A dual role for the histone methyltransferase PR-SET7/SETD8 and histone H4 lysine 20 monomethylation in the local regulation of RNA polymerase II pausing

Abstract

RNA polymerase II (Pol II) promoter-proximal pausing plays a critical role in postinitiation transcriptional regulation at many metazoan genes. We showed recently that histone H4 lysine 16 acetylation (H4K16Ac), mediated by the MSL complex, facilitates the release of paused Pol II. In contrast, H4 lysine 20 trimethylation (H4K20me3), mediated by SUV420H2, enforces Pol II pausing by inhibiting MSL recruitment. However, how the balance between H4K16Ac and H4K20me3 is locally regulated remains unclear. Here, we demonstrate that PR-SET7/SETD8, which monomethylates histone H4 lysine 20 (H4K20me1), controls both H4K16Ac and H4K20me3 and in doing so, regulates Pol II pausing dynamics. We find that PR-SET7-mediated H4K20me1 is necessary for the recruitment of the MSL complex, subsequent H4K16Ac, and release of Pol II into active elongation. Although dispensable for SUV420H2 recruitment, PR-SET7-mediated H4K20me1 is required for H4K20me3. Although depletion of SUV420H2 is sufficient to deplete H4K20me3 and relieve an H4K20me3-induced pause, pausing is maintained in the absence of PR-SET7 despite H4K20me3 depletion because of an inability to recruit the MSL complex in the absence of H4K20me1. These findings highlight the requirement for PR-SET7 and H4K20me1 in establishing both the H4K16Ac and H4K20me3 marks and point to a dual role in the local regulation of Pol II pausing.

Keywords: Chromatin Histone Modification; Chromatin Structure; Gene Regulation; H4K20me1; MSL Complex; PR-SET7/SETD8; RNA Polymerase; RNA Polymerase II Promoter-proximal Pausing; SUV420H2; Transcription Regulation.

FIGURE 1.

PR-SET7-mediated H4K20me1 up-regulates gene expression by preventing promoter-proximal Pol II pausing. A, schematic of the TMS1 and JUND gene loci. TMS1 consists of three exons (I-III, white boxes) and a CpG island that spans the promoter region and exons I and II. JUND consists of one exon (white box) that is encompassed by a CpG island. Nucleotide positions (bp) shown for both genes are relative to the transcription start site (arrows). Regions (1–9 for TMS1 and 1–6 for JUND) amplified by primer sets in real-time PCR following ChIP experiments are shown below the schematic. B, MCF7 cells were transfected with an empty pSUPERIOR.retro.puro vector (−) or a pSUPERIOR.retro.puro vector expressing shRNA against PR-SET7 (+) and grown for 4 days under puromycin selection. Cells were harvested and subjected to radioimmune precipitation assay buffer extraction. Protein lysates (100 μg) were resolved by SDS-PAGE electrophoresis, transferred to a PVDF membrane, and Western blotted with the indicated antibodies. C, cells from B were also used to extract mRNA, and mRNA levels were quantified by reverse transcription followed by real-time PCR. Shown are PR-SET7, TMS1, and JUND mRNA expression levels relative to that in MCF7 cells containing the empty vector after normalization to 18 S levels. Data are mean ± S.D. from two independent experiments. D, cells treated as in B were subjected to ChIP experiments with the indicated antibodies, and DNA immunoprecipitation was quantified by real-time PCR with primers that span regions 1–9 (TMS1) or 1–6 (JUND) shown in A. Immunoprecipitated DNA is expressed as a percentage of input DNA or relative to DNA isolated from histone H4 ChIP. Each experiment was conducted twice and gave similar profiles. Shown is the mean ± S.D. of triplicate determinants from a representative experiment.

FIGURE 2.

