©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Murine Transcription Factor A-crystallin Binding Protein I
COMPLETE SEQUENCE, GENE STRUCTURE, EXPRESSION, AND FUNCTIONAL INHIBITION VIA ANTISENSE RNA (*)

(Received for publication, October 12, 1994)

James P. Brady (§) Marc Kantorow Christina M. Sax David M. Donovan Joram Piatigorsky (¶)

From the Laboratory of Molecular and Developmental Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alphaA-crystallin binding protein I (alphaA-CRYBP1) is a ubiquitously expressed DNA binding protein that was previously identified by its ability to interact with a functionally important sequence in the mouse alphaA-crystallin gene promoter. Here, we have cloned a single copy gene with 10 exons spanning greater than 70 kb of genomic DNA that encodes alphaA-CRYBP1. The mouse alphaA-CRYBP1 gene specifies a 2,688-amino acid protein with 72% amino acid identity to its human homologue, PRDII-BF1. Both the human and the mouse proteins contain two sets of consensus C(2)H(2) zinc fingers at each end as well a central nonconsensus zinc finger. The alphaA-CRYBP1 gene produces a 9.5-kb transcript in 11 different tissues as well as a testis-specific, 7.7-kb transcript. alphaA-CRYBP1 cDNA clones were isolated from adult mouse brain and testis as well as from cell lines derived from mouse lens (alphaTN4-1) and muscle (C(2)C). A single clone isolated from the muscle C(2)C library contains an additional exon near the 5`-end that would prevent production of a functional protein if the normal translation start site were utilized; however, there is another potential initiation codon located downstream that is in frame with the rest of the coding region. In addition, we identified multiple cDNAs from the testis in which the final intron is still present. Finally, we used an antisense expression construct derived from an alphaA-CRYBP1 cDNA clone to provide the first functional evidence that alphaA-CRYBP1 regulates gene expression. When introduced into the alphaTN4-1 mouse lens cell line, the antisense construct significantly inhibited expression from a heterologous promoter that utilized the alphaA-CRYBP1 binding site as an enhancer.


INTRODUCTION

alphaA-CRYBP1 (^1)is a DNA binding protein that was identified by its ability to recognize the sequence 5`-GGGAAATCCC-3` at position -66/-57 of the gene encoding mouse alphaA-crystallin, a protein expressed almost exclusively in the ocular lens(1, 2) . Mutations in the alphaA-CRYBP1 binding site caused significant reductions in alphaA-crystallin promoter function in transient transfection assays, suggesting that factors that bind to this site are likely to play a critical role in regulating alphaA-crystallin gene expression(1, 3) . A partial 2.5-kbp alphaA-CRYBP1 cDNA encoding the carboxyl-terminal portion of a protein that contains two contiguous zinc fingers was isolated from a mouse lens-derived cell line library(1) . DNA sequence analysis revealed that alphaA-CRYBP1 is homologous to a rat protein, AT-BP2, that interacts with the alpha(1)-antitrypsin promoter (4) as well as to a human protein called PRDII-BF1, MBP-1, or HIV-EP1, that binds to similar sequences in the interferon beta gene promoter(5) , the MHC H2-K^b gene promoter(6) , and the HIV-1 viral enhancer(7) , respectively. The peptide sequence of PRDII-BF1 inferred from overlapping cDNAs revealed a 2,717-amino acid protein with one pair of consensus zinc fingers near the amino terminus and a similar pair near the carboxyl terminus corresponding to those in alphaA-CRYBP1(5) . The amino- and carboxyl-terminal zinc fingers of PRDII-BF1 can independently recognize the same DNA binding sites(5) . The alphaA-CRYBP1 transcript is approximately 9.5 kb long in newborn mice, suggesting that the mouse gene encodes a protein of comparable size with human PRDII-BF1(1) .

Aside from its ability to bind to DNA in a site-specific manner, nothing is known about the mechanisms by which alphaA-CRYBP1 might regulate gene expression in the mouse. In this report, we describe the cloning and characterization of the mouse alphaA-CRYBP1 gene. We chose to clone the gene for several reasons. First, the alphaA-CRYBP1/PRDII-BF1 binding site is very similar to the recognition sequence of the transcription factor NF-kappaB that has been implicated in the regulation of many human and murine genes(8) , raising the possibility that some of the promoters that are thought to interact with NF-kappaB might also interact with alphaA-CRYBP1/PRDII-BF1. Studies of the alphaA-CRYBP1 gene may help to elucidate additional pathways of regulation for an array of genes. Second, Kantorow et al.(9) showed that an anti-alphaA-CRYBP1 antibody recognizes proteins of different sizes on Western blots of mouse tissue culture cells, and Muchardt et al.(10) have identified alternatively spliced transcripts of PRDII-BF1 by polymerase chain reaction in human cell lines, suggesting that a thorough knowledge of the intron/exon structure of the alphaA-CRYBP1 gene is necessary to determine if alternative RNA splicing is involved in the generation of variant alphaA-CRYBP1 proteins with potentially different capabilities for transcriptional regulation. Finally, obtaining a clone of the alphaA-CRYBP1 locus will allow us to selectively mutate this gene through the process of homologous recombination in embryonic stem cells followed by the incorporation of those stem cells into mouse blastocysts(11) .


