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Fan Zhang,
Takeshi Kurosu, and
B. Matija Peterlin*
Department of Medicine, Microbiology and Immunology, Rosalind Russell Medical Research Center, University of California at San Francisco, San Francisco, California 94143-0703
Received 20 April 2005/ Returned for modification 30 June 2005/ Accepted 3 October 2005
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Positive transcription elongation factor b (P-TEFb) counteracts N-TEF and prepares RNAPII for elongation (29). P-TEFb contains a type C cyclin (CycT1, CycT2, or CycK) and cyclin-dependent kinase 9 (Cdk9). Although CycT2 is expressed in two isoforms (CycT2a and CycTb), no functional differences between them have been observed (29). In this study, we used CycT2b and called it CycT2. P-TEFb phosphorylates Spt5 in DSIF (12), RD in NELF (8), and serines at position 2 in 52 heptapeptide repeats that constitute the C-terminal domain (CTD) of RNAPII (29). This process converts DSIF into an elongation factor, dissociates NELF from nascent transcripts, and exchanges the Mediator for the cotranscriptional processing machinery on RNAPII (29). The latter contains 5' capping, intronic splicing, and 3' polyadenylation factors. P-TEFb is required not only for the expression of most eukaryotic genes but also for replication of the human immunodeficiency virus (HIV) (36). In this system, the viral transactivator Tat recruits P-TEFb to the transactivation response TAR RNA. The formation of this ternary complex stimulates high levels of transcription and replication of HIV.
Studies in our laboratory have found that activators and repressors bind CycT1 in different ways to exert their effects on transcription. For example, activators such as CIITA (13), NF-
B (1), and c-Myc (6, 14) bind cyclin boxes in CycT1 and recruit P-TEFb to transcription units. This binding allows P-TEFb to phosphorylate its targets. Since P-TEFb can increase rates of elongation of transcription from sequences far upstream and downstream of promoters and coding sequences, this finding also suggested that eukaryotic enhancers could function by this mechanism (37). In sharp contrast, repressors such as CTD analogs and PIE-1 bind the C-terminal histidine-rich stretch in CycT1 (43). This region in CycT1 is also required for the substrate recognition of Cdk9 (37). Thus, these repressors block enzymatic effects of P-TEFb. In this scenario, RNAPII still initiates on promoters but can no longer elongate on their cognate genes.
In this study, we wanted to extend our findings to proteins that can mediate active repression in hematopoietic cells. Runx1, which is also called acute myeloid leukemia 1, polyomavirus enhancer-binding protein 2
-B, or core-binding factor 2, belongs to a family of Runt domain proteins (11). Null mutations in Runx1 result in loss of definitive hematopoiesis due to the absence of hematopoietic stem cells (26). It is also the most frequent target of chromosomal rearrangements and/or point mutations in human leukemias (5, 22, 27). Although in certain contexts, such as the T-cell antigen receptor, it activates transcription (20); in other contexts, such as the CD4 gene in immature thymocytes, Runx1 functions as a potent transcriptional repressor (34). Negative effects of Runx1 are mediated via three distinct domains. The transducin-like enhancer of split (TLE) proteins, which are the mammalian homologues of the Groucho family of corepressors (Groucho/TLE), bind the VWRPY sequence at the extreme C terminus of Runx proteins (16). Sin3A binds sequences spanning positions 181 to 210 in Runx1 (19). Yet, an important inhibitory domain (ID; positions 371 to 411) next to the activation domain (positions 291 to 371) (15) and nuclear matrix targeting sequence (NMTS; positions 318 to 358) (42) in Runx1 has no known partner and no known mechanism of action. In this study, we demonstrated that this domain binds CycT1, inhibits effects of P-TEFb, and blocks transcriptional elongation by RNAPII.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids. Plasmid target pG6L6CAT was made by inserting two oligonucleotides into the plasmid reporter pHIVSCAT (18). The first one contained six modified LexA operator sequences (5'-GTACTGTATGTACATACAGTAC-3') and was inserted into the XmaI site, whereas the second one contained six modified Gal4 DNA-binding sequences (5'-CGGAGTACTGTCCTCCGAG-3') and was inserted into the KpnI site, which is located 442 bp upstream of LexA operator sites.
