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Department of Molecular Physiology and Biophysics,1 Department of Biochemistry and Molecular Biology Baylor College of Medicine, Houston, Texas 770302
Received 12 November 2002/ Returned for modification 23 December 2002/ Accepted 23 February 2003
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ABSTRACT |
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30-amino-acid motif at the N terminus of p220 that has the characteristics of a LisH motif. LisH motifs are found in a large number of proteins in the database but are of unknown function. Point mutations in conserved residues in the LisH motif of p220 block histone H4 transcriptional activity without affecting localization in Cajal bodies or phosphorylation on Cdk2 phosphorylation sites. These studies indicate that the ability of p220 to promote S phase is independent of its ability to promote histone H4 transcription and suggests that p220 may link cyclin E/Cdk2 to multiple independent downstream functions. |
INTRODUCTION |
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DNA synthesis is accompanied by additional transcriptional programs that promote completion of replication. One of these is the activation of core histone synthesis that occurs as cells enter S phase. Histones are the building blocks of nucleosomes and are required for packaging of newly synthesized DNA into chromatin. Histone synthesis is complex and is regulated by multiple mechanisms which together provide an 35-fold increase in the quantity of histone mRNA during S phase (20; for a review, see reference 21). Histone gene transcription is activated 5-fold as cells enter S phase (20). Linker histone (H1) and core histones (H2A, H2B, H3, and H4) are relatively unusual in that the cell requires a large number of gene copies to support adequate nucleosome formation during DNA replication, and it appears that gene clustering (3) provides a means by which to coordinate the expression of different classes of histone genes. There are both common and specific cis-acting regulatory elements in replication-dependent histone genes (21). Both subtype-specific consensus elements (SSCEs) and the common YY1 element located in the structural gene contribute to cell cycle-regulated transcription, and in some cases, trans-acting factors that interact with SSCEs have been identified (Oct1 in the case of H2B and IFR2 in the case of histone H4) (4, 5, 11, 12, 24, 29, 33-35). In addition, histone mRNA is stabilized during S phase. This process involves recognition of a stem-loop element in the 3' untranslated region of histone mRNAs by the stem-loop binding protein (SLBP1) followed by RNA processing and stabilization in the cytoplasm (13, 30). One level of cell cycle control comes from the fact that SLBP1 is translated only during S phase and is rapidly destroyed as cells complete DNA synthesis (36).
Histone synthesis is thought to be linked to the basic cell cycle machinery by the cyclin E/Cdk2 substrate p220NPAT. p220 was identified in screens for cyclin E substrates (22, 39) and demonstrated to promote S-phase entry from G1 when overexpressed (39). Like many other Cdk substrates (1, 22, 31, 41), p220 interacts with cyclin E through a cyclin-binding RXL motif located between residues 1062 and 1066 (Y. Wei and J. W. Harper, unpublished data). Insight into p220 function came from the finding that p220 is localized in discrete nuclear organelles called Cajal bodies in a cell cycle-regulated manner in normal diploid fibroblasts (23, 40). Although Cajal bodies were initially described in 1903 (8), their precise functions are still emerging (18, 27). Studies of both Xenopus and mammalian systems suggest that they function, at least in part, as sites of assembly of transcription and splicing complexes (17; reviewed in references 18 and 27). In particular, there is strong evidence that Cajal bodies are responsible for maturation of snRNPs and nuclear RNPs and may also be the sites of assembly of RNA polymerase complexes (18, 25, 27). In G1 normal diploid fibroblast cells, p220 is localized in two Cajal bodies, and these are physically tethered to histone gene clusters located at chromosome 6p21 (23, 40). As these cells enter S phase, two additional p220-containing Cajal bodies appear and are tethered to histone gene clusters at 1q21. Both sets of p220-positive Cajal bodies are maintained until the metaphase-anaphase transition, when Cajal bodies are disassembled (23). As cells exit mitosis and reenter G1, p220-containing Cajal bodies reform and colocalize at 6p21. Additional Cajal bodies that lack p220 are also seen in some cell types and are sometimes associated with other chromosomal loci, such as snRNA genes on chromosome 17 (16). Phospho-specific antibodies were used to demonstrate that p220 becomes phosphorylated at the G1/S transition as cyclin E/Cdk2 accumulates in Cajal bodies and that this phosphorylation is maintained until the metaphase-anaphase transition (23). In transformed cells, the number of Cajal bodies appears to be more variable. The mechanism underlying this is not clear, but the facts that additional p220-containing Cajal bodies exist in particular cell types and that these are also linked to histone gene clusters (40) suggest that aneuploidy may be responsible for some of the heterogeneity observed.
