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(Received for publication, March 23, 1995; and in revised form, July
12, 1995) From the
The human ATBF1 cDNA reported previously, now termed ATBF1-B,
encodes a 306-kDa protein containing 4 homeodomains and 18 zinc fingers
including one pseudo zinc finger motif. Here, we report the isolation
of a second ATBF1 cDNA, 12 kilobase pairs long, termed ATBF1-A. The
deduced ATBF1-A protein is 404 kDa in size and differs from ATBF1-B by
a 920-amino acid extention at the N terminus. Analysis of 5`-genomic
sequences showed that the 5`-noncoding sequences specific to ATBF1-A
and ATBF1-B transcripts were contained in distinct exons that could
splice to a downstream exon common to the ATBF1-A and ATBF1-B mRNAs.
The expression of ATBF1-A transcripts increased to high levels when P19
and NT2/D1 cells were treated with retinoic acid to induce neuronal
differentiation. Preferential expression of ATBF1-A transcripts was
also observed in developing mouse brain. Transient transfection assays
showed that the 5.5-kilobase pair sequence upstream of the
ATBF1-A-specific exon (exon 2) supported expression of the linked
chloramphenicol acetyltransferase gene in neuronal cells derived from
P19 cells but not in undifferentiated P19 or in F9 cells, which do not
differentiate into neurons. These results showed that ATBF1-A and
ATBF1-B transcripts are generated by alternative promoter usage
combined with alternative splicing and that the ATBF1-A-specific
promoter is activated during neuronal differentiation.
The ATBF1 (AT motif binding factor 1) cDNA was first isolated
from HuH-7 human hepatoma cells based on the ability of its product to
bind to an AT-rich enhancer element of the human Besides ATBF1, there are three
transcription factors that contain both homeodomain and zinc finger
motifs. Chicken Sequence comparison shows
that homeodomains I, II, and III of ATBF1 are 77, 69, and 61% identical
with the corresponding homeodomains of ZFH-2 (9, 10) .
Homeodomain IV of ATBF1 is 46% identical with homeodomain III of ZFH-2.
13 zinc fingers of ATBF1 and ZFH-2 show identities ranging from 22 to
89%. All of these homologous domains are colinearly arranged in the
ATBF1 and ZFH-2 molecules. These observations suggest that ATBF1 and
ZFH-2 may have similar functions, raising the possibility that ATBF1
plays a role in mammalian central nervous system development. In fact,
the level of ATBF1 transcripts is highly elevated in embryonic and
neonatal mouse brains(11) . In addition, ATBF1 expression is
activated during retinoic acid-induced neuronal differentiation of P19
embryonal carcinoma cells(11) . In this paper, we report the
isolation of a second ATBF1 cDNA, termed ATBF1-A, which is 3.3 kb (
Figure 1:
ATBF1-A
cDNA. Open bar, full-length ATBF1-A cDNA; solid bars,
partial ATBF1-A and ATBF1-B cDNA clones and an RT-PCR product. Short bars with vertical lines at both ends indicate
probes used for Northern blot analysis, RNase protection assays, and
screening genomic libraries.
Figure 4:
Analysis of ATBF1-A and ATBF1-B
transcripts in various cell lines. A, Northern blot analysis.
Total RNA (15 µg) of M426 (lane 1), HuH-7 (lane
2), and huH-1 (lane 3) cells was analyzed using human
ATBF1 cDNA I probe (Fig. 1). Total RNA (15 µg) of P19 cells
with (lane 5) or without (lane 4) treatment of
retinoic acid was analyzed using mouse ATBF1 cDNA I probe(11) . Arrowheads with A and B indicate expected
positions of ATBF1-A and ATBF1-B transcripts, respectively. GAPD, glyceraldehyde-3-phosphate dehydrogenase mRNA. B, RNase protection assays. Total RNA (5 µg) of M426 (lane 2), HuH-7 (lane 3), huH-1 (lane 4),
undifferentiated NT2/D1 (lane 5), and retinoic acid-treated
(neuronally differentiated) NT2/D1 (lane 6) cells was analyzed
using MD14 probe shown below the figure. Total RNA (5 µg)
of P19 cells with (lane 9) or without (lane 8)
treatment of retinoic acid was analyzed using BX151 probe shown below
the figure. Lanes 1 and 7 show MD14 and BX151 probes,
respectively. + and - indicate with and without retinoic
acid treatment, respectively. Arrowheads with A and B indicate ATBF1-A and ATBF1-B transcripts,
respectively.
