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. 2007 Dec;27(24):8466-79.
doi: 10.1128/MCB.00993-07. Epub 2007 Oct 8.

Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes

Gregory D Gregory  1 , Christopher R Vakoc, Tanya Rozovskaia, Xingwu Zheng, Shetal Patel, Tatsuya Nakamura, Eli Canaani, Gerd A Blobel
Affiliations

Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes

Gregory D Gregory et al. Mol Cell Biol. 2007 Dec.

Abstract

Histone lysine methylation regulates genomic functions, including gene transcription. Previous reports found various degrees of methylation at H3K4, H3K9, and H4K20 within the transcribed region of active mammalian genes. To identify the enzymes responsible for placing these modifications, we examined ASH1L, the mammalian homolog of the Drosophila melanogaster Trithorax group (TrxG) protein Ash1. Drosophila Ash1 has been reported to methylate H3K4, H3K9, and H4K20 at its target sites. Here we demonstrate that mammalian ASH1L associates with the transcribed region of all active genes examined, including Hox genes. The distribution of ASH1L in transcribed chromatin strongly resembles that of methylated H3K4 but not that of H3K9 or H4K20. Accordingly, the SET domain of ASH1L methylates H3K4 in vitro, and knockdown of ASH1L expression reduced H3K4 trimethylation at HoxA10 in vivo. Notably, prior methylation at H3K9 reduced ASH1L-mediated methylation at H3K4, suggesting cross-regulation among these marks. Drosophila ash1 and trithorax interact genetically, and the mammalian TrxG protein MLL1 and ASH1L display highly similar distributions and substrate specificities. However, by using MLL null cell lines we found that their recruitments occur independently of each other. Collectively, our data suggest that ASH1L occupies most, if not all, active genes and methylates histone H3 in a nonredundant fashion at a subset of genes.

