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Dynamic histone H3 methylation during gene induction: HYPB/Setd2 mediates all H3K36 trimethylation.

Edmunds JW, Mahadevan LC, Clayton AL - EMBO J. (2007)

Bottom Line: Upon stimulation, transcription-dependent increases in H3K4 and H3K36 trimethylation are seen across coding regions, peaking at 5' and 3' ends, respectively.Addressing molecular mechanisms involved, we find that Huntingtin-interacting protein HYPB/Setd2 is responsible for virtually all global and transcription-dependent H3K36 trimethylation, but not H3K36-mono- or dimethylation, in these cells.These studies reveal four distinct layers of histone modification across inducible mammalian genes and show that HYPB/Setd2 is responsible for H3K36 trimethylation throughout the mouse nucleus.

View Article: PubMed Central - PubMed

Affiliation: Nuclear Signalling Laboratory, Department of Biochemistry, Oxford University, Oxford, UK.

ABSTRACT
Understanding the function of histone modifications across inducible genes in mammalian cells requires quantitative, comparative analysis of their fate during gene activation and identification of enzymes responsible. We produced high-resolution comparative maps of the distribution and dynamics of H3K4me3, H3K36me3, H3K79me2 and H3K9ac across c-fos and c-jun upon gene induction in murine fibroblasts. In unstimulated cells, continuous turnover of H3K9 acetylation occurs on all K4-trimethylated histone H3 tails; distribution of both modifications coincides across promoter and 5' part of the coding region. In contrast, K36- and K79-methylated H3 tails, which are not dynamically acetylated, are restricted to the coding regions of these genes. Upon stimulation, transcription-dependent increases in H3K4 and H3K36 trimethylation are seen across coding regions, peaking at 5' and 3' ends, respectively. Addressing molecular mechanisms involved, we find that Huntingtin-interacting protein HYPB/Setd2 is responsible for virtually all global and transcription-dependent H3K36 trimethylation, but not H3K36-mono- or dimethylation, in these cells. These studies reveal four distinct layers of histone modification across inducible mammalian genes and show that HYPB/Setd2 is responsible for H3K36 trimethylation throughout the mouse nucleus.

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EGF-stimulated distribution of acetylated and methylated histone H3 across c-fos and c-jun. (A) Schematic representation of c-fos and c-jun showing positions analysed by real-time PCR. Primer positions indicate the 5′ position of the forward primer relative to the transcription start site. Exons are represented by boxes, unfilled for untranslated regions and filled for translated regions. Termination sites are shown as filled circles. (B) Quiescent cells were unstimulated (control) or stimulated with EGF (50 ng/ml) for 15, 30, 60 or 120 min and formaldehyde crosslinked mononucleosomes were prepared for ChIP. Aliquots of chromatin were immunoprecipitated with H3K9ac- (i), H3K4me3- (ii), H3K36me3- (iii) or H3K79me2- (iv) specific antibodies. Recovered DNA sequences were quantified by real-time PCR. Average % input recoveries from two independent experiments are plotted.
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f1a: EGF-stimulated distribution of acetylated and methylated histone H3 across c-fos and c-jun. (A) Schematic representation of c-fos and c-jun showing positions analysed by real-time PCR. Primer positions indicate the 5′ position of the forward primer relative to the transcription start site. Exons are represented by boxes, unfilled for untranslated regions and filled for translated regions. Termination sites are shown as filled circles. (B) Quiescent cells were unstimulated (control) or stimulated with EGF (50 ng/ml) for 15, 30, 60 or 120 min and formaldehyde crosslinked mononucleosomes were prepared for ChIP. Aliquots of chromatin were immunoprecipitated with H3K9ac- (i), H3K4me3- (ii), H3K36me3- (iii) or H3K79me2- (iv) specific antibodies. Recovered DNA sequences were quantified by real-time PCR. Average % input recoveries from two independent experiments are plotted.

Mentions: We first carried out a comprehensive mapping study to determine the distribution of histone modifications across c-fos and c-jun in control, EGF-stimulated (Figure 1) or anisomycin-stimulated (Supplementary Figure S1) mouse fibroblasts. A modified ChIP protocol, using MNase instead of sonication to generate predominantly mononucleosomal chromatin (MacDonald et al, 2005) was used to produce high-resolution quantitative comparative maps of the distribution and dynamics of H3K4 trimethylation (K4me3), K36 trimethylation (K36me3), K79 dimethylation (K79me2) and K9 acetylation (K9ac) across these genes (Figure 1 and Supplementary Figure S1). Each antibody was first titrated in ChIP to arrive at concentrations that recovered virtually all available epitopes, leaving little or none in the unbound fraction as monitored by western blotting. Peaks of distribution of each modification across these genes are shown graphically (Figure 1B and Supplementary Figure S1B) and as bar charts with error bars (Figure 1C and Supplementary Figure S1C). This yielded highly reproducible maps with striking differences in the amounts of each modification recovered. Their interpretation is extremely complex because of the continuous dynamic turnover of acetylation at these genes, the fact that methylation may occur transiently with the traverse of RNA pol II, and the phenomenon of microheterogeneity of histone modifications at any single position. Evaluating these maps in overview, common trends applying to both c-fos and c-jun emerge. First, K9ac (Figure 1B, panel i) and K4me3 (panel ii) have remarkably similar overlapping distributions across the start site of both genes. For both modifications, there is a striking dip precisely at the start site of both genes, and the positions of peaks of modification largely coincide. The stability of this colocalisation at all points analysed is consistent with sequential ChIP assays showing these modifications coexist on the same nucleosomes at these positions (Hazzalin and Mahadevan, 2005). The sharp dip in K4me3 and K9ac at the start site on both genes appears to be due to this position lacking a nucleosome and being more accessible, as indicated by MNase sensitivity maps across these genes (Figure 2 and Supplementary Figure S2). These show that the start sites of c-fos and c-jun are considerably more sensitive to MNase digestion with <5% (c-fos −79, c-jun −57) and <10% (c-jun −396) of the corresponding genomic DNA signal remaining after digestion (Figure 2 and Supplementary Figure S2).


