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The Thymocyte-Specific MAR Binding Protein, SATB1, Interacts in Vitro with a Novel Variant of DNA-Directed RNA Polymerase II, Subunit 11
doi:10.1006/geno.2002.6772, available online at http://www.idealibrary.com on IDEAL
Article
The Thymocyte-Specific MAR Binding Protein, SATB1, Interacts in Vitro with a Novel Variant of DNA-Directed RNA Polymerase II, Subunit 11 Linda K. Durrin and Theodore G. Krontiris* Division of Molecular Medicine, Beckman Research Institute of the City of Hope National Medical Center, Duarte, California 91010, USA *To whom correspondence and reprint requests should be addressed. Fax: (626) 301-8862. E-mail: [email protected]. Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession number AF468111.
A yeast two-hybrid screen of a Jurkat (T cell) derived cDNA library, using SATB1 (a matrix attachment region binding protein) as the bait, yielded four independent isolates of a novel variant of the DNA directed RNA polymerase II, subunit 11 (RPB11). Absence of lysine-17 from the amino terminus of this variant cannot be explained by alternative mRNA splicing. Instead, allele-specific PCR, combined with GenBank database searches, suggests that a recent gene duplication event has resulted in distinct loci encoding three variant forms of RPB11. Differential splicing of mRNA transcripts accounts for unique carboxy termini among the RPB11 proteins. The exclusive association of SATB1 with one form of RPB11 is influenced primarily by the N-terminal amino acid disparity, as deletion of the entire C terminus does not alter interaction affinity. Association of RPB11 with SATB1 maps between amino acids 58 and 222 of SATB1, a region that includes a PDZ-like dimerization motif. Key Words: SATB1, POLR2J family, protein interaction
INTRODUCTION SATB1 was originally identified as a matrix or scaffold attachment region (MAR/SAR) binding protein [1]. Eukaryotic chromosomes are typically arranged as 50- to 100-kb loop domains that are attached at their bases (MARs) to the intranuclear framework by nonhistone proteins [2,3]. MARs define the borders between chromatin domains, each of which harbors a gene, functional segment of a gene, or gene cluster [4]. MARs colocalize with transcriptional enhancers, promoters, and origins of replication [5,6]. The nuclear matrix near MARs may serve as a “sink” for organization of the proteins that regulate transcription, replication, RNA processing, recombination, and DNA repair [4]. SATB1 is a 95-kDa phosphoprotein expressed predominantly in thymic lymphocytes and pre-B cells [1]. A 150amino-acid MAR binding domain is centrally located within SATB1 [7,8]. At the carboxy-terminal region, an atypical homeodomain binds poorly and with low specificity to DNA; however, it assists the SATB1 MAR-binding domain specifically to recognize unwinding elements (for example, 5C/ATAATA-3) [9]. Two Cut-like repeats do not exhibit functional significance at MARs [9]. A dimerization domain with similarity to a PDZ motif has been identified within the amino
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terminus of SATB1 [10]. This motif is required for SATB1 interaction with MAR elements [10]. SATB1 interacts preferentially with double-stranded, AT-rich sequences (AT stretches interspersed with C’s but lacking G’s) that have a propensity to unwind in supercoiled plasmids [1]. SATB1 specifically recognizes MAR elements near CD8A [11] and HBG1 [12], the MAR 3 of the mouse and human IGH@ enhancer [1,13], and the MAR near the human BCL2 major breakpoint region [14]. Furthermore, SATB1 has been found associated with the nuclear matrix at the bases of many unidentified chromatin loops [15]. SATB1, in association with MARs, acts as a transcriptional repressor [16,17]. Recently, SATB1-null mice were generated and found to display dysregulation (mostly derepression) of up to 2% of the genes in thymocytes [18]. SATB1 apparently regulates gene expression at specific times and places during T-cell development. DNA-directed RNA polymerase II (pol II) is the fundamental enzyme involved in all aspects of eukaryotic mRNA synthesis. In association with additional, multi-subunit complexes, pol II recognizes specific promoters, transcribes the DNA into a fully processed message (capped, spliced, cleaved, and polyadenylated) and has a role in the regulation of these processes. As such, it is a focal point in cellular processes from
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FIG. 1. POLR2J and transcribed splice variants. The boxes (top) represent exons, with the filled and open regions indicating coding and noncoding domains, respectively. The line delineates intronic sequences. The bent arrow indicates the transcriptional start site. The RPB11B1 family is encoded at a locus on chromosome 7 that is differentially spliced at the C terminus, as shown (top). RPB11B1 (red solid line) and RPB11B1 (blue dashed line) are encoded by three, and RPB11B1 (brown dotted line), by four exons within approximately 6-kb of genomic sequences. Both RPB11-A and RPB11a are encoded by four exons and the first three exons are similar to those shown for the RPB11B1 family. These two proteins differ by alternative splicing of the third to the fourth exon, such that RPB11a encodes an extra 10 amino acids. An allelic variation near the first splice junction encodes an additional lysine in the RPB11-A family but not in the RPB11B1 family. At the bottom are shown splice variants of RPB11 that have been deposited in GenBank (acc. nos. L37127 and X98433 (RPB11-A; gene POLR2J1), AJ277928–AJ277930 and AJ277932 (RPB11a; gene POLR2J1), AJ277739 (RPB11B1; gene POLR2J2), AJ277740 (RPB11B1; gene POLR2J2), and AF468111 (RPB11B1; gene POLR2J2)). Alternative C termini are shown in color corresponding to the diagram at top.
differentiation to apoptosis. Human pol II is composed of 12 subunits ranging in size from 220 to 10 kDa [19]. Subunits RPB1, RPB2, RPB3, and RPB11, equivalent to the prokaryotic , , , and subunits, respectively, have roles in assembly of the holoenzyme [19–22]. The RPB3–RPB11 heterodimer nucleates assembly of pol II, by binding RPB2 and subsequently RPB1 to form a complete core enzyme. Other subunits assemble on this platform to form the catalytically active holoenzyme. In addition to their structural and catalytic roles, many of the subunits have regulatory roles through contact with DNA or heterologous proteins. The bacterial -subunit interacts with transcriptional regulators, which influences polymerase activity [reviewed in 23]. SATB1 has the potential to affect many cellular processes because of its interaction with specific sequence contexts at the bases of chromosomal loop domains. We therefore used the yeast two-hybrid assay to identify proteins, encoded by a Jurkat cDNA library (human T-cell leukemic cell line), that interact with SATB1. Among the distinct proteins isolated (L.K.D. et al., manuscript in preparation) were multiple independent clones of a unique variant of pol II, subunit 11 (RPB11B1). Comparison of this variant with the previously described RPB11 (RPB11-A) suggests that SATB1 recognizes structural differences in pol II, subunit 11, that may have functional significance.
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RESULTS Yeast Two-Hybrid Screen The MAR-binding protein SATB1, deleted of 57 N-terminal amino acids (SATB158–763) was used as bait in the yeast twohybrid assay to identify SATB1-interacting proteins from the T-cell derived Jurkat cell line. Of 92 positive clones identified, 7 encoded isolates of a novel variant of RNA polymerase II, subunit 11 (RPB11). Most of these yeast two-hybrid clones encoded the full-length RPB11 variant; only one lacked two N-terminal residues. Additional SATB1-interacting proteins encoded by Jurkat cDNA clones are discussed elsewhere (L.K.D. et al., manuscript in preparation). Here, we refer to the published form of RPB11 as RPB11-A [19,24] and the new clone as RPB11B1 as it seems to be a splice variant of one member of the POLR2J family (Fig. 1). These two RPB11 proteins differed by a single amino acid in their N termini (residue 17 was a lysine in RPB11-A but deleted in our clones) and extensively at the C terminus; RPB11-A and RPB11B1, respectively, diverged after the arginine at position 106/105, and encoded an additional 11 or 53 non-homologous residues (Fig. 1). A second published variant was identical to RPB11B1 at the N terminus except for an isoleucine in place of a lysine at position 18, but it
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FIG. 2. Genomic sequences encode variant forms of RPB11. Genomic sequences encoding POLR2J apparently can be transcribed into three alternative forms of exon 1 (top). Only two of these have sequences required for efficient translation (GenBank acc. nos. AC004084, AJ277931, and AJ277932), whereas transcripts from the last gene (GenBank acc. no. AC004951, NT_007766) might be translated less efficiently [28]. Gene sequences encoded identical amino acids for exon 2. Two versions of exon 3 were encoded by the gene loci. Amino acids are indicated below the nucleotide triplets and a dashed line indicates a deletion at residue 17 (top) or within the third exon (bottom). Intronic sequences are shown as lowercase letters. Amino acids that differ among the genes are shown in bold. The consensus sequences of the 5 and 3 splice junctions are indicated (the nearly invariant gt or ag is boxed and additional nucleotides essential for efficient message splicing are underlined).
variant of RPB11 was thus notable and may have reflected absence of RPB11-A and other variants in the Jurkat cDNA library, sampling limitations, or truly restricted recognition with possible functional significance. differed at the C terminus [25]. This C terminus appeared to be a cloning artifact as it encoded an internal cDNA fragment, in reversed orientation, of growth factor receptor-bound protein-2 (GRB2; GenBank acc. no. M96995) [19]. More recently, three additional variant cDNAs were deposited into GenBank (RPB11a, RPB11B1, RPB11B1; Mark Vigneron et al., unpublished data; Fig. 1). A search of GenBank cDNAs and expressed sequence tags (ESTs) further identified three homologous cDNAs that lacked a classical (ATG) translation initiation site (GenBank acc. nos. BC017250, AL526460, and AL554541). The exclusive interaction of SATB1 with a single
POLR2J Family and Splice Variants The N-terminal difference between RPB11-A and RPB11B1 (presence or absence of lysine-17) occurred near the mRNA splice junction of exon 1 but could not be explained by alternative splicing (Fig. 2, top). Instead, either a single gene encoded allelic variation at this site or, alternatively, multiple genes encoded the RPB11 variants. Examination of genomic sequences deposited in both GenBank and the high-throughput genomic sequence (htgs) databases identified three distinct configurations for the first exon, and two for the third exon, of POLR2J (Fig. 2). In addition to the
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FIG. 3. Allele-specific PCR within exon 1/intron 1 of the POLR2J loci distinguishes three genetic loci. (A) Genomic and EST sequences from GenBank were examined to identify gene/allele-specific differences between RPB11-A, RPB11B1, and a third variant. Primers were designed to distinguish between alleles and an additional alteration was incorporated into the penultimate nucleotide to increase specificity. Primer pairs were used in all combinations (A/C, A/D, B/C, and B/D) in a PCR assay. Products were sequenced to confirm specificity: A/C, RPB11-; A/D does not prime efficiently and produces non-specific products; B/C, RPB11-A; and B/D, RPB11B1. (B) Example of products analyzed on a 3% NuSieve GTG agarose gel. The arrow indicates the 97-bp RPB11-A or RPB11-, and the 96-bp RPB11B1 products. Product sizes were determined by fractionating a 100-bp DNA ladder in an adjacent lane. The control reactions (minus genomic DNA) gave no products.
