to examine the expression patterns of HA1R-62 mRNAs
in the presence and absence of tetracycline. In the presence of
tetracycline, HA1R-62 mRNA was not expressed in the HW-F9-pTA-N-Hoxa1-8
cells (Fig. 2
, Lane 3). HA1R-62 mRNA expression was
up-regulated by the removal of tetracycline from the medium (Fig. 2
,
Lane 4). This again suggests that the expression of HA1R-62
is under the control of Hoxa-1 homeodomain protein in F9 cells. We
included RNA samples from the F9 wild-type cells treated with 1
µM RA for 48 h on the same blot. The
HA1R-62 mRNA was induced by RA treatment (Fig. 2
, Lane 2),
as expected.
|
. Expression of the exogenous Hoxa-1 mRNA in response to
tetracycline removal also resulted in increased expression of the
endogenous Hoxa-1 mRNA (Fig. 2
, Lane 4, and arrow
at right). These data suggest that the Hoxa-1 gene contains
an autoregulatory element to which the Hoxa-1 protein can bind, similar
to the regulation of Hoxb-1 (15)
.
Expression of HA1R-62 in Hoxa-1 3' Enhancer (-/-) CCE
Cells.
One method for generating a cell line with reduced amounts of
Hoxa-1 was to remove both copies of the Hoxa-1 3' RA-inducible enhancer
(RAIDR5) by two rounds of homologous recombination (16
, 17)
. In these CCE-B30 ES cells, the coding regions of the two
alleles of the Hoxa-1 gene are intact, but the RA-inducible 3' enhancer
is deleted from both alleles. This prevents the induction of Hoxa-1
mRNA by RA (16
, 17)
.5
Northern blot analyses of total RNA from a mouse ES cell line, CCE
wild-type cells, and from a RAIDR5-/- CCE cell
line, CCE-B30, after a 48-h treatment with different concentrations of
RA, are shown in Fig. 3
. In the Hoxa-1 3' enhancer-/- CCE-B30 cells, RA
induction of Hoxa-1 gene expression is almost completely eliminated
(Fig. 3)
. The same blot was also hybridized to a HA1R-62 cDNA probe; in
the CCE-B30 cells, expression of HA1R-62 mRNA is reduced as compared to
that in CCE wild-type cells treated with the same concentrations of RA.
These data again show a correlation between the levels of Hoxa-1 mRNA
induced by RA and the levels of HA1R-62 mRNA.
|
. The thymus and ovary showed the highest levels of expression of
HA1R-62 mRNA (size, 4.3 kb).
|
. As illustrated at the top of the Fig. 5A
, the original cDNA
isolated from the cDNA subtraction screening was about 120 bp long.
This cDNA fragment was used to screen an F9 RACT cDNA library, and a
2.8-kb clone with the same 3'-end was isolated. To isolate the
full-length cDNA, 5'-RACE was used (primer design was as indicated in
Fig. 5
), and a product was obtained that extended 760 bp from the
5'-end of the clone isolated from the cDNA library. Alignment of the
sequences enabled us to delineate the entire cDNA sequence, and we
found a single long ORF starting with a methionine codon at nucleotide
505 and ending with a TGA stop codon at nucleotide 1260. Four in-frame
stop codons were found upstream from the putative initiation site at
positions 49, 169, 250, and 286. A putative polyadenylation signal,
AATATA, was found at nucleotide position 3528–3533.
|
A protein homology (Swissprot database) search of the peptide encoded by the HA1R-62 ORF revealed that a HA1R-62 protein segment (amino acids 51–83) is 51% homologous to one transmembrane region (amino acids 674–706) of Bride of Sevenless Protein, a Drosophila protein that is a transmembrane ligand of the sevenless transmembrane tyrosine kinase receptor (19) .
In Vitro Translation of HA1R-62 mRNA.
