The κB transcriptional enhancer motif and signal sequences of V(D)J recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study
© Allen et al; licensee BioMed Central Ltd. 2002
Received: 20 June 2002
Accepted: 22 August 2002
Published: 22 August 2002
The ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements. The amino-DNA binding domain (ZAS-N) and the carboxyl-DNA binding domain (ZAS-C) of a representative family member, named κB DNA binding and recognition component (KRC), were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing. The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains.
Both fusion proteins selected sequences that were similar to the κB motif or the canonical elements of the V(D)J recombination signal sequences (RSS) from a pool of degenerate oligonucleotides. Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer. In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS.
The RSS are cis-acting DNA motifs which are essential for V(D)J recombination of antigen receptor genes. Due to its specific binding affinity for RSS and κB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(D)J recombination.
The ZAS gene family is an emerging family of important transcriptional proteins that have been implicated in the regulation of gene expression of the HIV-1 long terminal repeat , and genes encoding αA-crystallin , somatostatin receptor type II , the small calcium binding protein S100A4/mts1 , and type II collagen  via specific promoter or enhancer elements. Three human genes, HIVEP1/Mbp1/PRDII-BF1 [6–9], HIVEP2/Mbp2 [10–12], and HIVEP3 , and their respective mouse counterparts αACRYBP1 , MIBP1 , and KRC [14, 15], as well as rat AGIE-BP1/MIBP1 [16, 17] have been cloned and characterized. In addition, a distant relative Schnurri (Shn) has been identified in Drosophila [18–20]. Although little is known about the physiological functions of the mammalian ZAS proteins, Shn has been shown to be an important transcription regulator during embryonic development. Shn modulates transcription by relieving the repression of the nuclear protein Brinker and, in association with SMAD, mediates transcription response of the decapentaplegic pathway [21, 22].
Each ZAS gene encodes large sequence-specific DNA-binding proteins with Mr >250,000 that contain two widely separated of C2H2-type zinc finger pairs. Smaller protein isoforms with a single zinc finger pair or with no zinc finger pairs can be generated by alternative RNA splicing [23, 24]. The amino acid sequence and relative location of the two zinc finger pairs are highly conserved among ZAS proteins from invertebrate to vertebrate [Reviewed in ]. Although the zinc finger is a major structural motif involved in protein-nucleic acid interactions and is present in the largest superfamily of transcription factors, few proteins contain separate zinc finger pairs. The ZAS proteins (with two zinc finger pairs), tramtrack (with one finger pair), and basonuclin (with three finger pairs), constitute a unique class of C2H2 zinc finger transcription factors. [Reviewed in ]. In addition, each ZAS protein contains a sequence similar to the serine stripe present in basonuclin, in which eight serines are located on one side of a putative α-helix [13, 27].
The ZAS domain is a protein structure unique to the ZAS protein family. A ZAS domain denotes a composite protein structure consisting of a pair of C2H2 z inc fingers, an a cidic region, and a s erine/threonine-rich sequence [15, 25]. Here, we name the amino-DNA binding domain ZAS-N and the carboxyl-DNA binding domain ZAS-C. The DNA binding specificity of the ZAS-N or ZAS-C domains from several ZAS members have been characterized by electrophoretic mobility shift assays, methylation interference experiments, and DNAse I footprinting experiments. The cumulative data show that individual ZAS domains bind a κB-like consensus sequence, GGGN(4–5)CC . However, mouse KRC, αACRYBP1 and mouse MIBP1 have also been shown to bind distinct DNA sequences. KRC binds to the signal sequences of V(D)J recombination (RSS) [14, 28]. αACRYBP1 binds to a sequence in the type II collagen gene enhancer . MIBP1 binds to a TC-rich element present in the somatostatin type II receptor gene enhancer .