Insight into a role for p220 in histone transcription came from the finding that ectopic expression of p220 can activate the expression of histone 2B (H2B) and H4 promoter constructs (40). This activity is potentiated by coexpression of cyclin E/Cdk2 (40) and is inhibited by mutations in Cdk2 sites in p220 (23). These data have led to a model wherein cyclin E/Cdk2 links the S-phase cell cycle machinery with the histone gene transcription program that is critical for completion of DNA replication. Precisely how p220 activates histone transcription is unknown. Although p220 can be cross-linked with histone promoters (40) and its ability to activate transcription requires known cell-cycle regulated promoter elements (23, 40), there is no evidence that it directly interacts with DNA sequences. In addition, the major location of p220, Cajal bodies, is devoid of DNA, indicating that if p220 functions directly in transcription through interaction with chromatin, then this occurs outside Cajal bodies. An alternative mechanism might involve a function for p220 within the Cajal body, such as the assembly or activation of transcription complexes that function to activate histone genes during S phase (23).
A major limitation in the analysis of p220 function is the fact that it lacks obvious functional motifs that would link it to histone transcription or cell proliferation. To begin to understand p220 function, we have performed an extensive mutagenic analysis of the protein using a recombination-based mutagenesis approach we refer to as "lox-scanning" mutagenesis. These studies reveal that the two known activities of p220—G1 acceleration and activation of histone gene transcription—primarily involve two independent functional elements in p220. Sequences at the extreme N terminus are required for activation of histone H2B and H4 transcription. This region displays similarities with an 33-amino-acid motif referred to as a lissencephaly type 1-like homology (LisH) motif. This motif was originally identified in a number of proteins in the database (15), including the protein lissencephaly 1 (Lis-1), which is required for neuronal migration. The precise function of LisH motifs in these proteins is not known, but several proteins containing LisH domains have been linked to microtubule and kinetochore function (15). We found that point mutations in conserved residues in the LisH-like motif of p220 abolish histone H4 transcriptional activation without affecting the localization in Cajal bodies or the ability of the protein to promote S-phase entry. In contrast, sequences in the C terminus are required for efficient G1 acceleration but not histone transcription. The identification of separation of function mutations in p220 indicates that its ability to activate transcription is not linked with its ability to promote G1 progression and suggests the possibility that activation of p220 by cyclin E/Cdk2 links upstream cell cycle signals with independent cellular outputs that both promote completion of S phase.
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MATERIALS AND METHODS |
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LisH, 5'-GGA TCC GCC ACC ATG TTG TTA CCC CAG ACT TTT ATT TTG GAA AGT TCA G-3'; for pCMV-p220VLVLE/A, 5'-GGA TCC GCC ACC ATG TTG TTA CCC TCG GAC GCA GCC CGG GCT GCA TTG GGT TAC GCA CAGCAAGCAAAC CTC ATT TCT AC-3'; for pCMV-p220-F/A, 5'-GGA TCC GCC ACC ATG TTG TTA CCC TCG GAC GTA GCC CGG CTT GTA TTG GGT TAC TTA CAG CAA GAA AAC CTC ATT TCT ACC TGC CAG ACT GCT ATT TTG GAA AGT TCA GAT TTA AAA G-3'; for pCMV-p220-FE/A, 5'-GGA TCC GCC ACC ATG TTG TTA CCC TCG GAC GTA GCC CGG CTT GTA TTG GGT TAC TTA CAG CAA GAA AAC CTC ATT TCT ACC TGC CAG ACT GCT ATT TTG GCA AGT TCA GAT TTA AAA G-3'. In all cases, the reverse primer was 5'-GTT TTC TGC TAG CTT TTC TTG-3'. PCR products were ligated into pCR2.1 (Invitrogen), and mutations were verified by sequence analysis. Mutant fragments were released from pCR2.1 with BamHI/NheI and subcloned into pCMV-p220 to generate the appropriate expression construct. Cell culture and cell cycle analysis. Human U2OS cells and rodent Rat1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C with 0.5% CO2. For transfection, U2OS cells were seeded in 60-mm dishes and cultured overnight to a confluence of 60 to 70%. pCMV-p220 or mutant plasmids (10 µg) and 0.3 µg of pCMV-CD20 plasmid were cotransfected with FuGENE 6 (Roche). The medium was changed 8 h later. Thirty-six hours after transfection, cells were washed with phosphate-buffered saline (PBS) and treated with PBS-0.1% EDTA at room temperature for 10 min. Cells were then collected and washed with PBS prior to incubation with fluorescein isothiocyanate-conjugated anti-CD20 (Becton Dickinson) in PBS on ice for 15 min. Cells washed with PBS-0.1% serum and then fixed with 80% ethanol-PBS at 4°C for at least 1 h. Fixed cells were washed with PBS once and incubated in PBS with 10 µg of propidium iodide/ml and 40 µg of RNase A/ml at 37°C for 30 min. The DNA content of CD20-positive cells was determined by flow cytometry using an EPICS XL-MCL (Beckman-Coulter) instrument.