Using the full-length cDNA clone of HA1R-62 in pBluescript plasmid
vector as a template, the corresponding mRNA was transcribed by T7 RNA
polymerase and translated in a rabbit reticulocyte lysate. As shown in
Fig. 6
, the mRNA programmed the synthesis of a peptide with a molecular mass
of about 26 kDa, which corresponds to the molecular mass estimated from
the amino acid sequence.
|
The cloning of a RA-responsive gene, HA1R-62, in F9 murine
teratocarcinoma cells is described. Interestingly, this gene was
initially cloned in our screening to identify genes that are
differentially expressed in F9-10 cells. F9-10 is a murine F9
teratocarcinoma stem cell line that expresses high levels of exogenous
Hoxa-1 in comparison to F9 wild-type stem cells, which do not express
endogenous Hoxa-1 mRNA in the absence of RA. The Hoxa-1 gene has been
shown to be transcriptionally activated by retinoids such as RA via a
RA-responsive element 3' of its coding region (13
, 16
, 17)
. The loss of both copies of RAR in F9 cells results in
the loss of Hoxa-1 induction by RA and in the partial loss of ability
of cells to differentiate in response to RA (20)
. It is
therefore likely that this RA-induced differentiation is mediated in
part through the actions of homeobox genes and specifically through the
action of Hoxa-1. The HA1R-62 gene shows a dramatically reduced
induction by RA in Hoxa-1 3' RARE-/- CCE cells
(Fig. 3)
. Conversely, in cell lines in which the Hoxa-1 gene is stably
overexpressed, HA1R-62 mRNA is elevated (Figs. 1
and 2)
. These data
strongly suggest that this novel RA-responsive gene, HA1R-62, is a
target gene of the Hoxa-1 protein, which is itself a direct target of
RARs (7
, 21)
.
We do not know whether HA1R-62 is a direct target of the Hoxa-1 protein. It is possible that HA1R-62 is indirectly regulated by Hoxa-1 as a result of Hoxa-1 changing the differentiation state of the cell. However, this is unlikely because the F9-10 and the HW-F9-pTA-N-Hoxa1-8 cell lines were designed to conditionally overexpress only the Hoxa-1 protein, and these cell lines also overexpress HA1R-62 mRNA.
It is difficult to speculate about the function of the HA1R-62 gene product based on the DNA sequence data. However, a protein homology search of the peptide encoded by the HA1R-62 ORF revealed that a HA1R-62 protein segment (amino acids 51–83) is 51% homologous to one transmembrane region (amino acids 674–706) of the Bride of Sevenless Protein, a Drosophila protein that is a transmembrane ligand of the sevenless transmembrane tyrosine kinase receptor (19) . Thus, it is possible that HA1R-62 encodes a transmembrane protein. HA1R-62 mRNA is expressed not only in the mouse embryo, but also in adult mouse thymus, lung, kidney, and ovary, suggesting that RA is involved in regulation of its expression in these adult organs. Much research needs to be done to determine the function of the HA1R-62 protein. We are currently further investigating the function and possible regulatory relationships between Hoxa-1 and HA1R-62 in the cascade of events that takes place during the cellular differentiation induced by RA.
Very few gene targets of the Hoxa-1 protein have been identified to date. This laboratory is in the process of identifying additional putative Hoxa-1 target genes in F9 cells.4 Future research aimed at identifying more target genes of Hox proteins and analyzing precisely how target genes work together to control proper organogenesis and differentiation in a variety of species should clarify many aspects of Hox complex gene function.
Materials and Methods
Cell Culture.
The murine F9 teratocarcinoma wild-type stem cell line and the
metallothionein promoter:Hoxa-1 stably transfected derivatives
JG-F9-MT-H1.6-993-10 (F9-10) were grown in DMEM supplemented with 10%
heat-inactivated bovine calf serum and 2 mM glutamine, as
described previously (22)
. This F9-10 cell line, in
response to zinc, expresses an exogenous Hoxa-1 mRNA that is about 1.5
kb because its 3'-untranslated region was truncated in this
metallothionein promoter:Hoxa-1 construct; these cells also express
only the longer form of the Hoxa-1 protein (331 amino acids) and not
the shorter Hoxa-1 protein (133 amino acids), which lacks the homeobox
domain (7
, 14 , 21)
. [The Hoxa-1 gene was formerly named
ERA-1 and Hox1.6 (21)
.] CCE murine ES cells were
maintained in DMEM supplemented with 10% heat-inactivated fetal bovine
serum, 2 mM glutamine, 1 mM sodium pyruvate,
100 µM minimal essential medium nonessential amino acids,
1 µM ß-mercaptoethanol, and 1 x
103 units/ml leukemia inhibitory factor (LIF), as
described previously (23)
.
cDNA Subtractive Hybridization.