The EF.Gal.CycT1 plasmid was described previously (37). EF.Gal.CycT2 was made by inserting the PCR product of the full-length CycT2b cDNA into EcoRI and XbaI sites of EF.Gal.CycT1 to replace CycT1.
The pCLex87 plasmid was described previously (43). To construct plasmids encoding full-length Lex.Runx1(1-451) and the mutant Lex.Runx1(1-274), Lex.Runx1(259-451), Lex.Runx1(259-446), Lex.Runx1(259-367), and Lex.Runx1(259-327) chimeras, various cDNA fragments of Runx1 were amplified by PCR and cloned subsequently into EcoRI and XhoI sites of the pCLex87 vector. The DNA template for PCR was from Gal.Runx1, a gift from Dan Littman.
To create the plasmid encoding the Flag epitope-tagged wild-type Runx1 protein, a Flag epitope sequence was incorporated into the 5' primer. PCR fragments amplified from Gal.Runx1 were inserted into HindIII and XhoI sites of the pcDNA3.1Hygro expression vector (Invitrogen, Carlsbad, CA).
Plasmids encoding the mutant Gal.Runx1(1-446) and Gal.Runx1(1-367) chimeras were constructed by deletion PCR from Gal.Runx1 with primers encompassing Runx1 amino acids 441 to 446 or 362 to 367 immediately followed by a stop codon and the vector sequences following the stop codon.
Plasmid target pUAS/Runx-CAT was a gift from Dan Littman (34).
Transient transfections and CAT assays. When using the pG6L6CAT plasmid target, HeLa cells were cotransfected with pG6L6CAT (0.4 µg) and different plasmid effectors (total, 1.6 µg) with Lipofectamine according to the manufacturer's instructions (Invitrogen). The ratio of plasmids encoding activators (Gal.CycT1 or Gal.CycT2b) versus repressors (LexA fusion proteins) was 1:1. All transfections were balanced to the total 2.0 µg of DNA with the appropriate empty vectors. When using the pUAS/Runx-CAT plasmid target, HeLa cells were cotransfected with pUAS/Runx-CAT (0.6 µg) and plasmids encoding Gal.Runx1 chimeras (0.6 µg) with Lipofectamine PLUS reagent according to the manufacturer's instructions (Invitrogen). At 48 h after transfection, cells were lysed in lysis buffer (0.1% Triton X-100, 0.25 M Tris-HCl, pH 7.5). Chloramphenicol acetyltransferase (CAT) enzymatic assays were performed as previously described (7). The activity of the reporter plasmid alone was set to 1. Three independent transfections were performed in duplicate.
In vitro GST pulldown assays. For glutathione S-transferase (GST) pulldown assays, the wild-type and mutant LexA.Runx1 fusion proteins were transcribed and translated in vitro in the presence of 35S-labeled methionine with the TNT T7-coupled rabbit reticulocyte lysate system as instructed by the manufacturer (Promega Biotech, Madison, WI). GST and GST.CycT1 fusion proteins were expressed in Escherichia coli strain BL21(DE3)/pLysS competent cells (Novagen Bioscience, San Diego, CA), purified, and bound to glutathione-conjugated Sepharose beads as previously described (13).
Twenty micrograms of GST or the GST fusion protein was incubated with 20 µl of 35S-labeled LexA.Runx1 chimera in 300 µl of binding buffer (20 mM HEPES at pH 7.9, 1% Triton X-100, 20 mM dithiothreitol, 5 mM EDTA, 0.5% bovine serum albumin, 100 mM KCl) at 4°C with gentle agitation for 2 h. After binding, beads were washed four times with binding buffer and bound proteins were eluted by boiling in sodium dodecyl sulfate (SDS) sample buffer and resolved by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE); the gel was dried and analyzed by autoradiography.