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Lys-17 variations, the translation initiation site was mutated on one gene/allele to a less commonly used GTG translation initiating triplet (valine; GenBank acc. no. NT_007766). In this same clone, sequence differences within the third exon (Fig. 2, bottom) specified an extra cysteine and replaced a leucine with an aspartic acid. Thus, initial examination of POLR2J sequences suggested that separate genetic loci encoded members of a POLR2J family; however, allelic variation was not ruled out. Therefore, to distinguish between genetic and allelic variations, PCR amplification primers were designed that specifically amplified the first exon and the 3 portion of intron 1 from genomic DNA samples (Fig. 3A). The 5 primers (A and B) differentiated between a C/A disparity at nucleotide position –10 (relative to the translation initiation site) that correlated with the initiating ATG (C) or GTG (A) (Fig. 3A, single arrow). The 3 primers (C and D) discriminated between the presence and absence of lysine-17 (Fig. 3A, three arrows). Primer pair B/C amplified sequences corresponding to RPB11-A (97 nt), primers B and D specifically amplified RPB11B1 (96 nt), and the primer pair A and C amplified a third variant (97 nt; designated RPB11-). Primers A and D primed nonspecifically and only large, unidentified products were observed (data not shown). A total of 20 patient DNA samples (40 chromosomes) were analyzed and the above three products were observed in all instances (Fig. 3B). This assay detected alleles present at a frequency of 10% or greater (90% confidence level) [27]. Thus, we identified a POLR2J gene family that consisted of at least three distinct loci
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FIG. 4. Quantification of RPB11-A and RPB11B1 message by the ribonuclease protection assay (RPA). (A) The thick lines depict the cDNAs encoding RPB11-A (top) and RPB11B1 (bottom). The hatched line at the end of RPB11B1 represents 3 UTR sequences included in the construct. Thin lines are vector sequences that contain a T7 promoter. RPB11-A was digested to completion with Sau3A (upstream Sau3A site is for reference only) and RPB11B1 was digested with BseRI, which precisely separated nearly conserved sequences (upstream) from unique sequences (downstream). The 185-nucleotide RPB11-A probe protected the 145-nucleotide RPB11-A-specific RNA, and it may protect a 113-nucleotide RPB11B1-specific RNA (not detected). The 450-nucleotide RPB11B1 probe protected a 420-nucleotide RNA. (B) Autoradiogram of RPB11-A, RPB11B1, and GAPDH protected fragments. Total RNA (20 g) was hybridized with an excess of the probe indicated, before analysis of the nuclease-protected fragments on a 6% denaturing polyacrylamide gel. The lanes marked + and – are an aliquot of the probe either treated or untreated, respectively, with ribonuclease. Cell lines from which the RNA was prepared are indicated. Exposure was for 24 hours on Kodak Biomax MR single emulsion film.
encoding RPB11-A (and RPB11a; Fig. 1), RPB11B1 (also RPB11B1 and RPB11B1; Fig. 1), and finally, a polymorphic third variant (RPB11-; Fig. 2). Comparison of POLR2J Family Member Message Levels Previous work has shown that POLR2J message is present in all tissues examined, including the thymus [24]. We used the ribonuclease protection assay to confirm and quantify RPB11A and RPB11B1 message levels in Jurkat, and also in HeLa and MCF-7, cell lines. Probes were prepared (Fig. 4A), and the assay performed as described in Materials and Methods. The control GAPDH mRNA was easily detected within 1 hour of exposure and low levels of RPB11-A message also were observed at this time (data not shown), however, transcripts encoding RPB11B1 were detected only after 24 hours of exposure (Fig. 4B). The RPB11-A probe also should have protected RPB11B1 mRNA, but a combination of degradation products and the low abundance of the RPB11B1 message made it impossible to detect it with this probe, even after 24 hours of exposure. Specific in Vitro Association of RPB11B1 with SATB1 To investigate the specificity of interaction of RPB11B1 with SATB1, we used the GST pull-down and yeast two-hybrid -galactosidase assays. RPB11B1 and RPB11-A were prepared in vitro as downstream fusion proteins with GST and then incubated with 35S-methionine-labeled SATB1. Fulllength and truncated SATB1 used in these experiments are
GENOMICS Vol. 79, Number 6, June 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
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Article FIG. 5. GST pull-down assays demonstrate preferential interaction of RPB11B1 (over RPB11-A) with SATB1. Full-length and deletion constructs of SATB1 are shown in Fig. 6 (marked a, b, c, and d on the left side, (e) is similar to (a) but deleted of 57 N-terminal residues). SATB1 and luciferase were labeled with [35S]-methionine in an in vitro TNT coupled transcription/translation system. An aliquot of each protein, equivalent to the amount used for the pull-down assay, is shown in the lanes marked a–f, only. These test proteins were incubated with GST–RPB11B1, GST–RPB11-A, or GST alone (labeled at top of autoradiogram) and subsequently fractionated on 6% denaturing polyacrylamide gels, dried, and exposed for 2 days. Lanes are as follows: a, SATB11–763; b, SATB11–677; c, SATB11–354; d, SATB11–222; e, SATB158–763; and f, luciferase.
illustrated in Fig. 6 (constructs marked with the letters a–d to pB42AD. After transformation of each construct, along with their immediate left; constructs a and e are equivalent except the reporter construct p8op-lacZ, into appropriate yeast, the for deletion of the N terminus of construct e). We loaded an cells were induced and assayed for interaction of the RPB11 aliquot of the input SATB1 (Fig. 5, lanes a–e) equivalent to the variant with SATB1 (Fig. 6). RPB11B1 associated with amount used in the GST pull-down assay. In adjacent lanes SATB158–763, SATB158–677, SATB158–576, SATB158–457, SATB158–354, is shown the SATB1 that specifically associated with the bait, SATB158–289, and SATB158–222, but not with SATB1223–763. This GST–RPB11B1, GST–RPB11-A, or GST alone (a control for precisely mapped its interaction with SATB1 to a domain nonspecific interaction of SATB1 with the bait). Full-length between amino acids 58 and 222 in the N terminus, which and truncated SATB1 (SATB11–763, SATB11–354, SATB11–222, and agreed with the results from the GST pull-down assay. RPB11-A, on the other hand, did not associate with SATB158–763) interacted significantly (at least tenfold) more strongly with RPB11B1 than with RPB11-A or GST (Fig. 5, SATB158–222 or SATB158–763 to any significant extent, although lanes a, c–e). SATB11–677 interacted only about twofold more strongly with RPB11B1 than with RPB11-A (Fig. 5, lane b), a discrepancy that may be attributable to some property of SATB1 because this result is also observed when interaction strength is measured with the yeast two-hybrid -galactosidase assay. a Possibly, the intact SATB1 C terminus medib ates protein folding that is critical in the regulation of the physiological interaction of RPB11B1 and SATB1. Deletion of this domain may permit adventitious interactions to occur. c Luciferase served as a control for nonspecific association of RPB11 with the test proteins. As expected, no interaction of RPB11B1, RPB11d A, or GST with this control was observed (Fig. 5, lane f). These in vitro results were confirmed using the cell-based, yeast two-hybrid -galactosidase assay. SATB1 deletion constructs, similar FIG. 6. Yeast two-hybrid -galactosidase assay quantification of interaction between SATB1 and RPB11 constructs. Functional domains of SATB1 are illustrated at the top as the dimerization domain to those used for the GST pull-down assays (PDZ motif), MAR binding domain (MAR-BD), two Cut repeats, and the homeodomain. A stretch (except deleted of the 57 N-terminal amino of 15 glutamines (Q15) may have transcriptional significance. The N- and C-terminal truncated pepacids, which are toxic in eukaryotic cells [16]), tides of SATB1 were prepared as fusions with the LexA DNA-binding domain. The numbers at the were prepared as fusions to the lexA DNA C terminus depict the last residue included in the peptide (all polypeptides initiated at amino acid 58 except where noted). Constructs encoding these SATB1 proteins were introduced, along with binding domain in the yeast vector pLexA. RPB11B1, RPB11-A, or RPB11-A/CT (encoded as fusions of the bacterial activation domain in cDNAs encoding full-length RPB11B1, pB42AD), into a yeast strain harboring the p8op-LacZ reporter construct. After induction for 4 RPB11-A, or RPB11-A/CT (RPB11-A deleted hours in galactose-containing selective medium, -galactosidase activity was measured (YPH). All of its non-homologous C-terminal amino values were normalized by setting the two-plasmid positive control equivalent to 1000 units activacids) were prepared as fusions with the bac- ity. The striped oval between SATB1 amino acids 58 and 222 represents the interaction site of RPB11B1 with SATB1. terial activation domain in the yeast vector
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interaction with SATB158–677 and SATB158–354 was above background (negative controls were between 15 and 22 units activity). These adventitious levels of interaction (see above for GST pull-down assays) were 14–25% of the activity measured for the RPB11B1 counterparts. Role of the C Terminus of RPB11B1 in SATB1 Recognition Because RPB11B1 and RPB11A differed significantly at their C termini, and by the presence or absence of lysine-17, we sought to investigate which of these differences resulted in altered binding affinity of the variants for SATB1. Initially, we hypothesized that the extensive differences at the C termini had a greater chance of altering binding affinity. A series of deletions of the C terminus of RPB11B1 were prepared as GST fusion proteins (Fig. 7B). The most extensive of the deletions, RPB11B11–105, eliminated all C-terminal sequences that differed from RPB11-A. The BseRI restriction enzyme site was used to precisely divide the N and C termini of the two forms of pol II, subunit 11). We used two [35S]-methionine labeled SATB1 constructs, SATB11–763 and SATB11–222, as representative test proteins in the GST pulldown assays. Deletions of 15, 33, or 53 amino acids from RPB11B1 (Figs. 7B and 7C) did not decrease its affinity for either of the test constructs (Fig. 7A, lanes a or d). None of the constructs interacted significantly with the [35S]-Metluciferase control (Fig. 7A, lane f). We also tested, in a yeast two-hybrid -galactosidase assay, whether deletion of the C terminus of RPB11-A (amino acids 107–117) significantly increased its association with SATB1, a result we would expect if differences in the C terminus were responsible for inhibiting RPB11-A binding. RPB11-A/CT did not interact with either SATB158–763 or SATB158–222 (Fig. 6, right column). Thus, the C terminus of RPB11B1 was not the region conferring binding specificity to SATB1.