To prepare
BX151 probe for mouse ATBF1 mRNA, 151 bp of mouse ATBF1 cDNA
corresponding to nucleotides 3353-3503 of human ATBF1-A cDNA was
inserted into pBluescript II KS(+) (Fig. 1). This was
digested with BamHI and transcribed by T3 RNA polymerase
(Boehringer Mannheim) in the presence of
[ Total
RNA (5 µg) was hybridized with 5
Figure 6:
Exon-intron organization of 5`-region of
the ATBF1 gene. Boxes indicate exons, and solid lines indicate introns. White boxes, noncoding sequences of
ATBF1-A mRNA; shaded boxes, noncoding sequences of ATBF1-B
mRNA; black boxes, coding sequences. Sizes of introns were not
determined except that between exons 1 and 2.
Figure 2:
Nucleotide sequence of ATBF1-A cDNA and
deduced amino acid sequence. Dotted lines, zinc finger motifs; solid lines, homeodomains; (a), 5`-end of
The ATBF1-A cDNA encodes a protein of 3703 amino acids
with a molecular mass of 404 kDa. This protein contains 4 homeodomains
and 23 zinc finger motifs including 1 pseudo zinc finger motif. ATBF1-A
is longer than ATBF1-B by 920 amino acids added at the N terminus. This
extended region contains five zinc fingers, two acidic domains (amino
acids 110-145 and 432-510), and one region rich in both
serine and threonine (39%, amino acids 396-431) (Fig. 3).
In addition, computer analysis (22) revealed several sequences
that are similar to consensus motifs of RNA and DNA helicases. They
include a DEAH box-like sequence, SfrVFDvrHk (amino acids
230-239)(23) , a DEAD box-like sequence, VvfDgAnRrnRLSF
(amino acids 559-572)(23, 24) , and an
RNA-binding motif, QphpRlaR (amino acids
708-715)(23, 25) . An ATP-binding motif,
ASGSAGKS(7) , at amino acids 2930-2937 and two lysine
residues at 2959 and 2962 (26) may also be involved in ATP
binding functions (in the amino acid sequences described above, the
residues indicated in upper case are those found in the consensus
sequences).
Figure 3:
Potential functional domains of ATBF1-A. Rectangles, homeodomains I-IV; solid circles, zinc
finger motifs; ovals, segments rich in glutamic acid (E),
aspartic acid (D), glutamine (Q), glycine (G), proline (P), serine (S),
and threonine (T). The positions of DEAH box- and DEAD box-like
sequences and an ATP-binding site are indicated by arrows. The
position and the length of ATBF1-B are shown below.
To analyze more precisely the relative amount of
ATBF1-A and ATBF1-B transcripts, we conducted RNase protection assays
using MD14 probe capable of distinguishing these two types of
transcripts. The results showed that the three human cell lines
described above expressed both types of transcripts (Fig. 4B, lanes 2-4). The amounts of
ATBF1-A transcripts were 5-10-fold higher than those of ATBF1-B
transcripts, although the absolute levels of these transcripts were
low. Similarly, small amounts of ATBF1-A and ATBF1-B transcripts were
detected in undifferentiated NT2/D1 human embryonal carcinoma cells (Fig. 4B, lane 5). The band representing
ATBF1-B transcripts was very weak but visible after long exposure. Upon
induction of neuronal differentiation from these cells by treatment
with retinoic acid, the level of ATBF1-A transcripts increased about
50-fold (Fig. 4B, lane 6). The level of
ATBF1-B transcripts also increased but to a much lesser extent. Thus
the ratio of ATBF1-A and ATBF1-B transcripts in differentiated NT2/D1
cells was 50:1 as compared to 5:1 in undifferentiated cells. Similar
assays of P19 cells using BX151 probe showed that, as in the case of
NT2/D1 cells, the level of ATBF1-A transcripts was 50-fold higher in
retinoic acid-treated cells than in untreated cells (Fig. 4B, lanes 8 and 9). Also, as in
the case of NT2/D1 cells, the increase in the level of ATBF1-B
transcripts was much less, giving rise to a 50:1 ratio of ATBF1-A and
ATBF1-B transcripts in retinoic acid-treated cells. These results
showed that neuronal differentiation of P19 and NT2/D1 cells is
accompanied by preferential synthesis of ATBF1-A transcripts. RNase
protection assays of ATBF1 transcripts were also conducted on embryonic
and neonatal mouse brains. The results showed high levels of ATBF1
expression, predominantly in the form of ATBF1-A, in brains of
15-day-old embryos and 1-day-old neonates (Fig. 5). Expression
of ATBF1 transcripts was decreased in brains of 5- and 7-day-old
neonates.