Western blotting and immunoprecipitations. To determine the expression levels of proteins, 293T cells were cultured for 30 to 35 h after transfection with various plasmids (2 µg). Cells were lysed in lysis buffer after harvesting and subjected to SDS-PAGE and Western blotting. Goat polyclonal anti-LexA antibody (sc-1726; Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect the expression of the wild-type and mutant Lex.Runx1 fusion proteins. Mouse monoclonal anti-Flag M2 antibody (F3165; Sigma-Aldrich, St. Louis, MO) and rabbit polyclonal anti-Myc antibody (sc-789; Santa Cruz Biotechnology) were used to detect the expression of the Flag epitope-tagged Runx1 and Myc epitope-tagged CycT1 proteins. Mouse monoclonal anti-Gal4 DNA-binding domain (DBD) antibody (sc-510; Santa Cruz Biotechnology) was used to detect the expression of the Gal.Runx1 chimeras. Western blotting was performed as previously described (7).
For immunoprecipitations, 293T cells, which expressed the Flag epitope-tagged Runx1 and Myc epitope-tagged CycT1 proteins, were lysed in lysis buffer (1% NP-40, 10 mM Tris-HCl at pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, proteinase inhibitor cocktail [Sigma-Aldrich]) and immunoprecipitated with the anti-Flag antibody at 4°C for 1 h. Following binding to the antibody, reaction mixtures were incubated with protein A-Sepharose beads at 4°C for 1 h. After extensive washes with lysis buffer, immunoprecipitated products were eluted and subjected to SDS-PAGE, followed by Western blotting with the anti-Myc antibody.
Chromatin immunoprecipitation (ChIP) assays. ChIP was carried out essentially as described previously (25). For HeLa cells, cross-linking was achieved by incubating cells on a 10-cm plate with 10 ml of 1% formaldehyde in phosphate-buffered saline for 10 min at 37°C. Cross-linking reactions were stopped by addition of glycine to a final concentration of 0.125 M. Cells were then washed with phosphate-buffered saline and pelleted in an Eppendorf tube. One milliliter of buffer A (5 mM PIPES at pH 8.0, 85 mM KCl, 0.5% NP-40) was added to the cells, and the mixture was incubated for 20 min on ice. After spinning, the isolated nuclei were lysed in 200 µl of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl at pH 8.0) for 10 min on ice. The 200 µl of lysate was diluted with immunoprecipitation buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl at pH 8.0, 167 mM NaCl) to a final volume of 2 ml and then subjected to sonication to obtain DNA fragments averaging approximately 200 to 500 bp in length.
One-tenth of the total chromatin solution was used in each ChIP. Chromatin solutions were precleared with protein A/G-Sepharose beads and then incubated with the rabbit polyclonal anti-RNAPII antibody (sc-899; Santa Cruz Biotechnology) at 4°C overnight. Protein A/G-Sepharose beads were then added, and the mixture was incubated for another 2 h. The beads were washed five times in TSE-150 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl at pH 8.0, 150 mM NaCl), TSE-500 (like TSE-150 but with 500 mM NaCl), and buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl at pH 8.0) and twice in Tris-EDTA buffer. Immunocomplexes were eluted from the beads with elution buffer (1% SDS and 0.5% NaHCO3) for 30 min at 65°C. The DNA-protein complexes were then treated with proteinase K, followed by reverse cross-linking at 65°C overnight. DNA was extracted with phenol-chloroform, precipitated with ethanol, and dissolved in 40 µl of Tris-EDTA buffer. Two microliters of DNA was used with appropriate primer sets to amplify specific DNA fragments. PCRs were carried out for various cycles. Those where amplification was in the linear range were quantified by densitometry. Relative intensity was calculated and normalized to the input.