MATERIALS AND METHODS

Cloning, Screening, and Sequence Analysis

Genomic clones were isolated from a library of partially digested DBA/2J mouse genomic DNA in the EMBL-3 cloning vector (Clontech) and from a library of 129SV mouse DNA in Fix II (Stratagene). Brain and testis cDNA clones were isolated from libraries of randomly primed poly(A) RNA from adult BALB/c mice; the brain clones were in the vector SWAJ-2, and the testis clones were in Max1, respectively (Clontech). A single clone in Zap II (Stratagene) was also obtained from a library of randomly primed poly(A) RNA isolated from the C(2)C mouse myoblast cell line. Finally, additional clones were isolated from the alphaTN4-1 lens cell line cDNA library previously described(1) .

DNA fragments and oligonucleotides used as hybridization probes for library screening were made radioactive by random priming and end labeling according to standard procedures(12) . Libraries were hybridized in 3 times SSC, 0.3 M Na(3) citratebullet2H(2)O, pH 7.0, 2% Ficoll, 2% bovine serum albumin, 2% polyvinylpyrrolidone, 1% SDS, 0.05 M HEPES, and 0.018 mg/ml denatured salmon sperm DNA. After hybridization, the filters were washed several times in 2 times SSC. Hybridization and washes were both performed at 65 °C when DNA fragments were used as probes and at 55 °C when oligonucleotides were used as probes. Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer.

Genomic and cDNA clones were subcloned into pBluescript/SK- (Stratagene) and sequenced using Sequenase (version 2, U. S. Biochemical Corp.). DNA sequences were analyzed using version 7 of the University of Wisconsin Genetics Computer Group software package.

Genomic Southern Blot Analysis

The insert from the C(2)C cell line cDNA clone, CC-1, was hybridized in 0.5 M sodium phosphate, 7% SDS, and 1 mM EDTA at 65 °C to a ``zoo blot'' containing 8 µg of EcoRI-digested genomic DNA from mice and humans and an amount of DNA corresponding to the same number of genome equivalents of other species (BIOS). The blot was initially washed with 1 times SSC, 0.1% SDS at 52 °C and exposed to x-ray film. After an initial exposure, the blot was washed again with 0.2 times SSC, 0.1% SDS at 65 °C and re-exposed to film.

Northern Blot Analysis

20 µg of total RNA isolated from tissues of 3-week-old adult mice were subjected to electrophoresis in 1% agarose gels containing 6.2% formaldehyde and transferred to nylon membranes (BIOS). The blot was prehybridized in 5 times SSC, 5 times Denhardt's solution, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA followed by hybridization in 50% formamide, 5 times SSPE, 5 times Denhardt's solution, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA at 42 °C. The blot was washed three times in 0.5 times SSC, 0.2% SDS at room temperature and then once in 0.25 times SSC, 0.2% SDS at 50 °C. Hybridization probes were restriction fragments excised from the C(2)C cDNA clone and a human glyceraldehyde-3-phosphate dehydrogenase cDNA clone (Clontech).

Antisense Transfections

An alphaA-CRYBP1 antisense construct, pCCRXV-1, was created by cloning the C(2)C cDNA insert in reverse orientation into the EcoRI site of pCDNA-1/Neo (Invitrogen). alphaTN4-1 cells were transfected by the calcium phosphate precipitation method(13) . Conditions for cell growth, transfection, and cell harvesting were previously described(14) . To select for cells that express the bacterial neomycin resistance gene, the medium was replaced 48 h after transfection with fresh medium containing 500 µg/ml G418 (Life Technologies, Inc.), and the cells were refed every other day. Assays for CAT activity and beta-galactosidase activity were previously described(15, 16) .


RESULTS

Cloning the NH(2)-terminal Coding Sequences of alphaA-CRYBP1

Before attempting to clone the alphaA-CRYBP1 gene, we wanted to obtain a cDNA corresponding to the 5`-end of the gene. Two overlapping oligonucleotides (Fig. 1A) representing the 5`-end of the open reading frame (ORF) of the human PRDII-BF1 cDNA (5) were synthesized and used to screen a cDNA library created from randomly primed poly(A) RNA of C(2)C mouse muscle cells. A single 1.2-kbp-long cDNA clone, CC-1, was isolated. DNA sequence analysis of CC-1 (Fig. 1B) revealed an ORF following a short 5`-untranslated region. The ORF shows 79% nucleotide sequence identity and 76% deduced amino acid sequence identity with the amino-terminal open reading frame of PRDII-BF1(5) . These data are consistent with the 77% nucleotide and 75% deduced amino acid sequence identity levels previously identified between the 3`-coding sequences of human PRDII-BF1 and mouse alphaA-CRYBP1(1) . The major difference between CC-1 and PRDII-BF1 is the presence of a 65-bp insert containing a termination codon in CC-1 (Fig. 1B). There is, however, another ATG located within the insert (Fig. 1B) that is in the same reading frame as the remainder of the protein coding sequence in this cDNA (Fig. 1B).


