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Transcriptional Corepressor TLE1 Functions with Runx2 in Epigenetic Repression of Ribosomal RNA Genes

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Transcriptional Corepressor TLE1 Functions with Runx2 in Epigenetic Repression of Ribosomal RNA Genes Transcriptional corepressor TLE1 functions with Runx2 in epigenetic repression of ribosomal RNA genes Syed A. Alia, Sayyed K. Zaidia, Jason R. Dobsona, Abdul R. Shakoorib, Jane B. Liana, Janet L. Steina, Andre J. van Wijnena, and Gary S. Steina,1 aDepartment of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA 01655; and bSchool of Biological Sciences, University of the Punjab, Lahore 54590, Pakistan Communicated by Sheldon Penman, Massachusetts Institute of Technology, Brookline, MA, January 20, 2010 (received for review December 24, 2009) Epigenetic control of ribosomal RNA (rRNA) gene transcription by conveyed to the next interphase have not been established. Here, cell type-specific regulators, such as the osteogenic transcription we show that the Runx2 coregulator TLE1 (21) forms a functional factor Runx2, conveys cellular memory of growth and differentia- complex with Runx2 and the Pol I transcription factor UBF. tion to progeny cells during mitosis. Here, we examined whether TLE1, Runx2, and UBF together occupy nucleolar organizing coregulatory proteins contribute to epigenetic functions that are regions (NORs) during mitosis and control histone modifications. mitotically transmitted by Runx2 in osteoblastic cells. We show that This mitotic regulation by a corepressor protein establishes a the transcriptional corepressor Transducin Like Enhancer-1 (TLE1) unique dimension to a Runx2-mediated epigenetic mechanism associates with rRNA genes during mitosis and interphase through that reinforces cell fate decisions as well as sustains protein syn- interaction with Runx2. Mechanistically, depletion of TLE1 relieves thesis capacity and cell growth in lineage committed cells. Runx2-mediated repression of rRNA genes transcription and selec- tively increases histone modifications linked to active transcription. Results and Discussion Biologically, loss of TLE-dependent rRNA gene repression coincides C-Terminal Regulatory Domain of Runx2 Mediates Transcriptional with increased global protein synthesis and enhanced cell prolifer- Repression of rRNA Genes. To characterize the mechanisms by ation. Our findings reinforce the epigenetic marking target genes by which Runx2 controls rRNA gene transcription, we examined phenotypic transcription factors in mitosis and demonstrate a rRNA expression in the presence of Runx2 mutant proteins in requirement for retention of coregulatory factors to sustain phys- which DNA binding (R182Q) (22) or transcriptional Delta C iological control of gene expression during proliferation of lineage terminus (ΔC) functions are abrogated. The R182Q mutation committed cells. affects the binding of Runx2 to the DNA without affecting nuclear or subnuclear localization of the proteins (23). Removal cell fate | mitotic NORs | cell cycle | interphase nucleoli | coregulatory of the Runx2 C terminus (ΔC) abrogates interactions with sev- factors eral coregulatory proteins and subnuclear targeting without affecting nuclear localization or DNA binding activity (Fig. 1A) idelity of transcriptional control determines progenitor com- (19, 20, 23–25). Human osteoblastic cells were infected with Fmitment toward specific lineages and concomitantly excludes lentiviral vectors expressing epitope-tagged wild type (WT) options for divergence toward other cell fates (1). In addition to Runx2 or mutants R182Q and ΔC. In addition, Runx2 down- genetically encoded information, epigenetic mechanisms that regulates pre-rRNA transcription levels (Fig. 1A), as has been include DNA methylation (2) and histone modifications (3) play shown (9). However, both R182Q and ΔC mutants failed to a critical role in physiological regulation of gene expression. suppress rRNA gene expression (Fig. 1B). Expression of several CELL BIOLOGY During mitosis, many transcription factors are displaced from Runx2 target genes (Cdc6, CyclinH, Cdc 46, CyclinB) (10) that – the chromosomes (4 6). However, recent studies indicate that are transcribed by RNA Pol II is up-regulated by Runx2 but not association of gene regulatory factors with target genes is an the ΔC mutant or R182Q (Fig. S1A). Western blot analysis epigenetic mechanism to maintain cellular memory of growth revealed that WT Runx2, R182Q and ΔC proteins are expressed – and differentiation (7 11). at significant and comparable levels after 48 h of infection (Fig. Epigenetic control applies to RNA polymerase (Pol) II-tran- 1C). Chromatin immunoprecipitation (ChIP) assays showed that scribed protein coding genes as well as to RNA Pol I-mediated Runx2 interacts with three representative regions of the rDNA transcription of ribosomal RNA (rRNA) genes (12). A complex repeat (indicated as primer sets A, B and C; Fig. 1D), but not network of interdependent biochemical events regulates rRNA with the unrelated Phox (GP91) promoter (Fig. S1B). Both WT transcription during interphase to modulate the overall capacity Runx2 and ΔC exhibited increased rDNA occupancy compared of the protein-synthesis machinery, which is linked to cell growth – with control cells (Fig. 1E). As expected, the DNA binding and cellular phenotype (13 15). For examples, mechanisms that mutant R182Q does not interact with the rDNA promoter coordinate rRNA transcription with cell growth and differ- (Fig. 1E). Taken together, these findings indicate the involve- entiation in lineage-committed cells include the association of ment of the Runx2 C terminus and associated activities in sup- the osteogenic transcription factor Runx2 with mitotic chromo- pression of rRNA expression. somes in osteoblasts (9). Runx2 epigenetically marks rRNA and other target genes during mitosis by directly occupying lineage- specific promoter regions (9, 10). Similarly, MyoD in myoblasts Author contributions: S.A.A., S.K.Z., J.R.D., A.R.S., J.B.L., J.L.S., A.J.v.W., and G.S.S. de- and C/EBP in adipocytes epigenetically mark rRNA genes (16). signed research; S.A.A. and J.R.D. performed research; S.A.A., S.K.Z., J.R.D., A.R.S., J.B.L., This mitotic retention provides a component of molecular J.L.S., A.J.v.W., and G.S.S. analyzed data; and S.A.A., S.K.Z., J.R.D., A.R.S., J.B.L., J.L.S., A.J.v. memory that maintains mesenchymal lineage commitment and W., and G.S.S. wrote the paper. competency for protein synthesis in progeny cells (17, 18). The authors declare no conflict of interest. Runx factors are scaffolding proteins that interact with a large 1To whom correspondence should be addressed. E-mail: [email protected]. cohort of coregulatory proteins in interphase (19, 20). However, This article contains supporting information online at www.pnas.org/cgi/content/full/ which cofactors associate with Runx2 during mitosis and are 1000620107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1000620107 PNAS | March 2, 2010 | vol. 107 | no. 9 | 4165–4169 Downloaded by guest on September 28, 2021 and regulates their expression in progeny cells (9), thereby, epigenetically maintaining a molecular memory of the cell growth-related capacity to synthesize rRNA. However, the cor- egulators that support its epigenetic activity have not been identified. Because the Runx2 C terminus is required for rRNA gene repression (Fig. 1B), we focused on corepressors (i.e., TLE1 and HDAC1) that are known to interact with the Runx2 C terminus and support Pol II-mediated transcription of Runx2 target genes (10, 21, 26, 27). Western blot analysis with lysates from mitotically synchronized cells establishes that both TLE1 and HDAC1 are present in mitotic cells (Fig. 2A). Both proteins are stable upon inhibition of protein synthesis by cycloheximide (Fig. S1C), indicating that preexisting levels of these proteins are retained during mitotic progression. Fig. 1. Runx2-mediated repression of rRNA gene expression requires the C To investigate whether TLE1 and HDAC1 are associated with terminus. (A) A schematic representation of full-length Runx2protein indicating the DNA binding runt homology domain(RHD) and the nuclear matrix targeting mitotic chromosomes, we analyzed actively proliferating osteo- signal (NMTS). The location of the DNA binding point mutation (R182Q) and the blastic cells at various mitotic stages by using immunofluorescence CterminusofΔC truncation protein is also marked. (B) Real-time qPCR dem- microscopy. In addition to Runx2, we included the Pol I tran- onstrates relative level of precursor-rRNA (pre-rRNA) and mature-rRNA (m28S- scription factor UBF, a marker for mitotic NORs (28–30), in our rRNA) in response to WT Runx2, R182Q, and ΔCmutants,normalizedtomito- analysis. Our key findings are that TLE1 is localized with UBF foci chondrial Cytochrome oxidase (mCox). (C) Western blot analysis of SaOS-2 cell Δ during all stages of mitosis and that TLE1 is present at NORs on lysates demonstrating equivalent levels of the Runx2, R182Q, and C proteins in metaphase chromosomes (Fig. 2B Upper and Figs. S1D and S2A). comparison with empty vector (EV) is shown. CdK2 levels were used as a control for equal protein loading. (D) Represents Ribosomal DNA (rDNA) repeat indi- Furthermore, TLE1 colocalizes with Runx2 (Fig. 2 B Lower and C cating the Runx2 binding sites (vertical lines) are shown. The position of three Left, and Figs. S2B and S3A). In contrast, the histone modifying primer pairs, A, B, and C, used in this study for ChIP analysis are also shown; enzyme HDAC1, which is a known cofactor of Runx2 (20, 27), is arrows indicate positions of primers at rDNA repeat. (E) Chromatin immuno- excluded from NORs and does not colocalize with Runx2 or UBF precipitation
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