For 3A9 and 1200M cells, cross-linking was achieved by incubating 50 million cells in 1% formaldehyde in medium for 10 min at room temperature. Cells were then pelleted in a conical tube and washed with cold phosphate-buffered saline. The cell pellets were then resuspended in 1 ml of homogenization buffer (10 mM HEPES at pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1% NP-40), incubated for 10 min on ice, and homogenized with 10 strokes of a B pestle in a Dounce homogenizer. After spinning, the isolated nuclei were lysed in 500 µl of lysis buffer for 10 min on ice and subjected to sonication to obtain DNA fragments averaging approximately 200 to 500 bp in length.
One-fifth of the total chromatin solution was used in each ChIP. Chromatin solutions were diluted 10-fold in TES-150 buffer, precleared with protein A/G-Sepharose beads, and then incubated with the rabbit polyclonal anti-RNAPII antibody at 4°C overnight. Protein A/G-Sepharose beads were added, and the mixture was incubated for another 2 h. The beads were washed as described above. Immunocomplexes were eluted from the beads with elution buffer for 15 min at room temperature. Reverse cross-linking was performed at 65°C for 4 h and followed by treatment with proteinase K. DNA was extracted with phenol-chloroform, precipitated with ethanol, and dissolved in 30 µl of Tris-EDTA buffer. Two microliters of DNA was used with appropriate primer sets to amplify specific DNA fragments. PCR products, taken at various cycle numbers, were separated on agarose gel and visualized with SYBR green. PCR products at cycles where amplification was in the linear range were quantified by densitometry. Relative intensity was calculated and normalized to the input.
Flow cytometry. One million 3A9 or 1200M cells were stained with fluorescein isothiocyanate-conjugated anti-CD4 antibody or rat immunoglobulin G control antibody (5 µg/ml; BD Biosciences, San Diego, CA) on ice for 20 min, washed twice in phosphate-buffered saline containing 2% FBS, and analyzed on a FACScalibur (Becton Dickinson, San Jose, CA).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When we coexpressed the Gal.CycT1 or Gal.CycT2 fusion protein alone with pG6L6CAT, CAT activities were increased 35- or 45-fold over basal levels, respectively, in HeLa cells (Fig. 1B, compare bars 1, 2, and 9). This observation is consistent with previous data showing that CycT2 forms a more potent P-TEFb complex (24). This activation was decreased fivefold when we coexpressed the Lex.PIE-1 fusion protein with our Gal.CycT1 or Gal.CycT2 chimera (Fig. 1B, compare bars 2 and 3 and bars 9 and 10). Interestingly, we found that the wild-type Lex.Runx1 fusion protein repressed effects of P-TEFb by greater than twofold (Fig. 1B, compare bars 2 and 4 and bars 9 and 11). To dissect these effects further, we coexpressed a series of mutant Lex.Runx1 fusion proteins with our activators and found that the mutant Lex.Runx1(259-451) fusion protein repressed transcription even better than PIE-1 (Fig. 1B, compare lanes 3 and 5 and lanes 10 and 12). When its C-terminal five residues (VWRPY) were deleted, the mutant Lex.Runx1(259-446) fusion protein still retained most of its repressing activity (Fig. 1B, compare lanes 5 and 6 and lanes 12 and 13). This finding revealed that Groucho/TLE is not required for this repression. In sharp contrast, further C-terminal deletions of Runx1 to positions 367 and 327 (mutant Lex.Runx1(259-367) and Lex.Runx1(259-327) proteins) resulted in the loss of its repressing activity (Fig. 1B, compare lanes 7, 8, 14, and 15 to lanes 3 to 6 and 10 to 13). We also observed that the N terminus of Runx1, spanning positions 1 to 274, did not inhibit transcription in this assay (data not presented). The expression levels of these Runx1 chimeras were comparable (Fig. 1B, bottom). We conclude that the Runx1 sequences spanning positions 367 to 446 block effects of P-TEFb on transcription. It is important to point out that these sequences do not bind Sin3A or Groucho/TLE but contain the ID defined by Kanno et al. (15).