This is the first study to evaluate DNA targets of both ZAS domains from a single protein independently. We used site selection amplification binding assays to select specific DNA targets recognized by KRC fusion proteins containing ZAS-N or ZAS-C from an initial oligonucleotide pool containing degenerate 25-mers. After cloning and DNA sequencing, the KRC-selected sequence datasets were analyzed by the computer program Multiple Expectation Maximum for Motif Elicitation (MEME version 3.0) to generate sets of position specific scoring matrices (PSSMs) or motifs . When the program was set to identify wider sequences (= 9 nucleotides) the PSSMs were homologous to Sb , the κB motif , and the canonical heptamer and nonamer elements of the RSS . However, shortening the width of the motifs to 5 nucleotides, the target length for the C2H2 zinc finger pairs of the tramtrack , the ZAS-N dataset yielded two motifs, "GGTAT" and "T(T/C)TT(T/G)G" and the ZAS-C dataset yielded a single motif, "TGTGG". Juxtaposition of the two pentamers of ZAS-N forms a sequence homologous to the canonical RSS nonamer, "GGTTTTTGT". Similarly, the ZAS-C pentamer together with its complement form the canonical RSS heptamer palindrome "CACTGTG".
The computer program Multiple Alignment Sequence Tool (MAST version 3.0)  was used to search human and mouse genome databases for DNA elements matching the KRC-selected PSSMs. The hits included sequence matching DNA regions located within or close to mobile genetic elements, including the diversity (D) gene segments of the variable region of the immunoglobulin (Ig) heavy chain , and break points of chromosomal translocation between Ig DH2-2 and the B cell lymphoma 1 BCL-1 gene , and between the ENL/MLLT1/LTG19 gene and myeloid lymphoid leukemia MLL gene .
Amplification of KRC's DNA targets with a site selection amplification binding assay
The formation of protein-DNA complexes was monitored throughout the site selection experiments (Fig. 1B and Fig). Analytical EMSAs were performed under more stringent conditions than in EMSAs used to purify protein-bound oligonucleotides in the site selection experiments, using much less fusion protein (~0.1 to 0.5 μg) and an excess non-specific DNA poly(dI-dC) (10 μg). Initially, the DNA-protein complexes formed between the degenerate oligonucleotide pool and KRC/ZAS-N or KRC/ZAS-C were barely detectable, indicating that both fusion proteins bound DNA selectively (Figs. 1B and 1C, lane 1). In the subsequent rounds, the yield of the DNA-protein complexes increased, suggesting successful enrichment of KRC binding sites in the recovered oligonucleotides during the selection procedures. After the fourth rounds of selection and amplification, no further increase in the amount of DNA-protein binding complexes was observed. The experiment, therefore, was stopped at the fifth round for both fusion proteins. Furthermore, in rounds four and five, a cluster of close migrating DNA-protein complexes were observed for KRC/ZAS-N (Fig. 1B, lanes 4 and 5). In EMSA, the gel mobility of DNA-protein complexes depends on the overall mass of the binding proteins  and on the possible protein induced bending angle of DNA . Since a single fusion protein was used in each binding reaction, the slight variation in the gel mobility of the DNA-protein complexes may reflect that KRC has more than one target, or that the targets were located at different positions within the 25-mer DNA. Similarly, two closely migrating DNA-protein complexes were clearly seen for KRC/ZAS-C at rounds three through five (labeled C, Fig. 1C, lanes 3, 4, and 5). In addition, another complex, labeled C', which was minor and had significantly slower gel mobility was observed in round four and round five (Fig. 1C, lanes 4 and 5). Previously, we showed that KRC/ZAS-C bound DNA as dimers, tetramers, and multiple of tetramers . The significant difference in the gel mobility between complex C and complex C' suggested that they were likely composed of KRC/ZAS-C dimer and tetramer, respectively. These data show that the site selection amplification binding assays using both KRC DNA-binding domains were efficient in selecting KRC targets and that KRC/ZAS-C readily formed highly ordered DNA-protein structures.
Motif discovery by MEME: ≥ 6 W ≤ 25 nucleotides
The fifty-three KRC/ZAS-N-selected sequences (ZAS-N dataset) and 49 KRC/ZAS-C-selected sequences (ZAS-C dataset) were first analyzed by the Motif Expectation Maximum for Motif Elicitation (MEME) computer program. MEME analyzes input sequences for similarities and produces a PSSM or motif for each pattern it discovers . We set the parameters of MEME as follows: (i) zero or one occurrences of a single motif per sequence; (ii) five as the maximum number of motifs to identify; (iii) 5 to 25 nucleotides as the range of motif size; and (iv) both DNA strands as input.