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Figures

FIG. 1.
FIG. 1.
ASH1L is associated with actively transcribed genes. (A) ChIP analysis, with HeLa cells, of RNA Pol II at the active genes RPLP0, PPIA, RPS2, EEF1A1, and RPL41 and the inactive genes CD4 and MYT1. (B) ChIP assay using anti-ASH1L antibodies 296 and ab4477 at the genes listed above. (C and D) Similar analyses were performed with undifferentiated K562 cells. (E and F) RNA Pol II and ASH1L occupancy at the inactive NKX2.2 and active PABPC1 genes in murine embryonic stem (ES) cells. Error bars represent standard deviations. Highly transcribed and ubiquitously expressed active genes were identified using the RNA expression database SymAtlas, v1.2.4, which is maintained by the Genomics Institute of the Novartis Research Foundation (http://symatlas.gnf.org/SymAtlas/) (see Fig. S1A in the supplemental material) (50). rab Ig, rabbit immunoglobulin G.
FIG. 2.
FIG. 2.
Histone methylation and ASH1L occupancy at the PABPC1 gene. ChIP assays of HeLa cells with antibodies against (A) ASH1L (296), (B) ASH1L (ab4477), (C) H3K4me3 and H3K9me3, (D) H4K20me1, and (E) RNA Pol II. All error bars represent standard deviations. Anti-ASH1L ChIP results are the averages of three to five independent experiments per primer pair. Nonimmune isotype-matched rabbit immunoglobulin G (rab Ig) was used as the control. ChIP assays using a third antibody specific for ASH1L (337ap) are shown in Fig. S1B in the supplemental material.
FIG. 3.
FIG. 3.
Recruitment of ASH1L correlates with onset of gene expression. (A) Diagram of the murine β-globin gene locus (not drawn to scale). ChIP analysis of (B) RNA Pol II, (C) ASH1L (antibody ab4477), and (D) H3K4me3 in murine G1E cells before or after differentiation with estradiol for 21 h. Error bars represent standard deviations for three independent experiments. HS, DNase 1 hypersensitive site; IVR, intervening region; TR, transcribed region; rab Ig, rabbit immunoglobulin G.
FIG. 4.
FIG. 4.
ASH1L occupancy persists following a transcription elongation block. ChIP with RNA Pol II, H3K4me3, and ASH1L (ab4477) antibodies at indicated sites at the PABPC1 gene in HeLa cells with (+ DRB) or without (no DRB) treatment with DRB for 6 h. Data were plotted as severalfold changes from levels for nontreated cells. Error bars represent standard deviations. Similar analyses were performed with G1E cells and are shown as Fig. S3 in the supplemental material.
FIG. 5.
FIG. 5.
The CsetC domain of ASH1L methylates H3K4 in vitro and is partially inhibited by preexisting H3K9 trimethylation. (A) HMTase activity of the SET domain of ASH1L (GST-ASH1L-CsetC), SUV39H1 (GST-SUV39H1), or GST alone with OGNS as the substrate. Four microliters of assembled OGNS, equating to a total of 108 ng of core histones, was used per 20-μl reaction. (B) HMTase assays with OGNS as the substrate were scaled up (6×), resolved on a 10% SDS-polyacrylamide gel, and treated with Autofluor before exposure to film for 9 days. (C) HMTase activity of the SET domain of ASH1L (GST-ASH1L-CsetC) or GST with histone H3 and H4 peptides as substrates. The value obtained with GST alone was set to 1. Premethylation of H3K4 completely blocks activity, whereas H3K9 premethylation partially inhibits activity. *, P < 0.001; **, P < 0.05; unmod, unmodified. (D) HMTase assay using GST-H3(1-46) and GST-H4(1-34) with indicated lysine-to-arginine substitutions as substrates. Graphs represent scintillation counts from filter assays. Error bars denote standard deviations (top). Part of the reaction mixture was analyzed by SDS-polyacrylamide gel electrophoresis and Colloidal blue staining (middle) prior to autoradiography (bottom).
FIG. 6.
FIG. 6.
Overlapping chromatin occupancies of ASH1L and MLL1 at housekeeping genes. (A) ChIP with anti-ASH1L (296), anti-MLL1 (antibody 456), and control rabbit immunoglobulin G (rab Ig) at PABPC1 in 293T cells. (B and C) ChIP analysis, with 293T cells, of MLL1 (456) (B) and H3K4me3 (C) at the active genes RPLP0, PPIA, RPS2, UBB, and RPL41 and the inactive genes CD4 and MYT1. Error bars represent standard deviations.
FIG. 7.
FIG. 7.
ASH1L is required for full Hox gene expression and H3K4 trimethylation. (A) ChIP with anti-H3K4me3 and anti-ASH1L (296) antibodies at the active HoxA10 gene. (B) ChIP analysis of MLL1 (456) across the HoxA10 gene. (C) ChIP with anti-H3K4me2 across the HoxA10 gene. Error bars represent standard deviations. rab Ig, rabbit immunoglobulin G. (D) Efficiency of knockdown of ASH1L mRNA, determined using primers directed towards the middle (#1) and 3′ (#2) regions of ASH1L cDNA. (E) mRNA levels of indicated genes, as determined by quantitative real-time RT-PCR with shCTRL and shASH1L 293T cells. (F) ASH1L occupancy and (G) H3K4me3 levels at +0.5 to 1.0 kb of indicated genes in shCTRL and shASH1L 293T cells.
FIG. 8.
FIG. 8.
ASH1L occupancy is independent of MLL1. (A) Real-time RT-PCR analysis of HoxA9, Meis1, and TubA2 mRNA expression in Mll1+/+ and Mll1−/− MEFs. (B and C) ChIP analysis of ASH1L (296), MLL1 (456), H3K4me3, and H3K4me2 at (B) MLL1-independent genes and (C) MLL1-dependent genes in Mll1+/+ and Mll1−/− MEFs. Error bars represent standard deviations. 2.5 UR, 2.5 kb upstream region from the TSS; 0.5 and 2.0 TR, 0.5 and 2.0 kb downstream from the TSS, respectively; prom, promoter; rab Ig, rabbit immunoglobulin G.
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