Figure 5:
Analysis of ATBF1-A and ATBF1-B
transcripts in developing mouse brain. RNase protection assays were
conducted using BX151 probe and 5 µg of total RNA from mouse brains
of 15-day-old embryo (lane 2), 1-day-old neonate (lane
3), 5-day-old neonate (lane 4), and 7-day-old neonate (lane 5). Lane 1 shows BX151 probe. Arrowheads with A and B indicate ATBF1-A and ATBF1-B
transcripts, respectively.
Southern blot and restriction analysis of these
clones showed that
Figure 7:
RNase protection mapping of the 5`-ends of
exons 1 and 2. A, exon 1 (ATBF1-B-specific exon). Lane
1, size marker for probe 1B; lane 2, 1B probe (680 bp); lane 3, size marker; lane 4, M426 RNA. The arrowhead indicates a 115-bp RNase protected band. B,
exon 2 (ATBF1-A-specific exon). Lane 1, size marker for probe
1A; lane 2, 1A probe (474 bp); lane 3, size marker; lane 4, M426 RNA. The arrowhead indicates a 177-bp
RNase protected band. Size markers were fragments released from
pBluescript II KS(+) by digestion with HpaII.
Figure 8:
Transient transfection assays of
transcriptional activity of the 5`-flanking sequences of exons 1 and 2. A, CAT constructs containing the 5`-flanking sequences of
exons 1 and 2. The ATBF1 genomic sequence is shown above. B,
CAT expression in P19 cells. Undifferentiated (lanes
1-4) and differentiated (lanes 5-8) P19 cells
were transfected with pSV2-CAT (lanes 1 and 5), the
vector plasmid (lanes 2 and 6), pA5.5-CAT (lanes
3 and 7), and pB3.5-CAT (lanes 4 and 8)
and assayed for CAT activity 2 days later. + and - indicate
with and without treatment of retinoic acid, respectively. Retinoic
acid-treated cells were transfected on day 7. CAT activity was assayed
using 40 µg of protein and 2 h of incubation and normalized for the
expression of
The 3.5-kb 5`-flanking sequence of
ATBF1-B-specific exon 1 (pB3.5-CAT), on the other hand, did not support
CAT expression in undifferentiated or differentiated P19 cells or any
other cell lines described above. We report here the isolation of a second human ATBF1 cDNA,
termed ATBF1-A. This cDNA differs from the previously reported ATBF1
cDNA, termed ATBF1-B, by an extra 3.3-kb sequence at the 5`-end. Since
the extended region can encode five additional zinc fingers,
helicase-related sequences, and domains rich in acidic amino acids and
serine and threonine, it is possible that ATBF1-A may have functions
not associated with the ATBF1-B isoform. RNase protection assays
detected two sizes of mRNAs corresponding to ATBF1-A and ATBF1-B in
various cell lines and mouse brain. In all cases, ATBF1-A transcripts
were present in larger amounts than ATBF1-B transcripts, but the
absolute levels of these transcripts were low in various cell lines and
adult mouse brain. Similarly, the amounts of these ATBF1 mRNAs were low
in undifferentiated P19 and NT2/D1 embryonal carcinoma cells. However,
neuronal cells derived from these cells by treatment with retinoic acid
contained much higher levels of these transcripts. The increased
expression was particularly pronounced with ATBF1-A transcripts,
relative to ATBF1-B transcripts. Preferential expression of the ATBF1-A
form was also observed in developing mouse brain. The isolation and
analysis of 5`-genomic sequences defined the basis for the generation
of the two species of ATBF1 mRNAs. We found that 5`-noncoding sequences
specific to ATBF1-A and ATBF1-B mRNAs were contained in distinct exons.
We also found a downstream exon that can splice to either the ATBF1-A-
or ATBF1-B-specific exon. These results showed that alternative
splicing is involved in the generation of the two ATBF1 isoforms.