Runx1 interacts with CycT1 in vivo. To investigate whether the repressing activity of Runx1 is due to its physical association with P-TEFb, we coexpressed the Flag epitope-tagged Runx1 and Myc epitope-tagged CycT1 proteins in 293T cells. After incubation for 36 h, cells were lysed and immunoprecipitated with the anti-Flag antibody and protein A/G-Sepharose beads. After washing, immunoprecipitated complexes were separated by SDS-PAGE and subjected to Western blotting with the anti-Myc antibody. As presented in Fig. 2, lane 2, Runx1 interacts with CycT1 in vivo.
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As presented in Fig. 3, the wild-type Lex.Runx1 and mutant Lex.Runx1(259-451) and Lex.Runx1(259-446) fusion proteins bound the GST.CycT1 chimera but not GST alone (top panel, compare lanes 1, 3, and 5 to lanes 2, 4, and 6). By contrast, the mutant Lex.Runx1(259-367) and Lex.Runx1(259-327) fusion proteins did not bind the GST.CycT1 chimera (Fig. 3, top panel, compare lanes 7 and 9 to 8 and 10). Interestingly, deletion of the C-terminal VWRPY motif did not decrease the binding between Runx1 and CycT1 (Fig. 3, compare lanes 3 and 5). Thus, our GST pulldown assays defined a region between positions 367 and 446 in Runx1 that is required for binding of CycT1. Equally important, this mapping correlated closely with our functional assays in HeLa cells (Fig. 1), where the mutant Runx1(259-451) and Runx1(59-446) chimeras, which contained these CycT1-binding sequences, possessed strong repressing activities. In sharp contrast, the mutant Runx1(259-367) and Runx1(259-327) chimeras, which lacked this region, had no effect on transactivation by CycT1 or CycT2. Thus, the same domain in Runx1, which was required for binding of CycT1 in vitro and contains the ID, was also required for its repressing activity in vivo.
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The DNA region to be investigated in pG6L6CAT is 2 kb long. Extensive sonication of DNA yielded fragments small enough to resolve promoter-bound and elongating transcription complexes. For each condition tested, three sets of primers complementary to the 1-kb upstream noncoding sequence (A), the promoter sequence (B), and the 700-nucleotide (nt) 3' downstream coding sequence (C) were used. Each primer set amplified a PCR fragment 120 to 150 nt in length (Fig. 4, schematic at the top). Among them, the set of primers amplifying upstream sequences was used as the internal background control (A).
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RNAPII, lanes 1, 4, 7, and 10). Thus, there was no run-through transcription upstream of our promoter. The promoter region of the reporter gene was always occupied by RNAPII (Fig. 4, RNAPII, lanes 2, 5, 8, and 11). This indicates that transcription initiation and promoter occupancy by RNAPII occurred in the absence of upstream activators. In the coding region of the CAT reporter gene (C), the amounts of RNAPII increased 10-fold upon expression of the Gal.CycT1 chimera (Fig. 4, RNAPII, compare lanes 3 and 6). In sharp contrast, coexpression of the Gal.CycT1 and mutant Lex.Runx1(259-451) chimeras decreased these amounts fivefold (Fig. 4, compare lanes 6 and 9). Importantly, in this system, coexpression of the mutant Lex.Runx1(259-367) chimera had no effect (Fig. 4, RNAPII, compare lanes 6, 9, and 12). We further quantified the relative intensities of the PCR products with the values of primer set B set to 1 (Fig. 4, Relative Intensity). These data are consistent with Fig. 1, where the mutant Lex.Runx1(259-451) chimera, but not the mutant Lex.Runx1(259-367) chimera, inhibited the transactivation by CycT1 and CycT2. Thus, Runx1 inhibits transcription by blocking elongation by RNAPII. Runx1 ID is required for CD4 silencing. The CD4 silencer contains two Runx-binding sites in close proximity that function cooperatively to repress CD4 transcription (34). One of these two sites, namely, site 2, is located in the 135-bp core silencer sequence and confers strong inhibitory activity in transient-transfection assays (35). To investigate if Runx1 ID plays a similar role in CD4 silencing, we performed transient-expression assays with this CD4 core silencer in cells. The plasmid target pUAS/Runx-CAT contained three copies of the core CD4 silencer, spanning positions 131 to 265, placed 5' to the CD4 enhancer-promoter (Fig. 5A). To isolate effects of Runx proteins, Runx-binding site 2 within each silencer was replaced with the Gal4 DNA-binding site (UAS). The wild-type Runx1 protein fused to the Gal4 DBD, Gal.Runx1(1-451), represses transcription from this reporter (34). To map the sequences in Runx1 that are required for this repression, we constructed additional plasmids that direct the synthesis of progressive C-terminal deletions of Runx1 fused to the Gal4 DBD: mutant Gal.Runx1(1-446) and Gal.Runx1(1-446) chimeras.