The MSL complex binds H4K20me1 in vitro, and PR-SET7-mediated H4K20me1 allows for MSL recruitment and H4K16 acetylation. A, chromatin from cells treated as in Fig. 1 was immunoprecipitated with the indicated antibodies. DNA isolated from the immunoprecipitation was subjected to real-time PCR with primer sets that span the TMS1 and JUND loci (Fig. 1A) and is expressed as a percent of input DNA or fraction of DNA immunoprecipitated with the histone H4 antibody. Shown is the mean ± S.D. of triplicate determinants from a representative experiment. Each experiment was carried out twice with reproducible results. B, MCF7 nuclear extracts were incubated with streptavidin-Sepharose beads (−) or beads conjugated to C-terminal biotinylated peptides representing amino acids 11–27 of histone H4 (H4K20 peptides) that were unmodified (me0) or mono-, di-, or trimethylated at lysine 20 (me1–3). Protein-bead complexes were isolated by centrifugation, solubilized in 2× SDS buffer, loaded on SDS-PAGE gels, and analyzed by Western blot analysis with antibodies against hMOF or hMSL3. Ten percent of input nuclear extract was also loaded.

FIGURE 3.

The PR-SET7 enzyme is required for the di- and trimethylation of H4K20 but is not required for the association of SUV420H2 with chromatin. MB231 cells transfected with either the empty pSUPERIOR.retro.puro vector (Control, DAC) or a pSUPERIOR.retro.puro vector expressing shRNA against PR-SET7 (shPR-SET7, DAC + shPR-SET7) were grown under puromycin selection, with or without DAC, for 4 days. Cells were harvested and used for ChIP with the antibodies shown. DNA isolated from ChIP was quantified by real-time PCR with primers that span the TMS1 (1–9) or JUND (1–6) regions shown in Fig. 1A. Quantified DNA is shown as a percentage of input DNA or normalized to DNA isolated from histone H4 ChIP. The mean ± S.D. of triplicate determinants from a sample experiment is shown. Each experiment was performed twice and yielded similar results.

FIGURE 4.

Down-regulation of PR-SET7 does not lead to Pol II pause release and gene up-regulation because MSL and H4K16Ac are not reestablished in the absence of PR-SET7. A, MB231 cells treated as in Fig. 3 were used to determine protein expression levels of PR-SET7, TMS1, JUND, and GAPDH (loading control) as described in Fig. 1B. B, MB231 cells treated as in Fig. 3 were used to determine RNA expression levels of PR-SET7, TMS1, and JUND as indicated in Fig. 1C. C, ChIP with the indicated antibodies, followed by real-time PCR of immunoprecipitated DNA with TMS1 or JUND genomic primers, was conducted with MB231 cells used in A and B. DNA quantity is expressed as a percentage of input DNA or a fraction of histone H4 ChIP. Each experiment was carried out twice with reproducible results, and shown are the means ± S.D. of triplicate determinants from a representative experiment.

FIGURE 5.

MB231 cells treated with either non-silencing scrambled (Control, DAC) or SUV420H2 (siSUV420H2, DAC + siSUV420H2) siRNA were grown with or without DAC treatment for 4 days. Chromatin from cells was immunoprecipitated with IgG (negative control), methylated H4K20, or SUV420H2 antibodies, and DNA was extracted from the immunoprecipitated samples. Immunoprecipitated DNA was subjected to real-time PCR with primer sets that span the TMS1 or JUND genomic regions (Fig. 1A) and is expressed as a percentage of input DNA or a fraction of DNA obtained from histone H4 ChIP. Data are mean ± S.D. from a representative experiment from triplicate determinants.

FIGURE 6.

Dual functions of H4K20me1 in the local regulation of Pol II pausing. H4K20me1 by PR-SET7 allows for active elongation at genes regulated by Pol II promoter-proximal pausing via the recruitment of the MSL complex. The MSL complex acetylates H4K16, which, in turn, allows for the association of the BRD4/pTEFb complex, subsequent phosphorylation of serine 2 in the C-terminal domain of the initiated and paused Pol II, and, ultimately, pause release. Alternatively, PR-SET7-dependent H4K20me1 also promotes Pol II pausing at the same genes because it serves as a template for SUV420H2-mediated (SUV) H4K20me3. The presence of H4K20me3 inhibits MSL recruitment, acetylation of H4K16Ac, and, consequently, active elongation.