Transient transfection experiments showed that the 5`-flanking region
of exon 2, the first exon specific to ATBF1-A, exhibited promoter
activity in neuronal cells derived from P19 cells but not in
undifferentiated P19 cells. No promoter activity was expressed in F9
embryonal carcinoma cells or other non-neuronal cells. These results
indicate that the 5`-flanking sequence of the ATBF1-A-specific exon
functions as a neuronal cell-specific promoter. It is likely that this
promoter is responsible for the observed increase in ATBF1-A
transcripts in association with neuronal differentiation of P19 or
NT2/D1 cells. Recent CAT assays showed that the 5`-flanking sequence of
exon 2 could be shortened to 300 bp without losing promoter activity in
neuronal cells. This indicates that the relatively short promoter
region is sufficient to confer neuronal cell specificity. Computer
search has revealed that the 300-bp region contains putative binding
sites for several transcription factors, including c-fos,
AP-2, SP-1, and zif268 (also known as egr-1, krox24, or NGF-A). Whether
these factors are in fact involved in promoter activation in neuronally
differentiating P19 cells is being investigated in our laboratory. Although it is possible that ATBF1-B transcripts are produced by a
differentially regulated promoter, we have not yet detected
transcriptional activity associated with the 5`-flanking region of exon
1 (ATBF1-B-specific exon). Our failure to detect promoter activity in
this region is likely due to very low levels of ATBF1-B expression in
the cell lines used for CAT transfection assays. Obviously, these cells
are deficient of certain transcription factors important for the
synthesis of the ATBF1-B isoform. We are currently searching for cell
lines expressing higher levels of ATBF1-B transcripts to be used for
determination of ATBF1-B-specific promoter activity.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
L32832[GenBank],
L32833[GenBank].
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 26840-26848
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-fetoprotein gene (AFP)(1) . This protein is characterized by a large
size (306 kDa) and the presence of four homeodomains (I-IV) and
18 zinc fingers including 1 pseudo zinc finger motif. Transient
transfection assays showed that ATBF1 suppressed the activity of the
AT-rich element of the enhancer and promoter of the AFP gene
but not those of the albumin gene(2) . This effect is thought
to be mediated by specific interaction between homeodomain IV of the
ATBF1 molecule and the AT-rich element of the AFP gene(1, 2) .EF1 has one homeodomain and nine zinc finger
motifs. This protein has been shown to repress activities of the DC
segment of the
1-crystallin enhancer (3, 4) and
the E2 element of the immunoglobulin
chain and muscle creatine
kinase enhancer(5) . Its expression pattern in chicken embryos
suggests that EF1 plays a role in mesoderm development and
embryonic myogenesis(3, 4, 5) . Drosophila ZFH-1 contains one homeodomain and nine zinc finger
motifs(6) . It is expressed in the embryonic mesoderm and
nervous system(7) , and phenotypic analysis of loss-of-function
mutant embryos has shown that ZFH-1 determines cell fate or
positioning(8) . Drosophila ZFH-2 contains 3
homeodomains and 16 zinc finger motifs (6) and is expressed
almost exclusively in the central nervous system of Drosophila embryos(7) . It binds to the RCS element of the opsin gene
through homeodomain III (6, 9) and activates the
SER
element of the DOPA decarboxylase gene through
homeodomain II(6, 9) .
)longer than the previously reported clone, now termed
ATBF1-B. We show that ATBF1-A transcripts are generated by alternative
splicing and alternative usage of a promoter region that is activated
in neuronally differentiating cells.
Cells
M426 human fibroblasts were grown
in Dulbecco's modified Eagle's medium with 10% fetal bovine
serum. P19 mouse embryonal carcinoma cells, kindly provided by Dr. M.
W. McBurney, were grown in MEM with 10% fetal bovine serum. To
induce neuronal differentiation, P19 cells were allowed to form
aggregates in bacteriological grade Petri dishes for 4 days in the
presence of 0.5 µM retinoic acid and transferred to tissue
culture grade Petri dishes without retinoic acid(12) . The day
of adding retinoic acid to the medium was assigned day 0. F9 mouse
embryonal carcinoma cells were grown in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum on gelatin-coated
dishes. Visceral endoderm-like cells were induced from these cells by
treatment with 0.05 µM retinoic acid(13) .