Figure 1: Structure of human PRDII-BF1, oligonucleotide probes derived from PRDII-BF1, and partial sequence of the mouse cDNA clone CC-1. A, schematic representation of PRDII-BF1. Zinc finger sequences (Z) and acidic domains (A) are indicated by shaded and blackboxes, respectively. The locations of start and stop codons are shown above the cDNA. Overlapping oligonucleotides derived from the PRDII-BF1 cDNA sequence that were used to screen a C(2)C muscle cell cDNA library are shown below the PRDII-BF1 cDNA; the initiation ATG is underlined. B, 5`-terminal sequence of the C(2)C cell cDNA clone CC-1. Sequences in CC-1 that show similarity to the open reading frame of PRDII-BF1 are conceptually translated below the nucleotide sequence. Amino acids that are identical to PRDII-BF1 are underlined; nucleotides that are inserted relative to the PRDII-BF1 cDNA sequence are shaded. A conceptual translation of the inserted sequence is shown in the two different reading frames (ORF1 and ORF2) that align with the PRDII-BF1 open reading frame.



Genomic Southern Analysis of alphaA-CRYBP1

To confirm that alphaA-CRYBP1 is encoded by a single copy gene and to test the possibility that alphaA-CRYBP1 homologues might also be present in other species, a Southern blot of genome equivalents of EcoRI-digested DNA from taxonomically diverse species was hybridized with the CC-1 cDNA clone. CC-1 does not contain any zinc finger sequences that might cross-hybridize to unrelated zinc finger-containing genes. A low stringency wash (Fig. 2A) revealed hybridization bands of 3.7, 4.3, 9.1, and 19 kbp in mouse. Intense bands are also present in human and frog, and fainter bands can be seen in mussel, fruit fly, and nematode DNA. The apparent background hybridization in chicken DNA makes individual bands difficult to discern. However, our previous isolation of a cDNA from the chicken lens that is clearly homologous to alphaA-CRYBP1 indicates that at least one gene is related to alphaA-CRYBP1 in this species(3) . When the blot was washed again under highly stringent conditions (Fig. 2B), only bands in the mouse and human lanes remained visible, suggesting that the alphaA-CRYBP1 gene has sustained appreciable sequence changes during the course of evolution.


Figure 2: Genomic Southern blot analysis of alphaA-CRYBP1-related sequences in various eukaryotic species. A blot containing genome equivalents of EcoRI-digested DNA from the indicated species was hybridized with the insert from the alphaA-CRYBP1 cDNA clone CC-1. The blot was exposed after an initial low stringency wash (A) and then re-exposed after a high stringency wash (B).



Isolation of Genomic Clones

Approximately one million plaques from a DBA/2J mouse genomic DNA library were screened with both CC-1, which encodes the amino terminus of alphaA-CRYBP1, and with the insert from pYTN-8, which encodes the carboxyl terminus of alphaA-CRYBP1(1) . Fig. 3A shows restriction maps of the clones that were isolated and the regions of each clone that were sequenced. These sequences have been placed in GenBank. We were unable to isolate a DBA/2J genomic clone corresponding to nucleotides 6706-6808 in PRDII-BF1, the human homologue of the mouse alphaA-CRYBP1(5) . However, we were able to obtain a clone (MG17-4) from a 129SV mouse genomic library encompassing this sequence (Fig. 3A). The inserted sequence that was observed in CC-1 relative to PRDII-BF1 (see Fig. 1B) is located in the genomic clone MG62-1 (Fig. 3A). The 65-bp insert is flanked by consensus splice donor and acceptor sequences (17) (data not shown). The intron/exon structure of the alphaA-CRYBP1 based on the sequences of genomic and cDNA (see below) clones is shown in Fig. 3B. Since the GC-rich nature of the 5`-end of the gene prevented the establishment of the transcription start site, the numbering of the exons must remain tentative. The sizes of the restriction fragments in EcoRI-digested mouse genomic DNA that are expected to hybridize to CC-1 based on restriction mapping of genomic clones MG10-1, MG62-1, and MG4-2 (Fig. 3A) are indicated in Fig. 3B. The 4.3-, 3.7-, and >9.9-kbp fragments are consistent with the bands seen in Fig. 2. A 2.8-kbp band is not seen in Fig. 2, but there is a hybridizing fragment of 9.1 kbp. These data are consistent with alphaA-CRYBP1 being encoded by a single copy gene with a polymorphism in one of its EcoRI restriction sites.