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FIG. 7. The C terminus of RPB11B1 is not required for its interaction with SATB1, in vitro. (A) Full-length SATB11–763 and one deletion, SATB11–222, were used as test proteins (Fig. 6). They and the control luciferase were labeled with [35S]-methionine and interacted with GST–RPB11B1 as described in Fig. 5. Positions of the test proteins are indicated in the autoradiograph. The first two lanes and the last lane contain an aliquot of the input proteins, only. The lanes are marked as described in Fig. 5 (a, d, or f) and also according to the GST or GST fusion protein used for each pull-down assay. (B) A series of constructs encoding C-terminal truncations of RPB11B1 was prepared as GST fusion proteins. The N terminus, which is conserved (except for lysine17), is depicted as a filled line. The non-conserved C terminus is shown as a hatched line. The BseRI restriction enzyme site precisely separates the two domains. RPB11B11–158 is full-length, whereas RPB11B11–143, RPB11B11–125, and RPB11B11–105 are deleted of 15, 33, and 53 residues, respectively. (C) Representative GST or GST-RPB11B1 fusion protein, fractionated on SDS-PAGE gels and stained with Coomassie blue, were used in the above assays. Sizes are given in kDa.
We next examined whether the N-terminal difference (presence or absence of a lysine at position 17), either alone or in association with the C terminus, influenced recognition of SATB1 by RPB11B1. Accordingly, we prepared chimeric molecules in which we interchanged the C termini between RPB11-A and RPB11B1 (Figs. 8B and 8C). These GST fusion proteins were incubated with the representative [35S]-MetSATB11–222, and interacting protein was analyzed on denaturing gels. The controls, full-length RPB11B1 and RPB11A, behaved as observed previously (Fig. 8A). RPB11B1 associated with almost 100% of the test protein and RPB11A with an insignificant amount. Because the C terminus of RPB11B1 was dispensable for its interaction with SATB1, we expected that the chimera RPB11B1/A also would associate with SATB1. This was observed, although the interaction was weaker, with approximately 50% of the test protein interacting (Fig. 8A; signals are overexposed, making it difficult to observe the true differences quantitated by PhosphorImage analysis). Finally, the RPB11-A/B1 chimera was not expected to associate with SATB1 if the Nterminal alteration was solely responsible for recognition of the test protein. RPB11-A/B1 interacted with approximately 20% of the input protein (Fig. 8A). We concluded that both N- and C-terminal domains of RPB11B1 contributed to its interaction with SATB1, although the N terminus seemed to have an influential role.
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FIG. 8. Presence or absence of Lys-17, in association with the C terminus, determines the strength of interaction of RPB11 with SATB1, in vitro. (A) Only the single truncation SATB11–222 and the luciferase control were used for this assay. They were labeled in an in vitro transcription/translation system with 35 S-methionine and incubated with GST–RPB11B1, GST–RPB11-A, GST–RPB11B1/A, RPB11-A/B1, or GST. The interacting SATB1 was analyzed as described for Fig. 5. The input protein is indicated (first and last lanes) and lanes are marked as described in Fig. 5 (d or f) and also according to the GST or GST fusion protein used for each pull-down assay. (B) Chimeric molecules, in which the N terminus of RPB11-A is switched with the C terminus of RPB11B1, and vice versa, were prepared as GST fusion proteins. The N terminus of RPB11-A is depicted as a filled line, RPB11B1 as a hatched line. As noted previously, the BseRI restriction enzyme site precisely separates the two domains. (C) Representative GST or GST–RPB11 chimeric fusion proteins, fractionated on an SDS-PAGE gel and stained with Coomassie blue, were used in the above assays. Sizes are given in kDa.
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DISCUSSION As a MAR binding protein with established roles in repression and activation of gene expression [11,16,17,19,26], SATB1 might be expected to associate with components of the transcription machinery. Thus, we were intrigued to identify a novel variant of an important subunit of the DNA-directed RNA polymerase II (RPB11B1) as an interaction partner of SATB1. RPB11 Is Encoded by a Multigene Family We were initially puzzled by the genetic origin of the RPB11 proteins because an N-terminal divergence (± lysine-17) could not be explained by differential splicing, even though differences at the C terminus did seem to result from alternative message processing. A search of GenBank identified in excess of 50 ESTs and cDNAs that exclusively encoded the RPB11A proteins (RPB11-A, a; Fig. 1). Another 20 or more ESTs and cDNAs in the DNA repository encoded solely the RPB11B1 family of proteins (RPB11B1, , ; Fig. 1). A third variant (RPB11-; Figs. 2 and 3) was represented in the databases by a single cDNA and two ESTs that distinguished it from the above protein families. These results suggested that gene duplication had occurred, as it would be difficult to postulate alternative splicing of a single-copy, allelic gene that exclusively joined the RPB11-A (or RPB11B1, RPB11-) N terminus to the RPB11-A (or RPB11B1, RPB11-) associated C terminus. Our results with allele-specific PCR confirmed that two distinct genetic loci encoded RPB11-A and RPB11B1. A third locus had accumulated a significant mutation load including conversion of its translation initiation codon (AUG) to a less efficiently used GUG [28]. This latter locus could
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represent a transcribed RPB11 pseudogene. A previous report [24] suggested that POLR2J is a single-copy gene. The high level of sequence conservation of gene family members, coupled with the assay method used by this group (restriction enzyme digestion coupled with Southern blot analysis), most likely explains the discrepancy between their work and ours. POLR2J Duplication It seems that POLR2J mutation/duplication events occurred recently in evolution. The RPB11 subunit is evolutionarily conserved in organisms from bacteria to higher eukaryotes. Yeast and human RPB11-A are 52% identical with the major differences mapping to the C terminus [24,29]. A single mouse cDNA encodes a protein that is 100% identical to human RPB11-A [24,30,31]. No variant proteins have been identified for species other than primates. SATB1–RPB11B1 Interaction Rarely does a mutation occur that enables a gene to perform a new function. However, when it does occur, it may impart a selective advantage to the organism harboring that mutation. This may be the situation with RPB11B1, with the selective advantage being conveyed through its exclusive association with the MAR binding protein SATB1. SATB1 recognizes RPB11B1, but not RPB11-A, through its polymorphic N terminus (possibly with assistance from the C terminus). Neither this region nor the C terminus is required for subunit–subunit interactions that lead to assembly of the pol II holoenzyme [29]. However, Lys17 is evolutionarily conserved between prokaryotes and eukaryotes [29] and thus may have a significant role in pol II function. Additionally, the C-terminal sequences of RPB11B1 share sequence homology (47% identity, 49%
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similarity over 49 amino acids) with a transcriptional regulatory protein (IE63) from human herpes simplex virus. Secondary structural analysis of the RPB11B1 C terminus predicts three -strands followed by an -helix. Known proteins with the same motifs consist of DNA binding proteins, including Escherichia coli topoisomerase I and methyl CpG-bindingprotein-2. Whether these similarities are significant or not is unknown. However, SATB1 association with RPB11B1 likely brings the transcription machinery to functionally significant sites at the bases of particular chromosomal loops. The unique C terminus of RPB11B1, which likely forms a discrete domain folded on the surface of pol II, may additionally recognize transcription factors or promoter elements and impart another level of specificity on gene regulation. RPB11B1–SATB1 Interaction RPB11B1 associates with SATB1 near an N-terminal PDZ motif [32] (at amino acids 58–222; the PDZ domain encompasses residues 90–204 [10]). PDZ motifs function as modular protein-binding domains. Proteins containing a PDZ domain form either heterodimers or homodimers (as seems to be the case for SATB1). Alternatively, the PDZ motif can bind a specific recognition sequence at the C terminus of target proteins [32]. This consensus sequence (S/TXV) is absent from RPB11B1, which suggests that SATB1 recognizes an alternative protein domain of RPB11B1. Chromatin Remodeling, Transcriptional Machinery, and MAR-Binding Proteins Another MAR binding protein, the ubiquitous SAF-B, associates with RNA pol II through the common C-terminal domain (CTD) of the largest subunit and with serine-/arginine-rich RNA processing factors (SR proteins) [33]. This latter observation is relevant because another of the proteins that associated with SATB1 in our yeast two-hybrid screen was the SR protein, TOPORS [34] (L.K.D. et al., manuscript in preparation). It has additionally been noted that the transcription machinery interacts with chromatin modifying and remodeling complexes, and we have identified several SATB1 interacting proteins that possibly are components of these multi-subunit complexes (L.K.D. et al., manuscript in preparation). Thus, these MAR-binding proteins share common classes of binding partners that form parts of the basal transcription apparatus and chromatin remodeling network. Future work may elucidate how such interactions alter the transcriptional patterns of specific genes.