Luciferase assay. U2OS cells were seeded in 12-well tissue culture dishes and cultured overnight to a confluence of 60 to 70%. pCMV-p220 or mutant plasmids (4 µg), pCMV-LacZ (0.1 µg), and pGL2-histone H4 promoter luciferase plasmids (0.1 µg) (40) were cotransfected by using calcium phosphate. In some experiments, a pGL2-histone H2B luciferase reporter containing the H2BFR promoter from chromosome 6 was used (23). The medium was changed 8 h later. Thirty-six hours after transfection, cells were collected and luciferase assays were performed with a luciferase assay kit (Promega). A ß-galactosidase assay was used to normalize the luciferase activities.
Antibodies and immunofluorescence assays. Generation of polyclonal antibodies against p220 and two phosphorylation sites (T1270 and T1350) were described previously (23). U2OS or Rat1 cells on coverslips were transfected with 4 µg of pCMV-p220 or mutant plasmids. In some cases, 0.5 µg of pCMV-EYFP-coilin/ml (28) was used included in the transfection. Cells were fixed with 4% paraformaldehyde for 10 min and then treated with 0.5% Triton X-100 in PBS for 10 min. p220 was detected with an antibody raised against the C terminus (23) For detection of p220, cells were stained with an anti-C-terminus antibody (37°C for 1 h). Detection was done with Cy3-labeled anti-rabbit antibodies (Amersham). Nuclear DNA was revealed with DAPI (4',6'-diamidino-2-phenylindole) staining. To determine the phosphorylation status of p220, Triton X-100-treated cells were incubated with 3% H2O2 in PBS for 10 min and subsequently incubated with antigen retrieval solution (Dako) at 95°C for 30 min. Cells were incubated with antibodies against P-T1270 or P-T1350 in PBS for 1 h and then with horseradish peroxidase-conjugated anti-rabbit antibody (Roche) in PBS for 1 h. A tyramide signal amplification system (NEN) was used to detect the secondary antibodies. Cells were then fixed with 4% paraformaldehyde in PBS for 10 min prior to staining with anti-p220 C-terminus antibodies as described above. DNA was revealed with DAPI staining. Microscopic analysis was performed with an Olympus BX-60 microscope fitted with a Hamamatsu camera. Images were captured with a 100x objective lens using single-pass filters and merged by using NIH Image Pro software or a Deltavision deconvolution microscope.
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RESULTS |
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100-amino-acid intervals along the entire length of p220 with a 25-amino-acid sequence encoded by a loxP cassette and flanking sequences (Fig. 1B). Replacement of functional elements contained within independent folded domains would be expected to alter the biological activity of p220, which is measured subsequently in cell-based assays. The approach takes advantage of recently developed methods that allow efficient homologous recombination of linear DNA fragments with circular plasmids in E. coli (38). PCR was used to generate recombination substrates containing a total of 120 bp of p220 sequences (corresponding to sequences 5' and 3' of the segment to be replaced) flanking a loxP-Tcr-loxP cassette encoding resistance to tetracycline. This linear template was transformed into recombination-proficient DH10ß cells harboring both pML104 (Spr; encoding 
exonuclease and bet and gam recombination functions as well as RecA) (38) and the recombination target, a pCMV-p220 plasmid (Apr) (Fig. 1A). Recombination events were selected for resistance to tetracycline and ampicillin. Plasmids were then recovered and transformed into pBNN132 cells expressing Cre recombinase in order to excise the Tcr cassette. The identification of appropriate clones was aided by the inclusion of a BamHI restriction site in the loxP cassette (Fig. 1A to C), and mutations were verified by sequence analysis. The resulting set of 12 plasmids encode p220 mutants in which desired sequence elements along the length of p220 have been replaced precisely by residues encoded by the loxP cassette (Fig. 2). Mutant p220 plasmids were tested for expression by immunoblotting after transfection into U2OS cells. All 12 p220 mutant proteins were expressed at levels equal to or higher than that found with wild-type p220 expressed in parallel (Fig. 1D).