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RNAPII is engaged at the CD4 promoter in CD4– CD8+ thymoma cells but does not elongate. To understand further the mechanism by which Runx1 silences CD4 expression, we performed ChIP assays to examine the distribution of RNAPII along the CD4 gene. As determined by fluorescence-activated cell sorter, whereas mouse 3A9 T hybridoma cells express CD4, 1200M thymoma cells do not (Fig. 6A). Moreover, 1200M cells were used extensively to study the CD4 silencer and express Runx1 (34). Fifty million cells from each cell line were cross-linked with 1% formaldehyde, and their chromatin was sonicated to obtain DNA fragments of 500 nt or less. Immunoprecipitations were performed with the anti-RNAPII antibody and protein A-Sepharose beads or protein A-Sepharose beads alone (as a negative control for antibody specificity). Immunoprecipitated DNA was amplified by PCR with specific primers complementary to the CD4 promoter (A) and 800 nt downstream in the gene (B) (Fig. 6B, schematic on top). PCR products were taken at various cycles. Agarose gels of PCR products at two different cycles (40 and 45 cycles for the promoter region, 35 and 40 cycles for the coding region), where amplifications were in the linear range, are presented in Fig. 6B (bottom left).
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RNAPII, lane 2). This indicates that RNAPII is engaged at the CD4 promoter whether or not CD4 is expressed. In contrast, PCR with primer set B resulted in products only in 3A9 cells and not in 1200M cells (Fig. 6B, RNAPII, lane 5). We further quantified the relative intensities of these PCR products by normalizing them to the input DNA (Fig. 6B, lanes 3 and 6). These data indicate that although the amounts of RNAPII at the CD4 promoter are comparable in 3A9 and 1200M cells, RNAPII is absent in the CD4 coding region in 1200M cells (Fig. 6B, Relative Intensity), suggesting that RNAPII is engaged at the CD4 promoter but is unable to elongate in CD4– CD8+ thymoma cells. These results are consistent with Runx1 repressing CD4 expression by blocking the elongation of transcription by RNAPII. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Similar results were obtained previously with a CD4 minigene, where the Gal.Runx1 and Gal.Runx3 chimeras mediated the effects of the CD4 silencer (34). This effect has been called active repression in immature double-negative (CD4– CD8–) and single-positive (CD4– CD8+) thymocytes, as contrasted with more permanent epigenetic silencing in mature single-positive (CD4– CD8+) T cells in the periphery and can be observed on naked, episomal plasmids in transient-expression assays (34). Our study adds a more precise mechanism where, via its ID, Runx1 was able to block the effects of CycT1 and CycT2 on the elongation of transcription.
Transcription of the CD4 gene is regulated by both a T-cell-specific enhancer located 13 kb upstream from the transcription initiation site and a silencer located in the first intron of the CD4 gene, 2 kb downstream from the transcription initiation site (30, 31, 33). Previous studies in our laboratory demonstrated that transcriptional activators recruit P-TEFb, which increases rates of elongation of transcription from sequences far upstream and downstream of promoters and coding sequences (1, 6, 13, 14, 37). Thus, our findings that Runx1 blocks P-TEFb functions and arrests RNAPII at the promoter suggest that transcriptional repressors can function by decoying P-TEFb from RNAPII.