Motif discovery by MEME: ≥ 6 W ≤ 15 nucleotides
A motif of 25-nucleotides was obtained in the above MEME analysis when widths of ≥ 6 W ≤ 25 nucleotides were set. This is longer than known transcription factor binding sites. In addition, the information content (measured in bits), which reflects the degree of conservation of each column (or position) in those PSSMs was relatively low, ranging from 0 to 1.7, with an overall average <0.5 per position (Figs. 3 and 4). To elucidate more biologically relevant motifs with higher information content, a second pass of MEME was performed with motif widths set to shorter lengths, ranging from 6 to 15 nucleotides. The PSSMs discovered for each width all had significantly higher overall bits per position and obtained a TG-rich core sequence (data not shown).
A similar pass of MEME discovered three motifs in the ZAS-C dataset. The major motif had a consensus sequence: (A/T)-T-(T/A)-T-T-G-T-G-G, with a llr of 125 and an E-value of 1.5e-2 (Figure 7). This consensus sequence aligns with canonical RSS nonamer. Notably, the terminal 5 nucleotides, "T-G-T-G-G", each had an information content of >1.5 bits and were nearly invariant in that sequence alignment. With respect to the heptamer (canonical sequence: CACAGTG), the CAC sequence bordering the recombination site is the most conserved segment of the sequence , and mutation of these nucleotides has been found to decrease V(D)J joining in transfection assays using recombination substrates . Because the sequence of the canonical RSS heptamer is palindrome, we speculate that the TGTG sequence may be sufficient for KRC/ZAS-C binding. A second motif also contained a "TGTG" core sequence (Figure 8). A third sequence was homologous to the RSS nonamer, κB or Sb sequences (Figure 9). In general, PSSMs generated from MEME passes looking for shorter motifs yielded more conserved sequences.
Motif discovery by MEME: W = 5 nucleotides
KRC-bound sequences match RSS elements within endogenous antigen receptor gene segments
KRC was independently cloned due to its ability to bind the RSS  and the κB  motifs. Subsequently, sequence analysis identified KRC as a member of the ZAS family of proteins which share the ability to bind κB-like motifs . DNA competition analysis showed that KRC fusion proteins containing the ZAS-C domain bind specifically to both the RSS and to the κB motif [14, 28]. DNA footprinting analysis further showed that KRC/ZAS-C binds to specific nucleotides within the κB and the heptamer of the RSS . In this study, using a PCR-based DNA-binding site-selection and amplification procedure, we demonstrated that both the N-terminal ZAS-N and the C-terminal ZAS-C domains are able to bind GT-rich DNA sequences, and confirmed that the RSS and κB motifs are the high-affinity targets of KRC.
In the site-selection experiment, the increasing yield of DNA-protein complexes in successive rounds of DNA amplification and purification suggest that KRC/ZAS-N and KRC/ZAS-C bound DNA specifically. Conceivably, repetitive binding, selection and amplification should have selected increasingly specific KRC targets as increasingly stringent binding conditions were established. As far as we know, this is the first DNA site-selection study to employ the MEME program to identify target consensus sequence. The program has been conventionally used to identify conserved motifs in proteins. It was chosen as a DNA motif search tool in this study due to its flexibility in recognizing several patterns within a set of sequences. It was able to identify multiple motifs that could not be recognized by other alignment programs, such as Pileup (GCG Software Package, ) or Clustal W  which have been used in other site-selection experiments to identify a single consensus sequence. The oligonucleotide pool presented in this study was composed of a random 25-mer flanked by specific primers. A relatively large target was used to accommodate ligands with a range of potential sizes and also to minimize the influence of flanking primer sequences. Inspection of the sequence alignments shows that all of the KRC-bound oligonucleotides align in the (+) orientation, suggesting that orientation of binding may have been influenced by the flanking sequence. However, the flanking sequences were constant throughout the oligonucleotide pool, which should have controlled for their relative contribution to consensus sequence.