MATERIALS AND METHODS Yeast two-hybrid screen. The Matchmaker LexA Two-hybrid system was used as described in the Users Manual and Yeast Protocols Handbook (YPH, Clontech; Palo Alto, CA). In brief, a Jurkat cDNA library (Clontech, Palo Alto, CA), prepared in the vector pB42AD (4 106 individual cDNAs), was amplified twofold. Subsequently, the cDNAs were introduced into the yeast strain EGY48– [p8oplacZ] that had previously been transfected with a construct (pLexA-SATB158–763) that encoded an N-terminally truncated SATB1 fused to the LexA DNA binding
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domain. Colonies that expressed Leu+, LacZ+ phenotypes were picked for further analysis. cDNAs were sequenced from their 5 ends and identified using the BLAST search engine (NCBI) [35]. Allele-specific PCR. Primers (Fig. 3) were designed to permit a distinction between variant POLR2J loci. Additional degeneracy was incorporated into the penultimate 3 nucleotide to impart greater priming specificity. The four primers were used in all combinations in PCR assays. Reactions were done in 50-l total volume with AmpliTaq Gold polymerase (Roche, Indianapolis, IN). The reactions included 5 l of 10 PCR buffer (15 mM MgCl2, final concentration), 20 pmoles of each primer, 12.5–25 ng human genomic DNA, and 1 l dNTP mixture (10 mM each). The genomic DNAs were obtained from three human cell lines (Jurkat, HL60, and MCF-7) and from 20 samples of patient leukocytes. Reactions were run on a PTC-100 Programmable Thermal Controller using the following conditions: 94 C for 5 minutes, then 40 cycles of 94 C for 30 seconds, 55 C for 60 seconds, and 72 C for 60 seconds. A final extension was done at 72 C for 5 minutes. Products were analyzed on 3% NuSieve GTG agarose gels prepared in TBE buffer. Products obtained from the cell lines were validated by sequence analysis. Ribonuclease protection assay. Total RNA was isolated using the Purescript isolation method (Gentra Systems, Minneapolis, MN). Briefly, cells were washed once with PBS followed by lysis in cell lysis solution. Protein and DNA were precipitated and total RNA was recovered from the supernatant by isopropanol precipitation. Cellular transcripts were detected by the RNase protection assay (RPA; Ambion, Austin, TX) using in vitro synthesized cRNA probes. Coding sequences of RPB11-A [19] were excised with NheI/XbaI and inserted into the XbaI site of pBS-K/S+. Both coding and 3 UTR sequences of RPB11B1 were excised with BamHI and EcoRI and inserted into the same sites in pBS-S/K+. Probes were prepared by digesting the former construct with Sau3A and the latter with BseRI (Fig. 2A) followed by in vitro synthesis using T7 RNA polymerase and 200 Ci/mmol of [-32P]UTP. The 185-nucleotide RPB11-A probe protected a 145nucleotide fragment specific to RPB11-A (and possibly a 113-nucleotide fragment of RPB11B1). The 450-nucleotide RPB11B1 probe protected a 420-nucleotide RPB11B1-specific fragment. As an internal loading control, a 120-nucleotide fragment of the GAPDH mRNA was detected using a 175-nucleotide riboprobe [36]. RNase protection assays were performed as described by the manufacturer (Ambion, Austin, TX), using 20 g total RNA. Protected fragments were analyzed on 6% polyacrylamide/8 M urea gels that were dried before exposure to Kodak Biomax MR, single emulsion film for 1 hour or 24 hours. Specific message levels were quantified by PhosphorImage analysis. Glutathione S-transferase pull-down assay. Complementary DNAs encoding RPB11B1 and RPB11-A were cloned downstream and in-frame with glutathione S-transferase (GST) in the vector pGEX-2T (Amersham-Pharmacia Biotech, Piscataway, NJ). The full-length cDNA encoding RPB11B1 (also included 3 UTR sequences) was excised from pB42AD with HincII and XhoI (the 5 overhang was filled in with Klenow large fragment). The cDNA was then ligated into the SmaI site of the vector (pGEX-RPB11B11–158). Deletions from the C terminus were prepared by digesting with BseRI or StuI to excise sequences encoding 52 or 33 amino acids, respectively, from the 3 end (pGEX-RPB11B11–106 and pGEXRPB11B11–125). A construct deleted of 15 C-terminal residues was prepared by PCR amplification from the Jurkat cDNA library, using the primer pair 5ACAAACGGATCCATGAACGCCCCTCCAGCCTT-3 and 5-GCCGCGGAATTCTCACCGCCGCCTCTCCCTGA-3. Unique restriction enzyme sites for cloning into the BamHI and EcoRI sites of pGEX-2T are shown in bold and the translation initiation and termination sites are underlined (pGEX-RPB11B11–143). RPB11-A cDNA was excised from pBSK/S+ with BamHI and XbaI (XbaI end-filled in with Klenow large fragment) and religated into the BamHI and SmaI sites of pGEX-2T (pGEX-RPB11-A). Chimeric molecules were prepared in which the C terminus of RPB11-A was replaced by the C terminus of RPB11B1, and vice versa. The constructs, pGEX-RPB11-A or pGEX-RPB11B1, were digested with BseRI and EcoRI, both 5 and 3 end fragments were gel purified, followed by ligation of the RPB11-A 3 to the RPB11B1 5 and vice versa. All constructs were sequenced in their entirety. GST fusion proteins were prepared from the pGEX-2T-cDNA constructs and purified on agarose beads using standard protocols (Amersham-Pharmacia Biotech, Inc., Piscataway, NJ). The GST pull-down assay was used as described [38] to confirm the interactions between SATB1 and target proteins. SATB1 and SATB1 deletion constructs were labeled with 35S-methionine in the TNT-coupled transcription/translation system (Promega, Madison, WI). This test protein (5 l) was incubated for 15
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minutes on ice with 200 l of E. coli (BL21) protein extract [38], in bead binding buffer (BBB; 50 mM potassium phosphate, pH 7.5, 150 mM KCl, 1 mM MgCl2, 10% glycerol, 1% Triton X-100). After centrifugation for 15 minutes, at 4 C, at 14,000 rpm in a microcentrifuge, the supernatants were transferred to a second set of tubes containing the GST fusion protein (bait) bound to agarose beads, in another 200 l of BL21 protein extract in BBB. The test and bait proteins were then incubated for 2 hours at 4 C on a rotary shaker. After the reaction was complete, the beads were washed three times in BBB at 4 C. After careful removal of all liquid, 20 l of 1 SDS sample buffer was added to the beads containing SATB1 and interacting RPB11, mixed, and boiled for 5 minutes. The samples were mixed again, centrifuged briefly, and then fractionated on denaturing polyacrylamide gels. The gels were dried and exposed to Kodak Biomax MR, single emulsion film for 2–5 days. -Galactosidase assay. The relative strengths of interaction between SATB158–763, or 3 and 5 deletions of SATB1, and RPB11-A and RPB11B1 were measured using the quantitative -galactosidase assay (YPH; Clontech, Palo Alto, CA). RPB11-A1–117 (full-length) and RPB11-A1–107 (deleted of the C terminus) were prepared by excising an EcoRI and XbaI fragment (XbaI end-filled in with Klenow large fragment followed by ligation of an EcoRI linker) or an EcoRI and BseRI fragment (BseRI end-filled in with T4 DNA polymerase followed by EcoRI linker attachment) from pBS-RPB11-A. These fragments were subsequently inserted, independently, into the EcoRI site of the yeast vector pB42AD. The constructs were transformed into the yeast strain EGY48-[p8op-LacZ] and resulting strains were mated with the yeast strain YM4271, which harbored the SATB1 full-length and deleted constructs in the vector pLexA. Independent colonies from representative yeast were grown overnight in selective medium using raffinose (a noninducing sugar) as the carbon source. The cells were washed and then induced for 4 hours in galactose-containing selective medium. Each independent colony was assayed in duplicate, using ONPG as the substrate. The assay was repeated at least three times, in duplicate, for each yeast two-hybrid clone. Positive controls were pLexA-Pos (a single plasmid control) and pLexA-53 + pB42AD-T (tests for interaction of p53 with the large T antigen). Negative controls were pLexA + pB42AD, pLexA-53 + pB42AD, and pLexA + pB42AD-T, none of which produces fusion proteins that can activate the -galactosidase promoter.
ACKNOWLEDGMENTS We thank Marc Vigneron (CNRS-INSERM-Universite Louis Pasteur) for the RPB11-A cDNA clone; Mark Sherman (Beckman Research Institute, City of Hope) for assisting with 3D modeling of the pol II, subunit 11; Louis Geller and Jan-Erik Gustaveson (Beckman Research Institute, City of Hope) for sequence analysis; Kimberly Karlsberg (Beckman Research Institute, City of Hope) for assisting with graphics; and members of the Krontiris laboratory for helpful discussions. This work was supported by a grant from the National Institutes of Health (CA51985) and by the Beckman Research Institute of the City of Hope. RECEIVED FOR PUBLICATION SEPTEMBER 17, 2001; ACCEPTED MARCH 13, 2002. Note added in proof. While this paper was undergoing review, Grandemange et al. [37] published a manuscript confirming and increasing the characterization of the POLR2J family described here.
REFERENCES 1. Dickinson, L. A., Dickinson, C. D., and Kohwi-Shigmatsu, T. (1997). An atypical homeodomain in SATB1 promotes specific recognition of the key structural element in a matrix attachment region. J. Biol. Chem. 272: 11463–11470. 2. Gasser, S. M., and Laemmli, U. K. (1987). A glimpse at chromosomal order. Trends Genet. 3: 16–22. 3. Nelson, W. G., Pienta, K. J., Barrack, E. R., and Coffey, D. S. (1986). The role of the nuclear matrix in the organization and function of DNA. Annu. Rev. Biophys. Biophys. Chem. 15: 457–475. 4. Boulikas, T. (1995). Chromatin domains and prediction of MAR sequences. Int. Rev. Cytol. 162A: 279–388. 5. Gasser, S. M., and Laemmli, U. K. (1986). The organization of chromatin loops: characterization of a scaffold attachment site. EMBO J. 5: 511–518. 6. Cockerill, P. N., and Garrard, W. T. (1986). Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44: 273–282. 7. Nakagomi, K., Kohwi, Y., Dickinson, L. A., and Kohwi-Shigematsu, T. (1994). A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1. Mol. Cell. Biol. 14: 1852–1860.