The ID in Runx1 is rich in alanines and prolines. Similar sequence motifs are found in many other repressors, such as the Drosophila melanogaster Krüppel, Engrailed, Knirps, Even-skipped, and paired repressors (10). This sequence motif is similar to the inhibitory sequences in PIE-1 and mutant CTD peptides that bind the histidine-rich stretch in these type C cyclins (43). This histidine-rich stretch in CycT1 is required for the recognition and targeting of Cdk9 in P-TEFb to its substrates (37). In its absence, transcription complexes remain unmodified and do not clear the promoter. Since RNAPII does not elongate in the presence of PIE-1, mutant CTD peptides, and Runx1, this finding argues strongly for an analogous mechanism of binding and inhibition for all three of these repressors.
Recently, the NMTS and its N- and C-terminal flanking sequences were found to be most important for active repression of CD4 on the cell surface in double-negative (CD4– CD8–) thymocyte cultures (38). In that study, domains in Runx1 that bind Groucho/TLE and Sin3A had no effect on the expression of the CD4 gene. Since the endogenous CD4 gene was examined, these findings suggest that an additional function of Runx1 is the targeting of specific sequences to the nuclear matrix, possibly to position its enhancer, promoter, and silencer in an arrangement that favors repression over activation. In this context, the ID in Runx1 might be positioned optimally to repress effects of P-TEFb from the enhancer. Further epigenetic silencing of the CD4 gene by Runx3 could entail greater distancing between enhancer and promoter sequences in these chromatin structures, thus obviating the need for the occupancy of the silencer (34, 35).
In conclusion, we confirmed the existence of the previously described ID in Runx1 and revealed its mechanism of action (15). Although it plays a major role in the prokaryotic world (9), such exquisite control at the step of transcriptional elongation represents a new paradigm in our understanding of gene regulation in eukaryotic systems. Indeed, recent studies demonstrated that RNAPII might be engaged on most regulated but not transcribed promoters even in the absence of active transcription in organisms as distantly related as Caenorhabditis elegans and D. melanogaster (17). In humans, RNAPII is arrested near the promoter of dihydrofolate reductase, 
-actin, and HIV genomes (4). Moreover, flavopiridol, a specific inhibitor of Cdk9, blocks the expression of greater than 90% of the genes transcribed by RNAPII (3). Of interest, even distal enhancer and locus control regions seem to act by this mechanism. For example, the ß-globin locus control region affects the transition from initiation to elongation of transcription (32) and activators found in enhancers, such as CIITA (13), NF-B (1), and c-Myc (6, 14), all bind and recruit P-TEFb to their respective transcription units.
As to repressors, the glucocorticoid receptor inhibits effects of NF-B by blocking the phosphorylation of serine 2 in the CTD, which is phosphorylated by P-TEFb (25). PIE-1 and mutant CTD peptides do the same (43). In this transcriptional arena, P-TEFb and its global inhibitors, the RNA-protein complex of HEXIM1 and 7SK snRNA, are the key players (21, 41). Indeed, hexamethylene bisacetamide, which activates the expression of HEXIM1 (named after this compound), is one of the most potent differentiating agents of hematopoietic cells (28). When P-TEFb is in the complex with HEXIM1 and 7SK snRNA, its kinase is also inactive (21, 41). In all these cases, active repression can be viewed analogously to the inhibition of a kinase by its "pseudosubstrate," in this case, the inhibition of Cdk9 by Runx1. The beauty of this simple regulatory mechanism is that it is sequence specific and can be reversed quickly, with RNAPII already engaged and poised for elongation on target genes.
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ACKNOWLEDGMENTS |
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F.Z. was supported by a fellowship from amFAR (70576-31-RFT). This work was supported by a grant from the NIH (RO1 AI49104).
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FOOTNOTES |
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H.J. and F.Z. contributed equally to this work.
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