(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 -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) .
Besides ATBF1, there are three
transcription factors that contain both homeodomain and zinc finger
motifs. Chicken 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) .
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 ()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.
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
[-
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).
Total
RNA (5 µg) was hybridized with 5 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.
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. 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.
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 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.
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 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.
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 -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.
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].