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The regulatory element 3 to the A-globin gene binds to the nuclear matrix and interacts with special A-T-rich binding protein 1 (SATB1), an SAR/MAR-associating region DNA binding protein. Blood 4: 1298–1308. 13. Oancea, A. E., Berru, M., and Shulman, M. J. (1997). Expression of the (recombinant) endogenous immunoglobulin heavy-chain locus requires the intronic matrix attachment regions. Mol. Cell. Biol. 17: 2658–2668. 14. Ramakrishnan, M., et al. (2000). Modulated binding of SATB1, a matrix attachment region protein, to the AT-rich sequence flanking the major breakpoint region of BCL2. Mol. Cell. Biol. 20: 868–877. 15. de Belle, I., Cai, S., and Kohwi-Shigematsu, T. (1998). The genomic sequences bound to special AT-rich sequence-binding protein 1 (SATB1) in vivo in Jurkat T cells are tightly associated with the nuclear matrix at the bases of the chromatin loops. J. Cell Biol. 141: 335–348. 16. Kohwi-Shigematsu, T., Maass, K., and Bode, J. (1997). A thymocyte factor SATB1 suppresses transcription of stably integrated matrix-attachment region-linked reporter genes. Biochemistry 36: 12005–12010. 17. Liu, J., et al. (1997). The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression. Mol. Cell. Biol. 17: 5275–5287. 18. Alverez, J. D., et al. (2000). The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during T-cell development. Genes Dev. 14: 521–535. 19. Acker, J., et al. (1997). Interactions between the human RNA polymerase subunits. J. Biol. Chem. 272: 16815–16821. 20. Kimura, M., Ishiguro, A., and Ishihama, A. (1997). RNA polymerase II subunits 2, 3, and 11 form a core subassembly with DNA binding activity. J. Biol. Chem. 272: 25851–25855. 21. Ishiguro, A., et al. (1998). Two large subunits of the fission yeast RNA polymerase II provide platforms for the assembly of small subunits. J. Mol. Biol. 279: 703–712. 22. 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Biol. 6: 1385–1388. 33. Nayler, O., et al. (1998). SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements. Nucleic Acids Res. 26: 3542–3549. 34. Haluska, P., Jr, et al. (1999). Interaction between human topoisomerase I and a novel RING finger/arginine-serine protein. Nucleic Acids Res. 27: 2538–2544. 35. Altschul, S. F., et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402. 36. Lagnado, C. A., Brown, G. J., and Goodall, G. J. (1994). AUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A). Mol. Cell. Biol. 14: 7984–7995. 37. Grandemange, S., et al. (2001). A human RNA polymerase II subunit is encoded by a recently generated multigene family. B. M. C. Mol. Biol. 2: 14. 38. Melcher, K., and Johnston, S. A. (1995). GAL4 interacts with TATA-binding protein and coactivators. Mol. Cell. 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The Thymocyte-Specific MAR Binding Protein, SATB1, Interacts in Vitro with a Novel Variant of DNA-Directed RNA Polymerase II, Subunit 11 Linda K. Durrin and Theodore G. Krontiris* Division of Molecular Medicine, Beckman Research Institute of the City of Hope National Medical Center, Duarte, California 91010, USA *To whom correspondence and reprint requests should be addressed. Fax: (626) 301-8862. E-mail: [email protected]. Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession number AF468111.
A yeast two-hybrid screen of a Jurkat (T cell) derived cDNA library, using SATB1 (a matrix attachment region binding protein) as the bait, yielded four independent isolates of a novel variant of the DNA directed RNA polymerase II, subunit 11 (RPB11). Absence of lysine-17 from the amino terminus of this variant cannot be explained by alternative mRNA splicing. Instead, allele-specific PCR, combined with GenBank database searches, suggests that a recent gene duplication event has resulted in distinct loci encoding three variant forms of RPB11. Differential splicing of mRNA transcripts accounts for unique carboxy termini among the RPB11 proteins. The exclusive association of SATB1 with one form of RPB11 is influenced primarily by the N-terminal amino acid disparity, as deletion of the entire C terminus does not alter interaction affinity. Association of RPB11 with SATB1 maps between amino acids 58 and 222 of SATB1, a region that includes a PDZ-like dimerization motif. Key Words: SATB1, POLR2J family, protein interaction
INTRODUCTION SATB1 was originally identified as a matrix or scaffold attachment region (MAR/SAR) binding protein [1]. Eukaryotic chromosomes are typically arranged as 50- to 100-kb loop domains that are attached at their bases (MARs) to the intranuclear framework by nonhistone proteins [2,3]. MARs define the borders between chromatin domains, each of which harbors a gene, functional segment of a gene, or gene cluster [4]. MARs colocalize with transcriptional enhancers, promoters, and origins of replication [5,6]. The nuclear matrix near MARs may serve as a “sink” for organization of the proteins that regulate transcription, replication, RNA processing, recombination, and DNA repair [4]. SATB1 is a 95-kDa phosphoprotein expressed predominantly in thymic lymphocytes and pre-B cells [1]. A 150amino-acid MAR binding domain is centrally located within SATB1 [7,8]. At the carboxy-terminal region, an atypical homeodomain binds poorly and with low specificity to DNA; however, it assists the SATB1 MAR-binding domain specifically to recognize unwinding elements (for example, 5C/ATAATA-3) [9]. Two Cut-like repeats do not exhibit functional significance at MARs [9]. A dimerization domain with similarity to a PDZ motif has been identified within the amino
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terminus of SATB1 [10]. This motif is required for SATB1 interaction with MAR elements [10]. SATB1 interacts preferentially with double-stranded, AT-rich sequences (AT stretches interspersed with C’s but lacking G’s) that have a propensity to unwind in supercoiled plasmids [1]. SATB1 specifically recognizes MAR elements near CD8A [11] and HBG1 [12], the MAR 3 of the mouse and human IGH@ enhancer [1,13], and the MAR near the human BCL2 major breakpoint region [14]. Furthermore, SATB1 has been found associated with the nuclear matrix at the bases of many unidentified chromatin loops [15]. SATB1, in association with MARs, acts as a transcriptional repressor [16,17]. Recently, SATB1-null mice were generated and found to display dysregulation (mostly derepression) of up to 2% of the genes in thymocytes [18]. SATB1 apparently regulates gene expression at specific times and places during T-cell development. DNA-directed RNA polymerase II (pol II) is the fundamental enzyme involved in all aspects of eukaryotic mRNA synthesis. In association with additional, multi-subunit complexes, pol II recognizes specific promoters, transcribes the DNA into a fully processed message (capped, spliced, cleaved, and polyadenylated) and has a role in the regulation of these processes. As such, it is a focal point in cellular processes from
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FIG. 1. POLR2J and transcribed splice variants. The boxes (top) represent exons, with the filled and open regions indicating coding and noncoding domains, respectively. The line delineates intronic sequences. The bent arrow indicates the transcriptional start site. The RPB11B1 family is encoded at a locus on chromosome 7 that is differentially spliced at the C terminus, as shown (top). RPB11B1 (red solid line) and RPB11B1 (blue dashed line) are encoded by three, and RPB11B1 (brown dotted line), by four exons within approximately 6-kb of genomic sequences. Both RPB11-A and RPB11a are encoded by four exons and the first three exons are similar to those shown for the RPB11B1 family. These two proteins differ by alternative splicing of the third to the fourth exon, such that RPB11a encodes an extra 10 amino acids. An allelic variation near the first splice junction encodes an additional lysine in the RPB11-A family but not in the RPB11B1 family. At the bottom are shown splice variants of RPB11 that have been deposited in GenBank (acc. nos. L37127 and X98433 (RPB11-A; gene POLR2J1), AJ277928–AJ277930 and AJ277932 (RPB11a; gene POLR2J1), AJ277739 (RPB11B1; gene POLR2J2), AJ277740 (RPB11B1; gene POLR2J2), and AF468111 (RPB11B1; gene POLR2J2)). Alternative C termini are shown in color corresponding to the diagram at top.
differentiation to apoptosis. Human pol II is composed of 12 subunits ranging in size from 220 to 10 kDa [19]. Subunits RPB1, RPB2, RPB3, and RPB11, equivalent to the prokaryotic , , , and subunits, respectively, have roles in assembly of the holoenzyme [19–22]. The RPB3–RPB11 heterodimer nucleates assembly of pol II, by binding RPB2 and subsequently RPB1 to form a complete core enzyme. Other subunits assemble on this platform to form the catalytically active holoenzyme. In addition to their structural and catalytic roles, many of the subunits have regulatory roles through contact with DNA or heterologous proteins. The bacterial -subunit interacts with transcriptional regulators, which influences polymerase activity [reviewed in 23]. SATB1 has the potential to affect many cellular processes because of its interaction with specific sequence contexts at the bases of chromosomal loop domains. We therefore used the yeast two-hybrid assay to identify proteins, encoded by a Jurkat cDNA library (human T-cell leukemic cell line), that interact with SATB1. Among the distinct proteins isolated (L.K.D. et al., manuscript in preparation) were multiple independent clones of a unique variant of pol II, subunit 11 (RPB11B1). Comparison of this variant with the previously described RPB11 (RPB11-A) suggests that SATB1 recognizes structural differences in pol II, subunit 11, that may have functional significance.
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RESULTS Yeast Two-Hybrid Screen The MAR-binding protein SATB1, deleted of 57 N-terminal amino acids (SATB158–763) was used as bait in the yeast twohybrid assay to identify SATB1-interacting proteins from the T-cell derived Jurkat cell line. Of 92 positive clones identified, 7 encoded isolates of a novel variant of RNA polymerase II, subunit 11 (RPB11). Most of these yeast two-hybrid clones encoded the full-length RPB11 variant; only one lacked two N-terminal residues. Additional SATB1-interacting proteins encoded by Jurkat cDNA clones are discussed elsewhere (L.K.D. et al., manuscript in preparation). Here, we refer to the published form of RPB11 as RPB11-A [19,24] and the new clone as RPB11B1 as it seems to be a splice variant of one member of the POLR2J family (Fig. 1). These two RPB11 proteins differed by a single amino acid in their N termini (residue 17 was a lysine in RPB11-A but deleted in our clones) and extensively at the C terminus; RPB11-A and RPB11B1, respectively, diverged after the arginine at position 106/105, and encoded an additional 11 or 53 non-homologous residues (Fig. 1). A second published variant was identical to RPB11B1 at the N terminus except for an isoleucine in place of a lysine at position 18, but it
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FIG. 2. Genomic sequences encode variant forms of RPB11. Genomic sequences encoding POLR2J apparently can be transcribed into three alternative forms of exon 1 (top). Only two of these have sequences required for efficient translation (GenBank acc. nos. AC004084, AJ277931, and AJ277932), whereas transcripts from the last gene (GenBank acc. no. AC004951, NT_007766) might be translated less efficiently [28]. Gene sequences encoded identical amino acids for exon 2. Two versions of exon 3 were encoded by the gene loci. Amino acids are indicated below the nucleotide triplets and a dashed line indicates a deletion at residue 17 (top) or within the third exon (bottom). Intronic sequences are shown as lowercase letters. Amino acids that differ among the genes are shown in bold. The consensus sequences of the 5 and 3 splice junctions are indicated (the nearly invariant gt or ag is boxed and additional nucleotides essential for efficient message splicing are underlined).