Figure 3: Restriction maps of alphaA-CRYBP1 genomic clones and intron/exon structure of the alphaA-CRYBP1 gene. Panel A, restriction maps of lambda clones isolated from a DBA/2J mouse genomic library (MG10-1, MG18-1, MG62-1, MG 4-2, and MG49-1) and from a 129SV mouse genomic library (MG17-4). Shadedbars indicate the regions of each clone that were sequenced. solidbars labeled with Romannumeralsabove the maps represent exons; the exon labeled with an asterisk in MG62-1 (exon III) corresponds to the inserted sequence in the cDNA clone CC-1 (see Fig. 1B). Regions of overlap between clones are indicated by verticaldottedlines. The locations of the initiation and termination codons based on a comparison with PRDII-BF1 are indicted in MG10-1/MG18-1 and MG49-1, respectively. B indicates BamHI site. PanelB, intron/exon structure of the alphaA-CRYBP1 gene. Filledboxes correspond to protein coding exons; the openbox represents a 5`-untranslated exon; stippledboxes with Z and Aunderneath denote the locations of consensus zinc fingers and acidic domains, respectively. The cDNA clones CC-1 and pYTN8-1 that were used to isolate the genomic clones are shown aligned with the corresponding portions of the alphaA-CRYBP1 gene. The sizes of restriction fragments in EcoRI-digested mouse genomic DNA that should hybridize to each exon in CC-1 are shown below.



In addition to the clones shown in Fig. 3A, we obtained three other overlapping genomic clones that hybridized to the pYTN-8 insert (data not shown). The sequences of these clones are very similar to the 3`-end of alphaA-CRYBP1 but contain a number of missense and nonsense mutations that would occlude the production of a functional protein. Further analysis of these clones revealed the presence of a retroviral long terminal repeat 1,082 bp upstream of a termination codon homologous to that in pYTN-8(1) . Therefore, it appears that these three clones represent a partial or complete alphaA-CRYBP1 pseudogene.

Sequence Comparisons between alphaA-CRYBP1 and PRDII-BF1

The deduced amino acid sequence of alphaA-CRYBP1 was compared with that of PRDII-BF1 (Fig. 4). The extra peptide at the NH(2)-terminal region of alphaA-CRYBP1 was not included in this comparison (see Fig. 1B). The overall identity between the two protein sequences is 72%. Like PRDII-BF1(5) , alphaA-CRYBP1 contains two sets of consensus zinc fingers at each end of the protein as well as one central nonconsensus zinc finger. The zinc finger sequences are very highly conserved between mouse and human, the only difference being a single serine-threonine substitution in one of the carboxyl-terminal fingers. The amino- and carboxyl-terminal zinc fingers are both followed by a conserved stretch of acidic amino acids that probably represents a domain of interaction with other proteins (18) . A potential nuclear localization signal in PRDII-BF1 that was identified by Van't Veer et al.(19) is also present in alphaA-CRYBP1.


Figure 4: Alignment of the alphaA-CRYBP1 and PRDII-BF1 amino acid sequences. The protein sequences of alphaA-CRYBP1 and PRDII-BF1, derived from conceptual translations of DNA, were aligned using the algorithm of Needleman and Wunsch (45) as adapted by version 7 of the University of Wisconsin Genetics Computer Group sequence analysis software. Verticallines indicate identical amino acids between the mouse (M) and human (H) sequences. Amino acids corresponding to the inserted sequence in Fig. 1B (exon III) were deleted from the alphaA-CRYBP1 sequence prior to alignment. Boxes indicate the zinc finger domains identified by Fan and Maniatis(5) . Arrows indicate the locations of introns in the mouse alphaA-CRYBP1 gene, and the exons from which the alphaA-CRYBP1 peptide sequence is derived are indicated by Romannumeralsabove the mouse sequence. Boldunderlines define stretches of acidic amino acids flanking the zinc finger regions; a putative nuclear localization signal is also underscored with a thinnerline.



