DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells

Abstract

The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recruitment is ubiquitously coupled with active transcription in diverse mammalian cell types. DOT1L preferentially occupies the proximal transcribed region of active genes, correlating with enrichment of H3K79 di- and trimethylation. Furthermore, Dot1l mutant fibroblasts lacked H3K79 di- and trimethylation at all sites examined, indicating that DOT1L is the sole enzyme responsible for these marks. Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. Conversion of H3K79 monomethylation into di- and trimethylation correlated with the transition from low- to high-level gene transcription. We also observed enrichment of H3K79 monomethylation at intergenic regions occupied by DNA-binding transcriptional activators. Our findings highlight several similarities between the patterning of H3K4 methylation and that of H3K79 methylation in mammalian chromatin, suggesting a widespread mechanism for parallel or sequential recruitment of DOT1L and MLL to genes in their normal "on" state.

FIG. 1.

Human DOT1L occupies transcribed regions in a ubiquitous fashion. (A) ChIP in HEK293T cells transfected with pFLAG-DOT1L or empty vector. (B, D, F, and G) ChIP with the indicated antibodies in HEK293T cells across the PABPC1 gene with PCR primers at the indicated locations relative to the TSS. CD4 is a negative control region. (C and E) ChIP-qPCR analysis of HeLa cells at genes with high or low expression with the indicated antibodies. The DOT1L antibody is A300-954A. Primers amplify within the +0.5-kb region relative to the TSS. IgG, immunoglobulin G.

FIG. 2.

DOT1L is required for H3K79 di- and trimethylation at active genes. (A to F) ChIP analysis of wild-type (wt) MEFs, homozygous Dot1l mutant MEFs, or Dot1l mutant MEFs infected with DOT1L-murine stem cell virus-IRES-GFP. Levels of histone modification were normalized to total histone H3. Primers amplify within the +0.5-kb region relative to the TSS, except for Hbb, which amplifies a noncoding segment of the β-globin locus (IVR16 primers) and serves as a negative control.

FIG. 3.

SDS treatment of cross-linked chromatin samples facilitates detection of H3K79 methylation by ChIP. (A to E) ChIP performed with HeLa chromatin prepared either by sonication or by MNase digestion with the indicated antibodies. (F) ChIP for H3K79me2 including sonicated chromatin treated with 3 mM MgCl₂ and 1.5 mM CaCl₂ or MNase-digested chromatin treated with 0.5% SDS. ChIP primers amplify within the +0.5-kb region relative to the TSS. IgG, immunoglobulin G.

FIG. 4.

Levels of H3K79 methylation correlate with gene expression in mammalian cells. (A to G) ChIP-chip analysis of methylation with the indicated antibodies. The graphs shown are composite representations of similarly expressed genes with a moving median of 25 adjacent data points (spanning 660 bp). Each location is relative to the TSS, measured in base pairs. All methylation signals were normalized to the input and total histone H3.

FIG. 5.

Dynamic acquisition or removal of H3K79 and H3K4 methylation reflects the transcriptional activity of genes regulated by adipogenesis. A summary of ChIP-chip data from 47 dynamic genes is shown. ↑ represents a 1.5- to 2.5-fold change. ↑↑ represents a 2.5- to 7-fold change. ↑↑↑ represents a >7-fold change. A minus sign represents no significant change. All methylation changes were normalized to the input and total histone H3.

FIG. 6.

H3K79 monomethylation is converted into di- and trimethylation upon induction of Acsl1 transcription. ChIP-chip analysis of H3K79 methylation at Acsl1 in either undifferentiated (preadipocytes) or differentiated (adipocytes) 3T3-L1 cells is shown. All methylation signals were normalized to the input and total histone H3.

FIG. 7.

Distal upstream locations of H3K79 monomethylation at Cd36 correlate with occupancy of PPARγ. (A to F) ChIP-chip mapping of H3K4 methylation and H3K79 methylation at the Cd36 locus. All methylation signals were normalized to the input and total histone H3. (G) PPARγ ChIP-qPCR performed at the indicated regions of Cd36 relative to the TSS. The positive control for PPARγ occupancy is −0.2 kb. −2.2 kb is a negative control site. IgG, immunoglobulin G.

FIG. 8.

GATA-1 is required for H3K79 monomethylation at the LCR of the β-globin locus. (A) Schematic of the murine β-globin locus with known GATA-1 binding sites indicated (18). (B to D) ChIP-qPCR with the indicated antibodies detecting occupancy at various regions of the murine β-globin locus in G1E cells expressing GATA-1-ER before or after 24 h of estradiol treatment. Signals were normalized to the total histone H3 and the input. IVR, intervening region; pro, promoter; in2, intron 2.

FIG. 9.

Intergenic transcription is not required to establish H3K79me1 at the LCR. ChIP was performed with G1E(Bcl-X_L) cells expressing GATA-1-ER with the indicated antibodies before or after 18 h of treatment with estradiol with or without DRB. (A) HS1 primers. (B) Hbb-b1 intron 2 primers. IgG, immunoglobulin G.