Using the ZAS-N- and ZAS-C-selected DNA sequences as input, the MEME program discovered motifs containing sequences similar to the κB or Sb transcriptional enhancer motifs as well as the conserved heptamer or nonamer elements of the RSS. Generally, the significance of the llr and E-values scores of a motif generated by MEME increased with its length whereas the information content per nucleotide position decreased (Figures 3,4,5,6,7,8,9,10,11,12). These are statistical values showing different parameters of a motif: llr and E-values reflect the likelihood of a motif being generated at random whereas the information content represents the degree of conservation. In our analysis, passes of MEME where the width was not set to a fixed length but to a given range of nucleotides always yielded the longest motif. The MEME program aims at generating motifs with the most probable occurrence, and the length of a motif may override other parameters in the algorithm. MEME is a useful tool with which to discover the best motif among DNA sequences provided the length is specified and determined experimentally.
Given that the crystal structure of the DNA-protein complex revealed that the C2H2 zinc finger pair of TTK, like the first two zinc fingers of DNA-Zif268, binds five base-pairs , we hypothesize that each zinc finger pair of KRC may also interact with five base-pairs. Passes of MEME for pentameric motifs yielded homologous TG-rich sequences for the KRC/ZAS-N and KRC/ZAS-C datasets: T(T/G)T(T/G)G and GGTAT for KRC/ZAS-N, and TGTGG/T for KRC/ZAS-C. We had previously shown by methylation interference analysis that KRC/ZAS-C bound specifically to the sequence TGTGG within the context of the canonical RSS heptamer plus the immediately flanking guanine . Because the pentamer motif for KRC/ZAS-C predicted by MEME completely matched with the empirical results, we conclude that the two pentameric motifs discovered by MEME are likely authentic binding sites for KRC/ZAS-N as well.
Based on the results here, we hypothesize that each DNA binding domain of KRC binds to pentameric TG-rich sequences. Two KRC binding sites when put together can form some longer known KRC targets. For example a copy of GGTTT and its complement can form a sequence GG(N5–6)CC, fulfilling the minimal DNA binding requirement for the ZAS proteins other than a lack of the 5' guanine . Similarly, the GT-rich RSS nonamer and the palindromic RSS heptamer can serve as binding sites of KRC. Furthermore, our hypothesis can explain why half-sites but not complete KRC targets were frequently found in the protein-selected datasets. Although previous results of protein titration experiments suggested that KRC/ZAS-C binds DNA in a cooperative manner for a given oligonucleotide, the presence of multiple binding sites might not be favored over a single site in our site selection assay which used a degenerate pool of oligonucleotides and limited rounds of amplification. The data suggest that both DNA binding domains of KRC are potentially capable of binding to either RSS heptamer or nonamer. Because the pentameric motifs derived from the KRC/ZAS-N dataset more closely resemble the canonical RSS nonamer and the motif derived from the KRC/ZAS-C dataset more closely resemble the canonical RSS heptamer and a canonical sequence was derived from the majority of sequences, we propose that the ZAS-N domain of KRC binds RSS nonamers more frequently than the ZAS-C domain, and vice versa for the RSS heptamer in vivo.
Our results suggest that KRC binds with individual endogenous RSS elements and transcriptional enhancer motifs. As the most abundant RSS-binding species detected in thymus , it is intriguing to propose a role for KRC in regulation of the V(D)J recombination process. Several studies have shown that the RSS themselves may act as cis-acting elements which influence recombination frequency [51–56]. Furthermore, affinity of KRC for the RSS has been shown to vary inversely with activation of the catalytic components of the V(D)J recombinase, RAG1 and RAG2 . It is possible that differential affinity of KRC for individual RSS influences RSS utilization by the recombinase, allowing differential recombination of gene segments.
Our finding that KRC binds to the RSS as well as the κB motif may also provide a link between transcription and recombination in the context of the accessibility model [58, 59]. Enhancer or promoter elements are important for the recombination process in cell lines and animal models . Similarly, expression of transcription factors, in conjunction with the recombination activating genes, has been shown to induce V(D)J recombination in non-lymphoid tissues by rendering RSS accessible to the recombinase . The κB motif, first found in the Igκ light chain , and later in the TCR β2 locus , has been shown to promote V(D)J recombination by modulating locus accessibility . In addition to influencing recombination by binding of RSS, KRC binding of the κB motif may modulate accessibility and transcription of target loci. The ability of KRC to promote transcription of target genes has been demonstrated for the S100/mts1 gene by binding at the Sb enhancer motif . Similarly, binding of κB-like motifs by other ZAS proteins has also been implicated in transcriptional regulation [1–3, 17]. Considering KRC's target sequences, the κB motif and the RSS, the two binding domains on a single KRC protein could theoretically bring together cis-acting DNA elements for gene regulation, V(D)J recombination, or both. Such a molecule could coordinate transcription of individual promoter or enhancer elements, and/or could physically connect different cellular machineries via distinct DNA elements. KRC could provide a link between the fundamental processes of DNA transcription and V(D)J recombination.