Northern Analysis of alphaA-CRYBP1 Transcripts

Previously, we showed that the 3`-alphaA-CRYBP1 cDNA clone, pYTN-8, hybridized to a single 9.5-kb transcript in RNA from newborn mouse lens, brain, spleen, thymus, and liver as well as RNA from alphaTN4-1 lens-derived tissue culture cells(1) . We have confirmed this result with the CC-1 5`-cDNA probe and showed also the presence of the 9.5-kb alphaA-CRYBP1 transcript in lung, kidney, skeletal muscle, and intestine (data not shown). In addition, there is a prominent 7.7-kb alphaA-CRYBP1 transcript that is found only in testis and not ovary (Fig. 5A). A control hybridization with a glyceraldehyde-3-phosphate dehydrogenase cDNA probe indicated that there was no generalized RNA degradation in the testis preparation (Fig. 5B). The rat homologue of alphaA-CRYBP1, AT-BP2, also produces this testis-specific transcript(4) , which can be detected using a cDNA probe corresponding exactly to the homologous sequence of pYTN-8(1, 4) . The fact that this RNA is not observed in ovaries (Fig. 5) indicates that the message is testis-specific rather than germ cell-specific. We also hybridized a blot containing total RNA from four different tissues of 2-day-old, 2-week-old, and adult rats with CC-1. These experiments revealed a single 9.5-kb transcript that is present throughout postnatal development (data not shown).


Figure 5: Northern blot analysis of alphaA-CRYBP1 message in adult mouse ovary and testis. A, a blot containing 20 µg of total RNA isolated from adult mouse ovaries (O) and testes (Te) was hybridized with the insert from the C(2)C muscle cell line cDNA clone, CC-1. B, the blot was reprobed with the human glyceraldehyde-3-phosphate dehydrogenase cDNA.



Analysis of alphaA-CRYBP1 cDNA Clones

To confirm that the coding sequences identified in the alphaA-CRYBP1 genomic clones are actually transcribed into mRNA, we isolated additional alphaA-CRYBP1 cDNA clones. Restriction fragments encompassing the entire alphaA-CRYBP1 putative ORF were used to screen cDNA libraries derived from the brain and testes of adult mice and from the alphaTN4-1 mouse lens-derived cell line. The cDNA clones that were isolated (Fig. 6) were sequenced either entirely or from each end. With the exception of two silent site substitutions, the protein coding sequences in the cDNAs are all identical to the genomic sequences, consistent with alphaA-CRYBP1 being encoded by a single copy gene ( (1) and see below).


Figure 6: alphaA-CRYBP1 cDNA clones. cDNA libraries made from RNA isolated from alphaTN4-1 mouse lens-derived cells, adult mouse brain, and adult mouse testes were screened with restriction fragments encompassing the protein coding regions of genomic clones MG4-2 and MG49-1 (Fig. 3A). The top of the figure depicts the locations of exons above a consensus alphaA-CRYBP1 cDNA. Note that exon III is present only in CC-1. Z/A indicates the location of consensus zinc finger/acidic domains; Z denotes the central nonconsensus zinc finger. cDNA clones isolated from the brain (MBC1-13), testis (MTC1-8), and alphaTN4-1 (p2A-5 and p4B-1) libraries are represented by diagonalstripes, verticalstripes, and solidblack, respectively, and are aligned with the consensus cDNA. Clone CC-1, isolated from C(2)C mouse muscle cells, and the initial alphaA-CRYBP1 cDNA clone, pYTN-8, are also shown. Sequences in cDNA clones that are not present in the consensus cDNA are indicated by openbars.



All of the cDNA sequences except for the testis clones MTC-1, MTC-6, and MTC-7 can be clearly aligned with the ORF of PRDII-BF1 and show no evidence of alternative splicing relative to the human cDNA. The 3`-end of MTC-1 and the 5`-ends of MTC-6 and MTC-7 diverge from the PRDII-BF1 sequence and correspond exactly to the intron between exons IX and X (Fig. 3) that has been spliced out of the other overlapping cDNAs. This sequence is flanked by consensus donor and acceptor splice sites (17) and contains multiple stop codons that would result in the production of a truncated protein if present in the mRNA (data not shown).

In addition to CC-1, we obtained two cDNAs (one of which was isolated seven times) from the brain that encompass the 5`-end of the alphaA-CRYBP1 gene. Both of the brain cDNAs lack exon III, which is found only in CC-1 (Fig. 1B and Fig. 3). Of the three 5`-cDNAs, CC-1 contains the shortest stretch of 5`-untranslated sequence, and this sequence is identical to that immediately preceding the initiation codon in the genomic clones MG10-1 and MG18-1 (Fig. 3). Comparison of the 5`-untranslated region of MBC-1 with the MG10-1 genomic sequence reveals the presence of a 3.3-kb intron located 104 bp upstream of the initiation ATG (Fig. 3). The sequence of the 5`-end of MBC-3,5,6,7,9,10,11 diverges from the genomic sequence downstream from this intron. The 5`-terminal sequence of this clone does not appear to be present in our genomic clones based on both DNA hybridization experiments and direct sequence comparisons. Furthermore, the point at which the genomic sequence diverges from that of MBC-3,5,6,7,9,10,11 does not correspond to a splice acceptor site (data not shown)(17) . Therefore, we believe that the unidentified 5`-terminal sequence of MBC-3,5,6,7,9,10,11 is a cloning artifact. The 5`-end of MBC-1 lies within a region of genomic DNA in MG10-1 that is extremely rich in guanosines and cytosines (data not shown). Attempts to verify the 5`-end of the alphaA-CRYBP1 message by primer extension have been unsuccessful, probably as a result of RNA secondary structures that are stabilized by these GC residues.