variant of RPB11 was thus notable and may have reflected absence of RPB11-A and other variants in the Jurkat cDNA library, sampling limitations, or truly restricted recognition with possible functional significance. differed at the C terminus [25]. This C terminus appeared to be a cloning artifact as it encoded an internal cDNA fragment, in reversed orientation, of growth factor receptor-bound protein-2 (GRB2; GenBank acc. no. M96995) [19]. More recently, three additional variant cDNAs were deposited into GenBank (RPB11a, RPB11B1, RPB11B1; Mark Vigneron et al., unpublished data; Fig. 1). A search of GenBank cDNAs and expressed sequence tags (ESTs) further identified three homologous cDNAs that lacked a classical (ATG) translation initiation site (GenBank acc. nos. BC017250, AL526460, and AL554541). The exclusive interaction of SATB1 with a single
POLR2J Family and Splice Variants The N-terminal difference between RPB11-A and RPB11B1 (presence or absence of lysine-17) occurred near the mRNA splice junction of exon 1 but could not be explained by alternative splicing (Fig. 2, top). Instead, either a single gene encoded allelic variation at this site or, alternatively, multiple genes encoded the RPB11 variants. Examination of genomic sequences deposited in both GenBank and the high-throughput genomic sequence (htgs) databases identified three distinct configurations for the first exon, and two for the third exon, of POLR2J (Fig. 2). In addition to the
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FIG. 3. Allele-specific PCR within exon 1/intron 1 of the POLR2J loci distinguishes three genetic loci. (A) Genomic and EST sequences from GenBank were examined to identify gene/allele-specific differences between RPB11-A, RPB11B1, and a third variant. Primers were designed to distinguish between alleles and an additional alteration was incorporated into the penultimate nucleotide to increase specificity. Primer pairs were used in all combinations (A/C, A/D, B/C, and B/D) in a PCR assay. Products were sequenced to confirm specificity: A/C, RPB11-; A/D does not prime efficiently and produces non-specific products; B/C, RPB11-A; and B/D, RPB11B1. (B) Example of products analyzed on a 3% NuSieve GTG agarose gel. The arrow indicates the 97-bp RPB11-A or RPB11-, and the 96-bp RPB11B1 products. Product sizes were determined by fractionating a 100-bp DNA ladder in an adjacent lane. The control reactions (minus genomic DNA) gave no products.
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Lys-17 variations, the translation initiation site was mutated on one gene/allele to a less commonly used GTG translation initiating triplet (valine; GenBank acc. no. NT_007766). In this same clone, sequence differences within the third exon (Fig. 2, bottom) specified an extra cysteine and replaced a leucine with an aspartic acid. Thus, initial examination of POLR2J sequences suggested that separate genetic loci encoded members of a POLR2J family; however, allelic variation was not ruled out. Therefore, to distinguish between genetic and allelic variations, PCR amplification primers were designed that specifically amplified the first exon and the 3 portion of intron 1 from genomic DNA samples (Fig. 3A). The 5 primers (A and B) differentiated between a C/A disparity at nucleotide position –10 (relative to the translation initiation site) that correlated with the initiating ATG (C) or GTG (A) (Fig. 3A, single arrow). The 3 primers (C and D) discriminated between the presence and absence of lysine-17 (Fig. 3A, three arrows). Primer pair B/C amplified sequences corresponding to RPB11-A (97 nt), primers B and D specifically amplified RPB11B1 (96 nt), and the primer pair A and C amplified a third variant (97 nt; designated RPB11-). Primers A and D primed nonspecifically and only large, unidentified products were observed (data not shown). A total of 20 patient DNA samples (40 chromosomes) were analyzed and the above three products were observed in all instances (Fig. 3B). This assay detected alleles present at a frequency of 10% or greater (90% confidence level) [27]. Thus, we identified a POLR2J gene family that consisted of at least three distinct loci
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FIG. 4. Quantification of RPB11-A and RPB11B1 message by the ribonuclease protection assay (RPA). (A) The thick lines depict the cDNAs encoding RPB11-A (top) and RPB11B1 (bottom). The hatched line at the end of RPB11B1 represents 3 UTR sequences included in the construct. Thin lines are vector sequences that contain a T7 promoter. RPB11-A was digested to completion with Sau3A (upstream Sau3A site is for reference only) and RPB11B1 was digested with BseRI, which precisely separated nearly conserved sequences (upstream) from unique sequences (downstream). The 185-nucleotide RPB11-A probe protected the 145-nucleotide RPB11-A-specific RNA, and it may protect a 113-nucleotide RPB11B1-specific RNA (not detected). The 450-nucleotide RPB11B1 probe protected a 420-nucleotide RNA. (B) Autoradiogram of RPB11-A, RPB11B1, and GAPDH protected fragments. Total RNA (20 g) was hybridized with an excess of the probe indicated, before analysis of the nuclease-protected fragments on a 6% denaturing polyacrylamide gel. The lanes marked + and – are an aliquot of the probe either treated or untreated, respectively, with ribonuclease. Cell lines from which the RNA was prepared are indicated. Exposure was for 24 hours on Kodak Biomax MR single emulsion film.
encoding RPB11-A (and RPB11a; Fig. 1), RPB11B1 (also RPB11B1 and RPB11B1; Fig. 1), and finally, a polymorphic third variant (RPB11-; Fig. 2). Comparison of POLR2J Family Member Message Levels Previous work has shown that POLR2J message is present in all tissues examined, including the thymus [24]. We used the ribonuclease protection assay to confirm and quantify RPB11A and RPB11B1 message levels in Jurkat, and also in HeLa and MCF-7, cell lines. Probes were prepared (Fig. 4A), and the assay performed as described in Materials and Methods. The control GAPDH mRNA was easily detected within 1 hour of exposure and low levels of RPB11-A message also were observed at this time (data not shown), however, transcripts encoding RPB11B1 were detected only after 24 hours of exposure (Fig. 4B). The RPB11-A probe also should have protected RPB11B1 mRNA, but a combination of degradation products and the low abundance of the RPB11B1 message made it impossible to detect it with this probe, even after 24 hours of exposure. Specific in Vitro Association of RPB11B1 with SATB1 To investigate the specificity of interaction of RPB11B1 with SATB1, we used the GST pull-down and yeast two-hybrid -galactosidase assays. RPB11B1 and RPB11-A were prepared in vitro as downstream fusion proteins with GST and then incubated with 35S-methionine-labeled SATB1. Fulllength and truncated SATB1 used in these experiments are
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Article FIG. 5. GST pull-down assays demonstrate preferential interaction of RPB11B1 (over RPB11-A) with SATB1. Full-length and deletion constructs of SATB1 are shown in Fig. 6 (marked a, b, c, and d on the left side, (e) is similar to (a) but deleted of 57 N-terminal residues). SATB1 and luciferase were labeled with [35S]-methionine in an in vitro TNT coupled transcription/translation system. An aliquot of each protein, equivalent to the amount used for the pull-down assay, is shown in the lanes marked a–f, only. These test proteins were incubated with GST–RPB11B1, GST–RPB11-A, or GST alone (labeled at top of autoradiogram) and subsequently fractionated on 6% denaturing polyacrylamide gels, dried, and exposed for 2 days. Lanes are as follows: a, SATB11–763; b, SATB11–677; c, SATB11–354; d, SATB11–222; e, SATB158–763; and f, luciferase.