Oligonucleotides were synthesized chemically (Life Technologies, Rockville, MD. BSS1: 5'-GACGGTATCGATAAGCTT-3'; BBS2: 5'-CCGGGCTGCAGGAATTC-3'; and BSS4: 5'-GACGGTATCGATAAGCTT(N)25GAATTCCTGCAGCCCGG-3' where N is A, T, C, or G.
The fusion proteins KRC/ZAS-N and KRC/ZAS-C were produced in E. coli and purified by affinity chromatography as described previously [24, 28]. The regions of KRC used to generate KRC/ZAS-N and KRC/ZAS-C are schematically shown in Fig. 1A.
Site selection amplification binding assay, DNA cloning and sequencing
Site selection amplification binding assay was performed as described  with modifications. In the first DNA-protein binding reaction, [32P]-labeled double stranded oligonucleotides were generated by first annealing BSS2 (50 ng) to the BSS4 oligonucleotide (500 ng) then end-filling with 250 μM each of dATP, dGTP, and dTTP, 50 μCi of 32P-dCTP and Klenow. The oligonucleotide pool (~500 ng) was incubated with KRC/ZAS-N or KRC/ZAS-C (100 μg each) and 10 μg of non-specific competitor DNA poly(dI-dC). DNA-protein complexes and free DNA were resolved on a 5% polyacryamide gel. After autoradiography, DNA-protein complexes were isolated from the gels and were eluted from the gel slices by incubation in 1 ml of 10 mM Tris (pH 8.0) and 1 mM EDTA at 42°C for 4 hours. DNAs were purified by phenol/chloroform extraction, followed by alcohol precipitation, then were amplified by PCR using the BSS1 and BSS2 primer set. Subsequently, a portion of the DNA was labeled with [32P]dCTP and used for the next round of site-selection. After the first round, the stringency of each succeeding round of site selection was increased by using successively less (0.5×) fusion proteins and more (4×) non-specific competitor DNA. Protein-bound oligonucleotides from the fifth round of selection were purified and subcloned into plasmid vectors pCR 2.1 (Invitrogen, Carlsbad, CA). Plasmid DNA was prepared from cohorts of bacteria colonies using a kit (Qiagen, Carlsbad, CA). The nucleotide sequences of the inserts were determined using automated DNA sequencing procedures performed by the DNA Sequencing Core Facility at the Ohio State University.
Sequence analysis was performed using the computer programs MEME (version 3.0)  and MAST (version 3.0) . The data of both programs were processed on the Cray T3E supercomputer at the San Diego Supercomputer Center accessed through the Internet: MEME http://www.sdsc.edu/meme. For MEME, the free parameters of the analysis were set as the following: (i) the occurrences of a single motif distributed among the sequences were zero or one per sequence; (ii) the maximum number of motifs to find was five; (iii) the optimum width of each motif ranged from 3 to 25 nucleotides; and (iv) both strands of DNA were searched. For MAST, only MEME PSSMs with an E-value < 1 were presented, and the reverse complement DNA strand was considered with the forward orientation in the search.
List of abbreviations
Motif Expectation Maximum for Motif Elicitation
Multiple Alignment Search Tool
position specific scoring matrix
electrophoretic mobility shift assay
T cell receptor
log likelihood ratio
This research was supported in part by grant GM48798 (LCW) from the National Institutes of Health and by grant P30 CA16058 from National Cancer Institute. CEA was funded by a T-32 pre-doctoral fellowship (National Cancer Institute, Bethesda, MD). We thank Dr. Michael Gribskov for assistance with the MEME and MAST analysis.
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