A comparison of the cDNA and genomic clones indicates that the alphaA-CRYBP1 gene comprises 10 exons (Fig. 2B). The cDNA clones show evidence for alternative RNA splicing involving the third exon, which is present only in CC-1, as well as differential splicing of the final intron, which is present in three of the testis cDNA clones but not in any of the other cDNAs that span this region. The introns separating the 10 alphaA-CRYBP1 exons are all bounded by consensus splice donor and acceptor sites (17) except for the intron that immediately precedes exon VII with the carboxyl-terminal zinc fingers (Fig. 3). In this intron, the splice donor sequence is ``GC'' rather than the consensus ``GT''.

The Effect of an alphaA-CRYBP1 Antisense Expression Construct on a Heterologous Promoter Containing alphaA-CRYBP1 Binding Sites

With knowledge of the alphaA-CRYBP1 gene structure in hand, we are ready to conduct functional studies on the role of alphaA-CRYBP1 in regulating gene expression and embryonic development. As an initial experiment, we attempted to interfere with alphaA-CRYBP1 production in tissue culture cells by transfecting them with an alphaA-CRYBP1 antisense construct. It has been shown for a number of eukaryotic genes that the expression of antisense RNA complementary to a particular mRNA will reduce the amount of the protein encoded by that message (see (20) for a review). To make an alphaA-CRYBP1 antisense construct, we ligated the CC-1 cDNA clone in reverse orientation downstream of the cytomegalovirus promoter in the vector pCDNA-1/NEO (Invitrogen). The resulting construct, pCCRXN-1, also contains the bacterial neomycin resistance gene linked to the Rous sarcoma virus promoter and should confer neomycin (G418) resistance to cells transfected with this construct(21) .

pCCRXN-1 was transfected into alphaTN4-1 mouse lens-derived cells, and stably transformed cell lines were established by selection with G418. To assay for reduced alphaA-CRYBP1 activity, the cells were transiently transfected with plasmids containing either one (PCS-15) or four (pCS-31) copies of the alphaA-CRYBP1 binding site placed upstream of the herpes simplex virus thymidine kinase promoter which, in turn, was fused to the bacterial CAT reporter gene. It was previously shown that these reporter constructs produced significant levels of CAT activity in transfected alphaTN4-1 cells and that the level of CAT activity was directly proportional to the number of alphaA-CRYBP1 binding sites present in the construct(14) . In the present experiments, we found that the pCCRXN-1 construct was not maintained intact in stably transformed lines of alphaTN4-1 cells. A likely explanation for this result is that the antisense construct was poisonous to the cells, and only cells with deletions in the alphaA-CRYBP1 portion of the plasmid could survive the G418 selection process.

To obviate the putative poisonous effect of the alphaA-CRYBP1 antisense construct, we measured CAT activity in alphaTN4-1 cells that were transiently cotransfected with pCCRXN-1 and either of the two reporter constructs pCS-15 or pCS-31. Fig. 7shows CAT activity levels normalized to beta-galactosidase activity from a cotransfected bacterial lacZ-expressing plasmid. In cells cotransfected with pTKCAT, a reporter plasmid lacking the alphaA-CRYBP1 binding site, there was almost no CAT activity irrespective of the presence of the antisense construct (pCCRXN-1). Cells transfected with the pCS-15, which has one alphaA-CRYBP1 binding site, had low levels of CAT activity when cotransfected with either the antisense (pCCRXN-1) or control (pCDNA-1/Neo) plasmid, although there is a slight but statistically significant reduction in CAT levels with the antisense plasmid. Cells cotransfected with pCS-31, which contains four alphaA-CRYBP1 sites, and the control plasmid (pCDNA-1/Neo) show almost 10 times as much CAT activity as the cells transfected with pCS-15. When cells were cotransfected with pCS-31 and the antisense-containing plasmid (pCCRXN-1), CAT activity was reduced by approximately 50%. Therefore, the alphaA-CRYBP1 antisense construct appears to have a significant inhibitory effect on the activity of a promoter in which the alphaA-CRYBP1 binding site is the only enhancer element.


Figure 7: alphaA-CRYBP1 antisense experiments. CAT activity levels normalized to beta-galactosidase activity in cotransfected alphaTN4-1 cells are shown for each set of transfections. alphaTN4-1 cells were transfected with 1 µg of the lacZ-expressing plasmid pCMVbeta (Clontech), 12 µg of either the pCS-15 or pCS-31 reporter constructs, and 20 µg of either pCDNA-1 or pCCRXN-1 (denoted as pCCR for simplicity). CAT activity is expressed in counts/min/beta-galactosidase activity. beta-galactosidase activity was measured in absorbance units at A. Each verticalbar represents an average of six separate transfections; the standard errors are indicated on each bar.