illustrated in Fig. 6 (constructs marked with the letters a–d to pB42AD. After transformation of each construct, along with their immediate left; constructs a and e are equivalent except the reporter construct p8op-lacZ, into appropriate yeast, the for deletion of the N terminus of construct e). We loaded an cells were induced and assayed for interaction of the RPB11 aliquot of the input SATB1 (Fig. 5, lanes a–e) equivalent to the variant with SATB1 (Fig. 6). RPB11B1 associated with amount used in the GST pull-down assay. In adjacent lanes SATB158–763, SATB158–677, SATB158–576, SATB158–457, SATB158–354, is shown the SATB1 that specifically associated with the bait, SATB158–289, and SATB158–222, but not with SATB1223–763. This GST–RPB11B1, GST–RPB11-A, or GST alone (a control for precisely mapped its interaction with SATB1 to a domain nonspecific interaction of SATB1 with the bait). Full-length between amino acids 58 and 222 in the N terminus, which and truncated SATB1 (SATB11–763, SATB11–354, SATB11–222, and agreed with the results from the GST pull-down assay. RPB11-A, on the other hand, did not associate with SATB158–763) interacted significantly (at least tenfold) more strongly with RPB11B1 than with RPB11-A or GST (Fig. 5, SATB158–222 or SATB158–763 to any significant extent, although lanes a, c–e). SATB11–677 interacted only about twofold more strongly with RPB11B1 than with RPB11-A (Fig. 5, lane b), a discrepancy that may be attributable to some property of SATB1 because this result is also observed when interaction strength is measured with the yeast two-hybrid -galactosidase assay. a Possibly, the intact SATB1 C terminus medib ates protein folding that is critical in the regulation of the physiological interaction of RPB11B1 and SATB1. Deletion of this domain may permit adventitious interactions to occur. c Luciferase served as a control for nonspecific association of RPB11 with the test proteins. As expected, no interaction of RPB11B1, RPB11d A, or GST with this control was observed (Fig. 5, lane f). These in vitro results were confirmed using the cell-based, yeast two-hybrid -galactosidase assay. SATB1 deletion constructs, similar FIG. 6. Yeast two-hybrid -galactosidase assay quantification of interaction between SATB1 and RPB11 constructs. Functional domains of SATB1 are illustrated at the top as the dimerization domain to those used for the GST pull-down assays (PDZ motif), MAR binding domain (MAR-BD), two Cut repeats, and the homeodomain. A stretch (except deleted of the 57 N-terminal amino of 15 glutamines (Q15) may have transcriptional significance. The N- and C-terminal truncated pepacids, which are toxic in eukaryotic cells [16]), tides of SATB1 were prepared as fusions with the LexA DNA-binding domain. The numbers at the were prepared as fusions to the lexA DNA C terminus depict the last residue included in the peptide (all polypeptides initiated at amino acid 58 except where noted). Constructs encoding these SATB1 proteins were introduced, along with binding domain in the yeast vector pLexA. RPB11B1, RPB11-A, or RPB11-A/CT (encoded as fusions of the bacterial activation domain in cDNAs encoding full-length RPB11B1, pB42AD), into a yeast strain harboring the p8op-LacZ reporter construct. After induction for 4 RPB11-A, or RPB11-A/CT (RPB11-A deleted hours in galactose-containing selective medium, -galactosidase activity was measured (YPH). All of its non-homologous C-terminal amino values were normalized by setting the two-plasmid positive control equivalent to 1000 units activacids) were prepared as fusions with the bac- ity. The striped oval between SATB1 amino acids 58 and 222 represents the interaction site of RPB11B1 with SATB1. terial activation domain in the yeast vector
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interaction with SATB158–677 and SATB158–354 was above background (negative controls were between 15 and 22 units activity). These adventitious levels of interaction (see above for GST pull-down assays) were 14–25% of the activity measured for the RPB11B1 counterparts. Role of the C Terminus of RPB11B1 in SATB1 Recognition Because RPB11B1 and RPB11A differed significantly at their C termini, and by the presence or absence of lysine-17, we sought to investigate which of these differences resulted in altered binding affinity of the variants for SATB1. Initially, we hypothesized that the extensive differences at the C termini had a greater chance of altering binding affinity. A series of deletions of the C terminus of RPB11B1 were prepared as GST fusion proteins (Fig. 7B). The most extensive of the deletions, RPB11B11–105, eliminated all C-terminal sequences that differed from RPB11-A. The BseRI restriction enzyme site was used to precisely divide the N and C termini of the two forms of pol II, subunit 11). We used two [35S]-methionine labeled SATB1 constructs, SATB11–763 and SATB11–222, as representative test proteins in the GST pulldown assays. Deletions of 15, 33, or 53 amino acids from RPB11B1 (Figs. 7B and 7C) did not decrease its affinity for either of the test constructs (Fig. 7A, lanes a or d). None of the constructs interacted significantly with the [35S]-Metluciferase control (Fig. 7A, lane f). We also tested, in a yeast two-hybrid -galactosidase assay, whether deletion of the C terminus of RPB11-A (amino acids 107–117) significantly increased its association with SATB1, a result we would expect if differences in the C terminus were responsible for inhibiting RPB11-A binding. RPB11-A/CT did not interact with either SATB158–763 or SATB158–222 (Fig. 6, right column). Thus, the C terminus of RPB11B1 was not the region conferring binding specificity to SATB1.
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FIG. 7. The C terminus of RPB11B1 is not required for its interaction with SATB1, in vitro. (A) Full-length SATB11–763 and one deletion, SATB11–222, were used as test proteins (Fig. 6). They and the control luciferase were labeled with [35S]-methionine and interacted with GST–RPB11B1 as described in Fig. 5. Positions of the test proteins are indicated in the autoradiograph. The first two lanes and the last lane contain an aliquot of the input proteins, only. The lanes are marked as described in Fig. 5 (a, d, or f) and also according to the GST or GST fusion protein used for each pull-down assay. (B) A series of constructs encoding C-terminal truncations of RPB11B1 was prepared as GST fusion proteins. The N terminus, which is conserved (except for lysine17), is depicted as a filled line. The non-conserved C terminus is shown as a hatched line. The BseRI restriction enzyme site precisely separates the two domains. RPB11B11–158 is full-length, whereas RPB11B11–143, RPB11B11–125, and RPB11B11–105 are deleted of 15, 33, and 53 residues, respectively. (C) Representative GST or GST-RPB11B1 fusion protein, fractionated on SDS-PAGE gels and stained with Coomassie blue, were used in the above assays. Sizes are given in kDa.
We next examined whether the N-terminal difference (presence or absence of a lysine at position 17), either alone or in association with the C terminus, influenced recognition of SATB1 by RPB11B1. Accordingly, we prepared chimeric molecules in which we interchanged the C termini between RPB11-A and RPB11B1 (Figs. 8B and 8C). These GST fusion proteins were incubated with the representative [35S]-MetSATB11–222, and interacting protein was analyzed on denaturing gels. The controls, full-length RPB11B1 and RPB11A, behaved as observed previously (Fig. 8A). RPB11B1 associated with almost 100% of the test protein and RPB11A with an insignificant amount. Because the C terminus of RPB11B1 was dispensable for its interaction with SATB1, we expected that the chimera RPB11B1/A also would associate with SATB1. This was observed, although the interaction was weaker, with approximately 50% of the test protein interacting (Fig. 8A; signals are overexposed, making it difficult to observe the true differences quantitated by PhosphorImage analysis). Finally, the RPB11-A/B1 chimera was not expected to associate with SATB1 if the Nterminal alteration was solely responsible for recognition of the test protein. RPB11-A/B1 interacted with approximately 20% of the input protein (Fig. 8A). We concluded that both N- and C-terminal domains of RPB11B1 contributed to its interaction with SATB1, although the N terminus seemed to have an influential role.
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FIG. 8. Presence or absence of Lys-17, in association with the C terminus, determines the strength of interaction of RPB11 with SATB1, in vitro. (A) Only the single truncation SATB11–222 and the luciferase control were used for this assay. They were labeled in an in vitro transcription/translation system with 35 S-methionine and incubated with GST–RPB11B1, GST–RPB11-A, GST–RPB11B1/A, RPB11-A/B1, or GST. The interacting SATB1 was analyzed as described for Fig. 5. The input protein is indicated (first and last lanes) and lanes are marked as described in Fig. 5 (d or f) and also according to the GST or GST fusion protein used for each pull-down assay. (B) Chimeric molecules, in which the N terminus of RPB11-A is switched with the C terminus of RPB11B1, and vice versa, were prepared as GST fusion proteins. The N terminus of RPB11-A is depicted as a filled line, RPB11B1 as a hatched line. As noted previously, the BseRI restriction enzyme site precisely separates the two domains. (C) Representative GST or GST–RPB11 chimeric fusion proteins, fractionated on an SDS-PAGE gel and stained with Coomassie blue, were used in the above assays. Sizes are given in kDa.
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DISCUSSION As a MAR binding protein with established roles in repression and activation of gene expression [11,16,17,19,26], SATB1 might be expected to associate with components of the transcription machinery. Thus, we were intrigued to identify a novel variant of an important subunit of the DNA-directed RNA polymerase II (RPB11B1) as an interaction partner of SATB1. RPB11 Is Encoded by a Multigene Family We were initially puzzled by the genetic origin of the RPB11 proteins because an N-terminal divergence (± lysine-17) could not be explained by differential splicing, even though differences at the C terminus did seem to result from alternative message processing. A search of GenBank identified in excess of 50 ESTs and cDNAs that exclusively encoded the RPB11A proteins (RPB11-A, a; Fig. 1). Another 20 or more ESTs and cDNAs in the DNA repository encoded solely the RPB11B1 family of proteins (RPB11B1, , ; Fig. 1). A third variant (RPB11-; Figs. 2 and 3) was represented in the databases by a single cDNA and two ESTs that distinguished it from the above protein families. These results suggested that gene duplication had occurred, as it would be difficult to postulate alternative splicing of a single-copy, allelic gene that exclusively joined the RPB11-A (or RPB11B1, RPB11-) N terminus to the RPB11-A (or RPB11B1, RPB11-) associated C terminus. Our results with allele-specific PCR confirmed that two distinct genetic loci encoded RPB11-A and RPB11B1. A third locus had accumulated a significant mutation load including conversion of its translation initiation codon (AUG) to a less efficiently used GUG [28]. This latter locus could
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represent a transcribed RPB11 pseudogene. A previous report [24] suggested that POLR2J is a single-copy gene. The high level of sequence conservation of gene family members, coupled with the assay method used by this group (restriction enzyme digestion coupled with Southern blot analysis), most likely explains the discrepancy between their work and ours. POLR2J Duplication It seems that POLR2J mutation/duplication events occurred recently in evolution. The RPB11 subunit is evolutionarily conserved in organisms from bacteria to higher eukaryotes. Yeast and human RPB11-A are 52% identical with the major differences mapping to the C terminus [24,29]. A single mouse cDNA encodes a protein that is 100% identical to human RPB11-A [24,30,31]. No variant proteins have been identified for species other than primates. SATB1–RPB11B1 Interaction Rarely does a mutation occur that enables a gene to perform a new function. However, when it does occur, it may impart a selective advantage to the organism harboring that mutation. This may be the situation with RPB11B1, with the selective advantage being conveyed through its exclusive association with the MAR binding protein SATB1. SATB1 recognizes RPB11B1, but not RPB11-A, through its polymorphic N terminus (possibly with assistance from the C terminus). Neither this region nor the C terminus is required for subunit–subunit interactions that lead to assembly of the pol II holoenzyme [29]. However, Lys17 is evolutionarily conserved between prokaryotes and eukaryotes [29] and thus may have a significant role in pol II function. Additionally, the C-terminal sequences of RPB11B1 share sequence homology (47% identity, 49%
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similarity over 49 amino acids) with a transcriptional regulatory protein (IE63) from human herpes simplex virus. Secondary structural analysis of the RPB11B1 C terminus predicts three -strands followed by an -helix. Known proteins with the same motifs consist of DNA binding proteins, including Escherichia coli topoisomerase I and methyl CpG-bindingprotein-2. Whether these similarities are significant or not is unknown. However, SATB1 association with RPB11B1 likely brings the transcription machinery to functionally significant sites at the bases of particular chromosomal loops. The unique C terminus of RPB11B1, which likely forms a discrete domain folded on the surface of pol II, may additionally recognize transcription factors or promoter elements and impart another level of specificity on gene regulation. RPB11B1–SATB1 Interaction RPB11B1 associates with SATB1 near an N-terminal PDZ motif [32] (at amino acids 58–222; the PDZ domain encompasses residues 90–204 [10]). PDZ motifs function as modular protein-binding domains. Proteins containing a PDZ domain form either heterodimers or homodimers (as seems to be the case for SATB1). Alternatively, the PDZ motif can bind a specific recognition sequence at the C terminus of target proteins [32]. This consensus sequence (S/TXV) is absent from RPB11B1, which suggests that SATB1 recognizes an alternative protein domain of RPB11B1. Chromatin Remodeling, Transcriptional Machinery, and MAR-Binding Proteins Another MAR binding protein, the ubiquitous SAF-B, associates with RNA pol II through the common C-terminal domain (CTD) of the largest subunit and with serine-/arginine-rich RNA processing factors (SR proteins) [33]. This latter observation is relevant because another of the proteins that associated with SATB1 in our yeast two-hybrid screen was the SR protein, TOPORS [34] (L.K.D. et al., manuscript in preparation). It has additionally been noted that the transcription machinery interacts with chromatin modifying and remodeling complexes, and we have identified several SATB1 interacting proteins that possibly are components of these multi-subunit complexes (L.K.D. et al., manuscript in preparation). Thus, these MAR-binding proteins share common classes of binding partners that form parts of the basal transcription apparatus and chromatin remodeling network. Future work may elucidate how such interactions alter the transcriptional patterns of specific genes.