NT2/D1(NTERA-2.cl.D1) human embryonal carcinoma cells were grown in
Dulbecco's modified Eagle's medium with 10% fetal bovine
serum. Neuronal differentiation was induced from NT2/D1 cells by
treatment with 10 µM retinoic acid for 3
weeks(14) . HuH-7 human hepatoma cells were maintained in a
chemically defined medium, ISE-RPMI 1640(15) . huH-1 human
hepatoma cells were grown in RPMI 1640 with 1% fetal bovine serum.Isolation of cDNA Clones
An M426 human
fibroblast cDNA library, pCEV15(16) , was screened using a
human ATBF1 cDNA,
488(1) , labeled with
[
-P]dCTP as a probe. This resulted in the
isolation of
B and
E clones. These clones were then used as
probes to screen a second M426 fibroblast cDNA library,
pCEV-lacZ(17) , to isolate
I and
ME clones. A
short sequence between
ME and
I was isolated by RT-PCR using
primers corresponding to
ME and
I sequences.
Isolation of Genomic DNA Clones
A WI38
human lung fibroblast genomic DNA library in FIX II (Stratagene)
was screened using three cDNA fragments, E, ME, and MEM, as probes (Fig. 1). E probe (0.5 kb) was prepared from the 5` portion of
E clone corresponding to a 5`-noncoding region of ATBF1-B. ME
probe (0.7 kb) was prepared from the 5` portion of
ME clone. It
consisted of 0.4 kb of the 5`-noncoding sequence and 0.3 kb of the
5`-coding sequence of ATBF1-A. MEM probe (0.7 kb) was prepared from a
coding region common to ATBF1-A and ATBF1-B. These DNA fragments were
labeled with [
-P]dCTP.
DNA Sequencing
Nucleotide sequences were
determined from both strands by the dideoxy chain termination procedure
with standard sequencing primers and custom-made primers using
Sequenase Kit (Stratagene) with [-S]dATP or
using Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied
Biosystems).
Northern Blot Analysis
Total RNA was
isolated by the guanidium isothiocyanate-cesium chloride
method(18) . ATBF1 transcripts were detected by hybridization
with I probe (0.9 kb) prepared from I clone (Fig. 1) for
human samples or the mouse ATBF1 cDNA clone (11) for mouse
samples. Glyceraldehyde-3-phosphate dehydrogenase mRNA was detected
using a 0.78-kb glyceraldehyde-3-phosphate dehydrogenase cDNA probe as
described previously(11) . The probes were labeled with
[
-P]dCTP.
RNase Protection Assays
To prepare MD14
probe for RNase protection assays for human ATBF1-A and ATBF1-B
transcripts, 256 bp of human ATBF1-A cDNA sequence from nucleotides
3353-3608 was inserted into pBluescript II KS(+)
(Stratagene) (Fig. 1). This was digested with BamHI and
transcribed by T7 RNA polymerase (Boehringer Mannheim) in the presence
of [-P]CTP to produce a radioactive 287-bp
fragment consisting of 256 bp of the ATBF1-A sequence and 31 bp of the
vector sequence. This probe yielded a 256-bp fragment with ATBF1-A mRNA
and a 216-bp fragment with ATBF1-B mRNA (Fig. 4).
-P]CTP to produce a radioactive 228-bp
fragment consisting of 151 bp of the mouse ATBF1 sequence and 77 bp of
the vector sequence. This probe yielded a 151-bp fragment with ATBF1-A
mRNA and a 111-bp fragment with ATBF1-B mRNA (Fig. 4).
10
cpm of
probe in 40 mM PIPES (pH 6.4), 80% formamide, 0.4 M NaCl, and 1 mM EDTA at 45 °C overnight. The reaction
mixture was digested with 40 µg/ml RNase A and 2 µg/ml RNase T1
at 30 °C for 30 min. The sample was then treated with 130 µg/ml
proteinase K, electrophoresed on a 5% polyacrylamide/urea gel, and
autoradiographed.Mapping of the 5`-End of mRNA
For the
determination of the 5`-end of ATBF1-B mRNA, the 603-bp StyI-DraI fragment consisting of a 5`-portion of exon
1 and its 5`-flanking sequence was cloned into pBluescript II
KS(+), digested with StyI, and transcribed by T3 RNA
polymerase. The resultant 680-bp fragment consisting of 603 bp of the
genomic sequence and 77 bp of the vector sequence was used as a probe
(1B probe, Fig. 6). For the determination of the 5`-end of
ATBF1-A mRNA, the 428-bp StuI-BamHI fragment
consisting of a 5` portion of exon 2 and its 5`-flanking sequence was
cloned into pBluescript II KS(+), digested with StuI, and
transcribed by T7 RNA polymerase. The resultant 474-bp fragment
consisting of 428 bp of the genomic sequence and 46 bp of the vector
sequence was used as a probe (1A probe, Fig. 6). Both probes
were labeled with [-P]CTP. Total RNA (10
µg) from M426 cells was hybridized with 5
10 cpm of RNA probes, digested with RNase A and RNase T1 followed by
proteinase K, and analyzed by gel electrophoresis as described for
RNase protection assays above.