Two µg of mRNA from the F9 wild-type cells and F9-10 cells, both
treated with 100 µM ZnCl2 for
24 h, were used to synthesize double-stranded cDNA. A new
PCR-based cDNA subtraction method, suppression subtractive
hybridization (24)
, was carried out using the PCR-Select
kit (Clontech, Palo Alto, CA). Products from the secondary PCR
reactions were inserted into the vector pCRII using a T/A cloning kit
(Invitrogen, Carlsbad, CA). The insert DNA of each clone was prepared,
radiolabeled, and used for Northern analysis.
Northern Analysis and DNA Sequence Analysis.
Total RNA samples were isolated from F9 wild-type and F9-10 cells and
then subjected to Northern analysis as described previously
(22)
. DNA sequences of the candidate clones were
determined at the Biotechnology Center (Cornell University, Ithaca,
NY). Sequence searches of GenBank were carried out using the
BLAST program (18)
.
cDNA Library Screening.
Approximately 400,000 plaques of a F9 RACT 3-day cDNA library in the
ZapExpress
vector6
were screened with the original cDNA fragment of HA1R-62 isolated from
the cDNA subtraction screening, and four positive clones were isolated
and sequenced.
RACE.
RACE (25
, 26)
, a procedure to amplify nucleic acid
sequences from a mRNA template between a defined internal site and
unknown sequences at 5'-end of mRNA, was performed by using a Marathon
cDNA Amplication Kit (Clontech) according to the supplier’s protocol.
The primer for reverse transcription and the two internal anchoring
primers for nested PCR were 5'-TCCCCACCAAAGCCACCTCCATT-3',
5'-GAAAAAGTGCGAAGATCCAAAACGAA-3' (AS1), and
5'-GCGAAGATCCAAAACGAAATGTAAAGT-3' (AS2), respectively.
In Vitro Translation.
In vitro transcription and translation were performed by
using a TNT-coupled reticulocyte lysate system (Promega,
Madison, WI) with [S35]methionine radiolabeled
protein labeling mix (DuPont New England Nuclear) according to
the manufacturer’s instructions. The synthesized proteins were
analyzed by 7.5% SDS-PAGE, followed by autoradiography.
Acknowledgments
We thank Dr. Ker Yu, Dr. Alex Langston, and Hong Wu, all of the Pharmacology Department, Cornell University Medical School, New York, NY, for Hoxa-1 genomic constructs and cell lines.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by NIH Grant R01CA39036 (to
L. J. G.). 
2 To whom requests for reprints should be
addressed, at Department of Pharmacology, Cornell University Medical
College, 1300 York Avenue, New York, New York 10021. Phone: (212)
746-6250; Fax: (212) 746-8858; E-mail: ljgudas{at}mail.med.cornell.edu
3 The abbreviations used are: RA, retinoic acid;
EST, expressed sequence tag; ORF, open reading frame; RXR, retinoid X
receptor; RAR, retinoic acid receptor; ES, embryonic stem; RACE, rapid
amplification of cDNA ends; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
4 J. Shen and L. J. Gudas. Cloning of genes
differentially expressed in cells which overexpress Hoxa-1, a homeobox
gene, submitted for publication.
5 A. W. Langston and L. J. Gudas.
Functional analysis of the retinoic acid inducible enhancer 3' of the
Hoxa-1 gene, submitted for publication.
6 J. R. Thompson and L. J. Gudas,
unpublished data.
Received for publication 8/17/99. Revision received 11/ 3/99. Accepted for publication 11/ 3/99.
References
function in F9 cells by gene disruption results in aberrant Hoxa-1 expression and differentiation upon retinoic acid treatment. Proc. Natl. Acad. Sci. USA, 90: 9601-9605, 1993. and retinoid X receptor . J. Biol. Chem., 273: 2409-2415, 1998.This article has been cited by other articles:
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  E. Martinez-Ceballos, P. Chambon, and L. J. Gudas Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal J. Biol. Chem., April 22, 2005; 280(16): 16484 - 16498. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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