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We tested the activity of our p220 lox-scanning mutants in both of these assays. The data are summarized in Fig. 2. Consistent with previous results (39), expression of p220 led to an 15% increase in S-phase cells from 25 to 40%, with only a small (2%) increase in the percentage of G2/M cells, as determined by using a CD20 cotransfection flow cytometry assay (Fig. 3A and B). The majority of lox-scanning mutants, including mutants 1, 2, 3, 4, 6, 8, and 10, were also found to elevate the percentage of S-phase cells to an extent comparable to that seen with wild-type p220 (8 to 14% increase), as measured over five replicate experiments (Fig. 3A to C). However, five mutants—5 (LoxP629-653), 7 (LoxP828-853), 9 (LoxP1039-1054), 11 (LoxP1128-1252), and 12 (LoxP1325-1349)—displayed substantially reduced activity in this assay. Mutant 5 showed the most dramatic reduction, as it increased S phase by only 3% over five independent experiments (P < 0.01), while mutants 7, 9, 11, and 12 displayed 5 to 7% increases in S phase (P < 0.05) (Fig. 3A to C). These data indicate that much of the C terminus of p220 is critical to its ability to promote S phase when overexpressed.
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30%), but when it was combined with a mutation in the highly conserved Glu-30 residue, activity was reduced to 20% (Fig. 6F). In stark contrast, the LisH p220 mutant displayed essentially wild-type activity in the G1 acceleration assay (Fig. 6G). Moreover, all of the LisH mutant p220 proteins localized in Cajal bodies when expressed in Rat1 cells (Fig. 5 and data not shown). These data strongly argue that the LisH-like motif at the N terminus of p220 is critical to its ability to activate histone H4 reporter constructs.
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LisH and lox-scanning mutant 1 were compared with that of wild-type p220. Consistent with previous results (23, 40), expression of p220 led to an 3-fold increase in reporter activity (Fig. 6H). In contrast, lox-scanning mutant 1 displayed reporter activity comparable to that found in the absence of p220. The LisH mutant displayed intermediate activity, consistent with the results found with the H4 reporter (Fig. 6H). These data indicate that the N terminus of p220 also contributes to activation of the H2B promoter.
LisH mutants in p220NPAT are not defective in Cdk-mediated phosphorylation.
Previous studies have demonstrated that phosphorylation of p220 is important for its ability to activate histone transcription and to promote S-phase entry in U2OS cells. Overexpression of cyclin E/Cdk2 potentiates both the activation of histone H4 luciferase reporter constructs and the accumulation of S-phase cells by p220 (39, 40), while mutation of five Cdk2 phosphorylation sites reduces activity in histone H4 reporter assays (23). Thus, we considered the possibility that defects in p220 function observed with LisH and lox-scanning mutants might reflect defects in Cdk2-mediated phosphorylation which could possibly be rescued by coexpression of cyclin E/Cdk2. To begin to address this question, we transfected pCMV-p220 expression plasmids with and without plasmids expressing cyclin E and Cdk2 into U2OS cells, and histone H4-luciferase activity was then measured (Fig. 7A). While the luciferase activity of wild-type p220 was stimulated 6-fold by coexpressed cyclin E/Cdk2, the activities of the LisH motif mutants were increased less than 2-fold. In empty vector control experiments (Fig. 7A), cyclin E/Cdk2 cotransfection also led to a 2-fold increase in background luciferase activity. These data indicate that defects in p220 LisH mutant protein function cannot be substantially enhanced by high levels of cyclin E/Cdk2.