DISCUSSION

The genomic and cDNA clones that we have isolated indicate that the alphaA-CRYBP1 protein is encoded by a gene with 10 exons spanning greater than 70 kbp of DNA. Several other cDNAs have been isolated from rat, human, and mouse that contain zinc fingers very similar to those of alphaA-CRYBP1 and PRDII-BF1. The human cDNA, MBP-2, encodes a 2,500-amino acid protein with two sets of zinc fingers spaced comparably with those of PRDII-BF1(19) . However, MBP-2 lacks a central, nonconsensus zinc finger, and although there are patches of similarity between MBP-2 and PRDII-BF1 (Fig. 8), the overall sequence identity between these two proteins is only 33%. A partial cDNA clone, Rc-1, isolated from mouse cells encodes the carboxyl-terminal portion of a protein with one set of alphaA-CRYBP1-like zinc fingers adjacent to a conserved acidic domain, but the only similarity between the remainder of the encoded proteins of the two mouse cDNAs is confined to a stretch of 37 amino acids (22) (Fig. 8). Partial cDNAs isolated from rat (AT-BP1/AGIE-BP1) and human (KBP-1) appear to be homologous to MBP-2 and Rc-1, respectively(4, 23, 24) . Fig. 8also shows an alignment of the zinc finger sequences of alphaA-CRYBP1, PRDII-BF1, MBP-2, and Rc-1. The similarity between the zinc finger sequences in Fig. 8is reflected in the ability of the corresponding proteins to recognize the same DNA binding sites in gel mobility shift assays(1, 4, 5, 10, 21, 22, 23, 24) . However, each of these proteins preferentially binds to specific DNA sequences that differ for each protein. Possibly, several different mammalian proteins have evolved from an ancestral alphaA-CRYBP1-like protein, and during the course of evolution, these factors acquired subtle variations in DNA binding specificity and, perhaps, changes in their ability to interact with other transcription factors. Our genomic hybridization data suggest that homologues of these genes might be present in non-mammalian species. Structural and functional studies of alphaA-CRYBP1-like proteins from distantly related species may elucidate the utility of having multiple factors that recognize related DNA binding sites. Such studies may also reveal evidence for co-evolution between these factors and the gene promoters with which they interact.


Figure 8: Comparison of mouse and human cDNA clones that have related zinc finger sequences. The amino acid (a.a.) sequences of human clones MBP-2 and PRDII-BF1 and mouse clones alphaA-CRYBP1 and Rc-1 were aligned using the algorithm of Needleman and Wunsch (45) as adapted by version 7 of the University of Wisconsin Genetics Computer Group. The regions of highest similarity in the resulting alignments are indicated by the lines that connect the paired cDNAs. The numbers represent the percentage of amino acid sequence identity in each region. ZF/A indicates zinc finger/acidic regions; ZF indicates a nonconsensus zinc finger that is present in alphaA-CRYBP1 and PRDII-BF1 but not in the other two cDNAs. The zinc finger sequences from each clone are aligned at the bottom of the figure.



The intron/exon structure of the alphaA-CRYBP1 gene has several interesting features. The first is the presence of exon III that is found only in the one cDNA clone, which was isolated from the C(2)C muscle cell line library. The inclusion of exon III sequences in mRNAs would prevent the formation of a functional protein if the normal translation start site were utilized. There is another ATG, located further downstream, that is in the same ORF as the rest of the alphaA-CRYBP1 protein coding sequence. However, other studies have shown that reinitiation of translation at downstream ATGs usually occurs with very low efficiency(25) . Perhaps the inclusion of exon III sequences by alternative splicing serves to modulate the amount of alphaA-CRYBP1 protein without changing the level of mRNA. There are precedents for employing alternative exons as a means of down-regulating protein expression. For example, the Drosophila sex-lethal gene produces a male-specific transcript containing an in-frame stop codon that results in a non-functional protein only in male flies(26) , and a majority of the transcripts produced by the cH-ras gene contain an extra exon that leads to the production of a truncated, non-functional protein(27) . The C(2)C cell culture from which the cDNA library was constructed consisted largely of differentiating myoblasts. (^2)It has been shown that C(2)C cells undergo extensive changes in gene expression during differentiation into myotubes(28) , and it is possible that a reduction in alphaA-CRYBP1 levels may be necessary for affecting some of these changes.