MATERIALS AND METHODS Yeast two-hybrid screen. The Matchmaker LexA Two-hybrid system was used as described in the Users Manual and Yeast Protocols Handbook (YPH, Clontech; Palo Alto, CA). In brief, a Jurkat cDNA library (Clontech, Palo Alto, CA), prepared in the vector pB42AD (4 106 individual cDNAs), was amplified twofold. Subsequently, the cDNAs were introduced into the yeast strain EGY48– [p8oplacZ] that had previously been transfected with a construct (pLexA-SATB158–763) that encoded an N-terminally truncated SATB1 fused to the LexA DNA binding
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domain. Colonies that expressed Leu+, LacZ+ phenotypes were picked for further analysis. cDNAs were sequenced from their 5 ends and identified using the BLAST search engine (NCBI) [35]. Allele-specific PCR. Primers (Fig. 3) were designed to permit a distinction between variant POLR2J loci. Additional degeneracy was incorporated into the penultimate 3 nucleotide to impart greater priming specificity. The four primers were used in all combinations in PCR assays. Reactions were done in 50-l total volume with AmpliTaq Gold polymerase (Roche, Indianapolis, IN). The reactions included 5 l of 10 PCR buffer (15 mM MgCl2, final concentration), 20 pmoles of each primer, 12.5–25 ng human genomic DNA, and 1 l dNTP mixture (10 mM each). The genomic DNAs were obtained from three human cell lines (Jurkat, HL60, and MCF-7) and from 20 samples of patient leukocytes. Reactions were run on a PTC-100 Programmable Thermal Controller using the following conditions: 94 C for 5 minutes, then 40 cycles of 94 C for 30 seconds, 55 C for 60 seconds, and 72 C for 60 seconds. A final extension was done at 72 C for 5 minutes. Products were analyzed on 3% NuSieve GTG agarose gels prepared in TBE buffer. Products obtained from the cell lines were validated by sequence analysis. Ribonuclease protection assay. Total RNA was isolated using the Purescript isolation method (Gentra Systems, Minneapolis, MN). Briefly, cells were washed once with PBS followed by lysis in cell lysis solution. Protein and DNA were precipitated and total RNA was recovered from the supernatant by isopropanol precipitation. Cellular transcripts were detected by the RNase protection assay (RPA; Ambion, Austin, TX) using in vitro synthesized cRNA probes. Coding sequences of RPB11-A [19] were excised with NheI/XbaI and inserted into the XbaI site of pBS-K/S+. Both coding and 3 UTR sequences of RPB11B1 were excised with BamHI and EcoRI and inserted into the same sites in pBS-S/K+. Probes were prepared by digesting the former construct with Sau3A and the latter with BseRI (Fig. 2A) followed by in vitro synthesis using T7 RNA polymerase and 200 Ci/mmol of [-32P]UTP. The 185-nucleotide RPB11-A probe protected a 145nucleotide fragment specific to RPB11-A (and possibly a 113-nucleotide fragment of RPB11B1). The 450-nucleotide RPB11B1 probe protected a 420-nucleotide RPB11B1-specific fragment. As an internal loading control, a 120-nucleotide fragment of the GAPDH mRNA was detected using a 175-nucleotide riboprobe [36]. RNase protection assays were performed as described by the manufacturer (Ambion, Austin, TX), using 20 g total RNA. Protected fragments were analyzed on 6% polyacrylamide/8 M urea gels that were dried before exposure to Kodak Biomax MR, single emulsion film for 1 hour or 24 hours. Specific message levels were quantified by PhosphorImage analysis. Glutathione S-transferase pull-down assay. Complementary DNAs encoding RPB11B1 and RPB11-A were cloned downstream and in-frame with glutathione S-transferase (GST) in the vector pGEX-2T (Amersham-Pharmacia Biotech, Piscataway, NJ). The full-length cDNA encoding RPB11B1 (also included 3 UTR sequences) was excised from pB42AD with HincII and XhoI (the 5 overhang was filled in with Klenow large fragment). The cDNA was then ligated into the SmaI site of the vector (pGEX-RPB11B11–158). Deletions from the C terminus were prepared by digesting with BseRI or StuI to excise sequences encoding 52 or 33 amino acids, respectively, from the 3 end (pGEX-RPB11B11–106 and pGEXRPB11B11–125). A construct deleted of 15 C-terminal residues was prepared by PCR amplification from the Jurkat cDNA library, using the primer pair 5ACAAACGGATCCATGAACGCCCCTCCAGCCTT-3 and 5-GCCGCGGAATTCTCACCGCCGCCTCTCCCTGA-3. Unique restriction enzyme sites for cloning into the BamHI and EcoRI sites of pGEX-2T are shown in bold and the translation initiation and termination sites are underlined (pGEX-RPB11B11–143). RPB11-A cDNA was excised from pBSK/S+ with BamHI and XbaI (XbaI end-filled in with Klenow large fragment) and religated into the BamHI and SmaI sites of pGEX-2T (pGEX-RPB11-A). Chimeric molecules were prepared in which the C terminus of RPB11-A was replaced by the C terminus of RPB11B1, and vice versa. The constructs, pGEX-RPB11-A or pGEX-RPB11B1, were digested with BseRI and EcoRI, both 5 and 3 end fragments were gel purified, followed by ligation of the RPB11-A 3 to the RPB11B1 5 and vice versa. All constructs were sequenced in their entirety. GST fusion proteins were prepared from the pGEX-2T-cDNA constructs and purified on agarose beads using standard protocols (Amersham-Pharmacia Biotech, Inc., Piscataway, NJ). The GST pull-down assay was used as described [38] to confirm the interactions between SATB1 and target proteins. SATB1 and SATB1 deletion constructs were labeled with 35S-methionine in the TNT-coupled transcription/translation system (Promega, Madison, WI). This test protein (5 l) was incubated for 15
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minutes on ice with 200 l of E. coli (BL21) protein extract [38], in bead binding buffer (BBB; 50 mM potassium phosphate, pH 7.5, 150 mM KCl, 1 mM MgCl2, 10% glycerol, 1% Triton X-100). After centrifugation for 15 minutes, at 4 C, at 14,000 rpm in a microcentrifuge, the supernatants were transferred to a second set of tubes containing the GST fusion protein (bait) bound to agarose beads, in another 200 l of BL21 protein extract in BBB. The test and bait proteins were then incubated for 2 hours at 4 C on a rotary shaker. After the reaction was complete, the beads were washed three times in BBB at 4 C. After careful removal of all liquid, 20 l of 1 SDS sample buffer was added to the beads containing SATB1 and interacting RPB11, mixed, and boiled for 5 minutes. The samples were mixed again, centrifuged briefly, and then fractionated on denaturing polyacrylamide gels. The gels were dried and exposed to Kodak Biomax MR, single emulsion film for 2–5 days. -Galactosidase assay. The relative strengths of interaction between SATB158–763, or 3 and 5 deletions of SATB1, and RPB11-A and RPB11B1 were measured using the quantitative -galactosidase assay (YPH; Clontech, Palo Alto, CA). RPB11-A1–117 (full-length) and RPB11-A1–107 (deleted of the C terminus) were prepared by excising an EcoRI and XbaI fragment (XbaI end-filled in with Klenow large fragment followed by ligation of an EcoRI linker) or an EcoRI and BseRI fragment (BseRI end-filled in with T4 DNA polymerase followed by EcoRI linker attachment) from pBS-RPB11-A. These fragments were subsequently inserted, independently, into the EcoRI site of the yeast vector pB42AD. The constructs were transformed into the yeast strain EGY48-[p8op-LacZ] and resulting strains were mated with the yeast strain YM4271, which harbored the SATB1 full-length and deleted constructs in the vector pLexA. Independent colonies from representative yeast were grown overnight in selective medium using raffinose (a noninducing sugar) as the carbon source. The cells were washed and then induced for 4 hours in galactose-containing selective medium. Each independent colony was assayed in duplicate, using ONPG as the substrate. The assay was repeated at least three times, in duplicate, for each yeast two-hybrid clone. Positive controls were pLexA-Pos (a single plasmid control) and pLexA-53 + pB42AD-T (tests for interaction of p53 with the large T antigen). Negative controls were pLexA + pB42AD, pLexA-53 + pB42AD, and pLexA + pB42AD-T, none of which produces fusion proteins that can activate the -galactosidase promoter.
ACKNOWLEDGMENTS We thank Marc Vigneron (CNRS-INSERM-Universite Louis Pasteur) for the RPB11-A cDNA clone; Mark Sherman (Beckman Research Institute, City of Hope) for assisting with 3D modeling of the pol II, subunit 11; Louis Geller and Jan-Erik Gustaveson (Beckman Research Institute, City of Hope) for sequence analysis; Kimberly Karlsberg (Beckman Research Institute, City of Hope) for assisting with graphics; and members of the Krontiris laboratory for helpful discussions. This work was supported by a grant from the National Institutes of Health (CA51985) and by the Beckman Research Institute of the City of Hope. RECEIVED FOR PUBLICATION SEPTEMBER 17, 2001; ACCEPTED MARCH 13, 2002. Note added in proof. While this paper was undergoing review, Grandemange et al. [37] published a manuscript confirming and increasing the characterization of the POLR2J family described here.
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GENOMICS Vol. 79, Number 6, June 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
Article
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