Consistent with these data, we found that all p220 LisH mutant proteins retain the capability of being phosphorylated on Cdk sites. We performed immunofluorescence assays using anti-phospho-T1270 and anti-phospho-T1350 antibodies after transfection of p220 mutants in U2OS cells (Fig. 7B). These antibodies have been demonstrated to react with p220 in Cajal bodies during S phase in normal diploid fibroblasts (23). Cells expressing ectopic wild-type or mutant p220 proteins display anti-p220 staining throughout the nucleoplasm (Fig. 7B), as expected (16). All of the LisH mutant proteins were found to be phosphorylated on both T1270 and T1350, while p220Cdk (23), used as a control for phospho-antibody specificity, was devoid of staining with either phosphorylation site antibody (Fig. 7B). In contrast, certain lox-scanning mutants, in particular, mutants 9 to 12, were found to be defective in T1350 phosphorylation but not in phosphorylation of T1270 (Fig. 2 and 7B). In the case of mutant 12, the loxP replacement cassette is located adjacent to T1350 and could interfere directly in kinase recognition. However, mutants 9 to 11 are located some distance from T1350 in the linear sequence. Interestingly, mutant 10 was not phosphorylated on T1350 but was active in both the G1 acceleration assay and the histone transcription assay, suggesting that phosphorylation at this site is not critical to either of these activities. In addition, p220(LoxP629-653), which is the most severely affected with respect to G1 acceleration activity was found to be phosphorylated on both T1270 and T1350, suggesting that defects in phosphorylation are not responsible for the absence of S-phase-promoting activity with this p220 mutant.
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DISCUSSION |
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In this study, we have begun to explore how p220 functions in histone transcription and cell proliferation through the identification of functional domains. p220 contains a RXL-like motif (residues 1062 to 1066) (Fig. 6) that in other Cdk substrates is known to facilitate interaction with the cyclin (1, 31, 41). We found that replacement of the RVL sequence by AVA leads to a loss of cyclin E/Cdk2 binding in vitro (data not shown). In addition, short peptides containing wild-type (but not mutant) sequences from the E2F-1 RXL motif block association of p220 with cyclin E/Cdk2 in vitro. However, p220AVA is still phosphorylated by cyclin E/Cdk2 in vitro on at least a subset of sites, and it is not substantially defective in either S-phase-promoting activity or histone H4 reporter activation when transiently expressed (data not shown). Whether endogenous levels of p220 would require an intact RXL motif for function remains unknown.
Outside of this RXL motif, p220 lacks obvious functional domains that would link it mechanistically to known regulatory processes. Moreover, p220 is a large protein, making systematic analysis of functional domains difficult. We developed an approach we call lox-scanning mutagenesis (Fig. 1) as an approach to systematically analyze p220 sequences for functional activity. The approach is analogous to linker-scanning mutagenesis, which is frequently employed with smaller proteins, in that the LoxP replacement would be expected to disrupt the structure of folded domains and would therefore be expected to reveal regions of the protein important for function. Using this approach, we have identified multiple elements important for p220 function and have found that the ability of p220 to promote histone transcription can be genetically uncoupled from its ability to promote S phase.
The ability of p220 to promote the G1/S transition depends heavily upon sequences located in the C-terminal half of p220 (Fig. 2 and 3). lox-scanning mutants that have replacements of residues 629 to 653, 1228 to 1252, and 1325 to 1349 displayed reduced activity in cell cycle assays. Residues 629 to 653 appear to be most critical in this regard. This region is proximal to two previously identified Cdk sites, S775 and S779 (23). Whether this mutation affects phosphorylation at these sites is unknown, as phospho-specific antibodies against these sites are not available, but phosphorylation at T1270 and T1350 was not affected by this mutation. Two additional mutations that affect the G1 acceleration activity (LoxP1228-1252 and LoxP1325-1349) are located at the extreme C terminus of p220. Both of the mutant proteins are defective in phosphorylation of T1350 but not T1270. However, the fact that p220(LoxP1125-1149) is active in the G1 acceleration assay but is also not phosphorylated on T1350 indicates that this event is not required for S-phase promotion.
In contrast with the cell cycle function of p220, the histone H4 transcriptional activity appears to depend primarily on the N terminus (Fig. 2, 4, and 6). lox-scanning mutant 1, affecting residues 121 to 145, displayed the greatest reduction in activity toward the histone H4 promoter (20-fold). Importantly, this mutant displayed activity in cell cycle assays that was similar to that found with wild-type p220 (Fig. 3), indicating that transcriptional activity toward the H4 promoter is not required for the G1 acceleration function of p220. Additional lox-scanning mutants, particularly those affecting residues 325 to 349 (mutant 3) and 629 to 653 (mutant 5), also displayed significantly reduced activity (3-fold). Interestingly, mutant 5 was also defective in G1 acceleration. These data suggest that the region defined by mutant 5 may contribute to both cell cycle acceleration and histone transcription.