FIX-17 and -13 are
genomic clones used to analyze the exons 1-4. 1A and 1B probes
used for RNase protection assays of the 5` boundaries of exons 1 and 2
are drawn below.
CAT Plasmids
To construct pUMS-CAT, the
CAT gene and the poly(A) signal were removed from pSV2-CAT (19) and inserted into pBluescript II KS(+). The upstream
mouse sequence (UMS) of the c-mos gene was removed from
pUBT-luc (20) and inserted upstream of the CAT gene. To
construct pA5.5-CAT, the 5.5-kb (SalI-BamHI)
5`-flanking sequence of exon 2 (the first exon specific to ATBF1-A
mRNA) was inserted between UMS and the CAT gene of pUMS-CAT. To
construct pB3.5-CAT, the 3.5-kb (NotI-DraI)
5`-flanking sequence of exon 1 (the first exon specific to ATBF1-B
mRNA) was inserted between UMS and the CAT gene of pUMS-CAT.CAT Assays
Cells were transfected with 10
µg of chimeric CAT plasmids by the calcium phosphate precipitation
method. pCH110 
galactosidase expression plasmid (10 µg)
(Pharmacia Biotech Inc.) was cotransfected to monitor for transfection
efficiency to normalize CAT activity. Cells were harvested 48 h later
and lysed by five cycles of freezing and thawing. Part of the lysate
was used to assay -galactosidase activity(21) . The
remaining lysate was heated at 65 °C for 10 min and centrifuged at
12,000 g for 5 min, and the supernatant was analyzed
for CAT activity(19) .
Isolation of ATBF1-A cDNA
Screening of
pCEV15 cDNA library with the
488 human ATBF1 cDNA (1) as a probe resulted in the isolation of
B and
E
clones (Fig. 1). These clones were then used as probes to screen
pCEV-lacZ cDNA library to isolate
I and
ME. Sequence
analysis showed that
I and
B were identical, representing the
3`-half of the ATBF1 cDNA previously reported(1) .
E and
ME represented the sequence upstream of
I, but 527
nucleotides at the 5`-end of
E were entirely different from the
corresponding region of
ME. This segment of
E contained the
5`-noncoding sequence followed by the coding sequence of the ATBF1 cDNA
reported previously. In
ME, we found a reading frame extending
upstream of the translation initiation codon in
E. Thus,
ME
represented a new cDNA sequence with a longer coding sequence. To
complete sequence analysis of the new ATBF1 cDNA, we isolated a short
region between
ME and
I by RT-PCR. The sequence of the RT-PCR
product was identical with the corresponding region of ATBF1 previously
reported. Combining
ME,
I, and the RT-PCR product, the
sequence of the new ATBF1 cDNA was constructed (Fig. 2). This
cDNA is referred to as ATBF1-A and the ATBF1 cDNA reported previously
as ATBF1-B.
ME
cDNA; (b), exon 2-exon 3 junction; (c), DEAH box-like
sequence; (d), vitamin K-dependent carboxylase recognition
motif; (e), casein kinase II phosphorylation motif; (f), DEAD box-like sequence; (g), SAT box-like
sequence; (h), RNA-binding motif; (i), exon 3-exon 4
junction (alternative splicing site); (j), translation
initiation codon of ATBF1-B; (k), ATP-binding site; (l), nuclear targeting sequence; (m), 24 nucleotides
deleted in HuH-7 ATBF1 cDNA(1) ; (n), variable number
of GGC triplet. The termination codon and two potential polyadenylation
signals are underlined.