The large size of alphaA-CRYBP1 exon V is another striking feature of the alphaA-CRYBP1 gene. The average vertebrate exon size is 137 nucleotides, and very few are greater than 600 nucleotides in length(29) . However, it has been recently shown that increasing the size of an exon in vivo does not necessarily interfere with RNA splicing(30) . In light of the belief that many genes have evolved by a process of exon duplication and shuffling(31) , exon V may represent the structure of an ancestral alphaA-CRYBP1 gene. The recruitment of the other smaller exons during the course of evolution might have occurred to alter or extend the functional role of the alphaA-CRYBP1 protein in regulating gene expression as postulated above for differentiating myoblasts.

It is also interesting that the second (3`) set of zinc fingers in alphaA-CRYBP1 is encoded by a separate exon, and the nucleotides corresponding to the acidic domain adjacent to these fingers are located on yet another exon. This structure imbues the alphaA-CRYBP1 gene with the potential flexibility to produce functionally distinct protein isoforms via alternative splicing of these two domains. In addition, the intron that immediately precedes the carboxyl-terminal zinc finger-containing exon (exon VII) has a nonconsensus 5`-splice site in which ``GC'' replaces the almost universal ``GT'' at this position(17) . The mouse alphaA-crystallin gene also has an intron with the same noncanonical 5`-splice site(32) . In the case of alphaA-crystallin, the intron is spliced inefficiently, resulting in two forms of the alphaA-crystallin protein that differ by the presence or absence of the exon that precedes the aberrant splice site.

None of the cDNA clones that we isolated showed evidence of differential splicing involving the zinc finger domains. There are multiple, cell type-specific forms of alphaA-CRYBP1 as judged by Western blots where an antibody specific for the carboxyl-terminal half of alphaA-CRYBP1 recognizes 50-, 90-, and 200-kDa proteins, which may involve alternative RNA splicing in those cell lines(9) . Moreover, Muchardt et al.(10) detected in human cells, using a sensitive polymerase chain reaction technique, rare PRDII-BF1 transcripts that contained only the amino- or the carboxyl-terminal zinc fingers. One of these transcripts results from a deletion in which a nucleotide corresponding to bp 13 of alphaA-CRYBP1 exon VI is joined to the sequence beginning with nucleotide 204 in alphaA-CRYBP1 exon IX. The other transcript contains a deletion corresponding exactly to exon V of alphaA-CRYBP1. These results also suggest that there is at least some conservation of exon structure between the mouse gene and its human homologue that has not yet been isolated.

The testis-specific transcript described for the rat AT-BP2 gene (4) is also produced by the alphaA-CRYBP1 gene in the mouse. A number of other mammalian genes produce different sized mRNAs that are detected only in the testis. These messages may arise from testis-specific sites of transcription initiation(33, 34, 35, 36) , polyadenylation(37, 38) , or alternative splicing(39, 40, 41) . In several cases, it has been demonstrated that the testis-specific transcript is produced specifically by spermatogenic cells(37, 40, 42) . We isolated a number of alphaA-CRYBP1 cDNA clones from a mouse testis library. Three of these clones were derived from mRNAs in which the last intron had not been removed. Since the testis-specific transcript observed on Northern blots is smaller than the ubiquitous alphaA-CRYBP1 transcript, there must be another difference between the two mRNAs that is not reflected in the cDNAs that we isolated.

The antisense experiments provide direct functional in vivo evidence that alphaA-CRYBP1 is involved with regulating gene expression via the promoter sequence GGGAAATCCC. Although the antisense construct produces a significant decrease in CAT activity, there is still a substantial amount of CAT activity remaining in the cotransfected cells. This residual CAT activity probably results, in part, from incomplete inhibition of alphaA-CRYBP1 by the antisense message, but it might also reflect the ability of other proteins to regulate transcription through this 10-base pair sequence. Besides the related zinc finger proteins mentioned above, NF-kappaB and members of the c-rel family of transcription factors bind to a consensus sequence (GGGRNNYYCC) that corresponds to the site in our reporter constructs(8) . Furthermore, the promoter sequence, PRDII, to which PRDII-BF1 binds in the human interferon-beta gene, is recognized by at least five different factors including NF-kappaB, PRDII-BF1, and HMG I(Y) (43, 44) . However, our experiments provide the first functional evidence that alphaA-CRYBP1 is at least one of the proteins that is involved in regulating gene expression using this sequence from the promoter of the mouse alphaA-crystallin gene.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L36825[GenBank]-L36829[GenBank].

§
Partially supported by a Human Frontiers Science Program research grant from the international collaborative program.

To whom correspondence should be addressed: Tel.: 301-496-9467; Fax: 301-402-0781.

(^1)
The abbreviations used are: alphaA-CRYBP1, alphaA-crystallin binding protein I; kb, kilobase(s); bp, base pair(s); kbp, kilobase pair(s); ORF, open reading frame; CAT, chloramphenicol acetyltransferase.

(^2)
R. Gopal-Srivastava, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Rashmi Gopal-Srivastava for providing the C(2)C muscle cell cDNA library.


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