Sequence analysis of the extreme N terminus of p220 led to the identification of a LisH-like motif in p220 (Fig. 6). This 33 amino acid motif contains a series of conserved largely hydrophobic residues (9). To examine whether this motif might be involved in p220 function, we performed further mutagenic studies focusing on residues that are conserved in this motif. Deletion of this domain decreased activity toward histone H4 reporter constructs to an extent similar to that found with lox-scanning mutant 1. Additional point mutations in conserved residues reduced activity at least 10-fold. In contrast, none of the LisH mutations significantly affected the G1 acceleration activity of p220. These data indicate that this LisH domain is critical to the ability of p220 to activate histone transcription but is not required for its ability to promote S-phase entry. Consistent with this notion, we found that a p220 deletion mutant lacking residues 121 to 426 was fully active in promoting S phase and localized in Cajal bodies yet was defective in H4 reporter activation (data not shown). Given the close proximity of the LisH motif and sequences disrupted in lox-scanning mutant 1 (residues 121 to 145), it is possible that they act together to promote histone transcription. Alternatively, residues 121 to 145 may represent a second N-terminal domain that is involved in histone transcription independently of the LisH motif. Structural studies are required to determine if these two regions form a single functional domain.
p220 is localized in small nuclear organelles called Cajal bodies (23, 40). p220-containing Cajal bodies are associated with histone gene clusters on chromosome 6p21 during G1, S, and G2 phases of the cell cycle and with histone gene clusters on chromosome 1q21 during S and G2 phases. How p220 is targeted to these organelles and whether this localization is required for function is unknown. One potential explanation for the absence of activity of p220 mutants is that they do not localize properly to Cajal bodies. We tested each of the mutants examined here for their localization in Cajal bodies by transfection into Rat1 cells (Fig. 5). Surprisingly, all of the mutants examined localized specifically in Cajal bodies, as determined by colocalization with EYFP-coilin, analyzed in parallel. Thus, effects on transcription and cell cycle function do not appear to reflect an inability of the mutant proteins to localize in these organelles. We cannot, however, rule out the possibility that there are redundant sequences that target p220 to Cajal bodies and that within the Cajal body, the mutant p220 proteins are defective in association with other proteins that could independently serve to tether the p220 protein. Given the extent to which our panel of lox-scanning mutants explore p220 structure, it seems likely that a short linear sequence in p220 is involved in targeting it to Cajal bodies. The mechanisms responsible for localization of other Cajal body constituents is largely unknown, although it is clear that many Cajal body proteins display dynamic association with this organelle (reviewed in reference 27). Coilin itself requires its N terminus to associate with Cajal bodies, and this region is subject to specific arginine methylation (reviewed in reference 27). It is unclear whether p220 itself might be recruited to Cajal bodies in a coilin-dependent manner or whether it might require particular modifications for proper localization.
A major finding of this study is that p220 employs a LisH-like motif to promote histone transcription. LisH motifs are found in bacteria, fungi, and plant and animal proteins. Although LisH-containing proteins frequently have additional domains (15), such as WD40 repeats found in the human Lis1 and Drosophila Ebi proteins, there are a number of proteins in the database which, like p220, contain only LisH domains. Interestingly, p220 is not the only protein with a LisH domain that is involved in transcriptional regulation. Human TBLR1/IRA1 (Fig. 6A) and budding yeast Sif2p both contain an N-terminal LisH domain and C-terminal WD40 repeats. TBLR1/IRA1 is a component of an HDAC3 corepressor complex (37), while Sif2p is a component of the Sir4 histone deacetylase transcriptional silencing complex. Deletion of Sif2 in budding yeast leads to increased telomeric silencing (10). The precise functions of LisH motifs are unknown. In Lis1, the LisH motif is located on the N terminus of the protein and is adjacent to a coiled-coil domain, and this N-terminal fragment is known to be involved in Lis1 dimerization (2, 7). Given the known role of coiled-coil domains in dimerization, it seems likely that this motif is involved in the dimerization process, although a role for the LisH domain cannot be ruled out. It seems likely that LisH motifs function as protein interaction surfaces. Given the diverse array of proteins that contain this domain (Fig. 6B), it is probable that these different motifs confer binding to distinct targets in various settings. The identification of a functionally important LisH-like motif in p220 provides a starting point for the identification of proteins that collaborate with p220 to control histone gene transcription.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Institutes of Health (GM54137) and the Welch Foundation to J.W.H. Y.W. was supported by a Department of Defense predoctoral fellowship. J.J. was supported by a Department of Defense postdoctoral fellowship.
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FOOTNOTES |
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