Expression of ATBF1-A and ATBF1-B Transcripts in Cell
Lines and Developing Mouse Brain
Northern blot analysis
using the I clone as a probe detected faint bands in the region of
10-13 kb in M426, HuH-7, and huH-1 human cell lines (Fig. 4A, lanes 1-3). These bands
corresponded to ATBF1-A and ATBF1-B transcripts, but the levels of
their expression were very low in all these cell lines. Similarly, P19
embryonal carcinoma cells contained small amounts of ATBF1-A and
ATBF1-B transcripts (Fig. 4A, lane 4).
However, when P19 cells were treated with retinoic acid to induce
neuronal differentiation, there were large increases in the levels of
these transcripts (Fig. 4A, lane 5) (11) .
Identification of Alternatively Spliced
Exons
To explore the mechanism by which ATBF1-A and ATBF1-B
mRNAs are generated, we analyzed 5`-genomic sequences encompassing the
5`-ends of these transcripts. Two genomic clones were isolated. One
clone, FIX-17 (Fig. 6), could hybridize to ME and E probes
representing the 5`-noncoding sequences of ATBF1-A and ATBF1-B mRNAs,
respectively (Fig. 1). The other clone,
FIX-13 (Fig. 6), could hybridize to ME probe and also to MEM probe,
which represents a coding region common to ATBF1-A and ATBF1-B (Fig. 1).
FIX-17 contained exons 1 and 2, and
FIX-13
carried exons 3 and 4 (Fig. 6). Exons 1 and 2 in
FIX-17
were further characterized by sequence analysis and RNase protection
assays to define the 5` boundaries (Fig. 7). The results showed
that exon 1 carried most of the 5`-noncoding sequence of ATBF1-B mRNA
and exon 2 that of ATBF1-A mRNA (Fig. 2). Exon 3 in
FIX-13
contained the remaining ATBF1-A noncoding sequence (49 bp) followed by
the ATBF1-A protein coding sequence. Exon 4 contained the remaining
ATBF1-B noncoding sequence (23 bp) followed by the ATBF1-B protein
coding sequence. Analysis of exon-intron junctions confirmed the
presence of the consensus splice donor (GT) sites and acceptor (AG)
sites (results not shown). In addition, it was found that exon 4 could
splice to either exon 1 or exon 3 (Fig. 6). In the former case,
the ATBF1-B mRNA sequence is generated, and in the latter case, the
ATBF1-A mRNA sequence is generated with the ATBF1-B noncoding sequence
in exon 4 becoming a part of the ATBF1-A coding sequence (Fig. 2). These results are consistent with the mechanism that
ATBF1-A and ATBF1-B mRNAs are generated by alternative splicing.
Determination of Promoter Activity
The
presence of distinct 5` exons specific to ATBF1-A and ATBF1-B sequences
suggested that two different promoters may be used to initiate
transcription of the two types of ATBF1 mRNAs. To examine whether the
5`-flanking sequences of exons 1 and 2 have transcriptional activity,
they were linked to the CAT reporter gene (Fig. 8A) and
transfected into undifferentiated and neuronally differentiated P19
cells. The 5.5-kb 5`-flanking sequence of ATBF1-A-specific exon 2
(pA5.5-CAT) supported CAT expression in differentiated P19 cells (Fig. 8B, lane 7) but not in undifferentiated
P19 cells (Fig. 8B, lane 3). No CAT activity
was expressed in F9 embryonal carcinoma cells with or without retinoic
acid treatment (Fig. 8C). Treatment of F9 cells with
retinoic acid resulted in induction of endodermal cells but not
neuronal cells. pA5.5-CAT expressed CAT activity in other neuronal
cells, such as Neuro-2a and retinoic acid-treated NT2/D1 cells, but not
in non-neuronal cells, such as HepG2 (results not shown). These results
showed that the 5`-flanking sequence of exon 2 has neuronal
cell-specific promoter activity.
-galactosidase activity. CAT activity of each CAT
construct is expressed as percentage of that of pSV2-CAT. C,
CAT expression in F9 cells. Undifferentiated (lanes 1-4)
and differentiated (lanes 5-8) F9 cells were transfected
with pSV2-CAT (lanes 1 and 5), the vector plasmid (lanes 2 and 6), pA5.5-CAT (lanes 3 and 7), and pB3.5-CAT (lanes 4 and 8) and
assayed for CAT activity 2 days later. + and - indicate with
and without treatment of retinoic acid, respectively. CAT activity was
assayed, normalized, and expressed as described above for P19
cells.
)
We thank Dr. A. Moriyama, H. Chan, and T. Scheidl for
excellent technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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