Dynamic histone H3 methylation during gene induction: HYPB/Setd2 mediates all H3K36 trimethylation.

Edmunds JW, Mahadevan LC, Clayton AL - EMBO J. (2007)

EGF-stimulated distribution of acetylated and methylated histone H3 across c-fos and c-jun. (A) Schematic representation of c-fos and c-jun showing positions analysed by real-time PCR. Primer positions indicate the 5′ position of the forward primer relative to the transcription start site. Exons are represented by boxes, unfilled for untranslated regions and filled for translated regions. Termination sites are shown as filled circles. (B) Quiescent cells were unstimulated (control) or stimulated with EGF (50 ng/ml) for 15, 30, 60 or 120 min and formaldehyde crosslinked mononucleosomes were prepared for ChIP. Aliquots of chromatin were immunoprecipitated with H3K9ac- (i), H3K4me3- (ii), H3K36me3- (iii) or H3K79me2- (iv) specific antibodies. Recovered DNA sequences were quantified by real-time PCR. Average % input recoveries from two independent experiments are plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2168397&req=5

f1a: EGF-stimulated distribution of acetylated and methylated histone H3 across c-fos and c-jun. (A) Schematic representation of c-fos and c-jun showing positions analysed by real-time PCR. Primer positions indicate the 5′ position of the forward primer relative to the transcription start site. Exons are represented by boxes, unfilled for untranslated regions and filled for translated regions. Termination sites are shown as filled circles. (B) Quiescent cells were unstimulated (control) or stimulated with EGF (50 ng/ml) for 15, 30, 60 or 120 min and formaldehyde crosslinked mononucleosomes were prepared for ChIP. Aliquots of chromatin were immunoprecipitated with H3K9ac- (i), H3K4me3- (ii), H3K36me3- (iii) or H3K79me2- (iv) specific antibodies. Recovered DNA sequences were quantified by real-time PCR. Average % input recoveries from two independent experiments are plotted.
Mentions: We first carried out a comprehensive mapping study to determine the distribution of histone modifications across c-fos and c-jun in control, EGF-stimulated (Figure 1) or anisomycin-stimulated (Supplementary Figure S1) mouse fibroblasts. A modified ChIP protocol, using MNase instead of sonication to generate predominantly mononucleosomal chromatin (MacDonald et al, 2005) was used to produce high-resolution quantitative comparative maps of the distribution and dynamics of H3K4 trimethylation (K4me3), K36 trimethylation (K36me3), K79 dimethylation (K79me2) and K9 acetylation (K9ac) across these genes (Figure 1 and Supplementary Figure S1). Each antibody was first titrated in ChIP to arrive at concentrations that recovered virtually all available epitopes, leaving little or none in the unbound fraction as monitored by western blotting. Peaks of distribution of each modification across these genes are shown graphically (Figure 1B and Supplementary Figure S1B) and as bar charts with error bars (Figure 1C and Supplementary Figure S1C). This yielded highly reproducible maps with striking differences in the amounts of each modification recovered. Their interpretation is extremely complex because of the continuous dynamic turnover of acetylation at these genes, the fact that methylation may occur transiently with the traverse of RNA pol II, and the phenomenon of microheterogeneity of histone modifications at any single position. Evaluating these maps in overview, common trends applying to both c-fos and c-jun emerge. First, K9ac (Figure 1B, panel i) and K4me3 (panel ii) have remarkably similar overlapping distributions across the start site of both genes. For both modifications, there is a striking dip precisely at the start site of both genes, and the positions of peaks of modification largely coincide. The stability of this colocalisation at all points analysed is consistent with sequential ChIP assays showing these modifications coexist on the same nucleosomes at these positions (Hazzalin and Mahadevan, 2005). The sharp dip in K4me3 and K9ac at the start site on both genes appears to be due to this position lacking a nucleosome and being more accessible, as indicated by MNase sensitivity maps across these genes (Figure 2 and Supplementary Figure S2). These show that the start sites of c-fos and c-jun are considerably more sensitive to MNase digestion with <5% (c-fos −79, c-jun −57) and <10% (c-jun −396) of the corresponding genomic DNA signal remaining after digestion (Figure 2 and Supplementary Figure S2).

Bottom Line: Upon stimulation, transcription-dependent increases in H3K4 and H3K36 trimethylation are seen across coding regions, peaking at 5' and 3' ends, respectively.Addressing molecular mechanisms involved, we find that Huntingtin-interacting protein HYPB/Setd2 is responsible for virtually all global and transcription-dependent H3K36 trimethylation, but not H3K36-mono- or dimethylation, in these cells.These studies reveal four distinct layers of histone modification across inducible mammalian genes and show that HYPB/Setd2 is responsible for H3K36 trimethylation throughout the mouse nucleus.

View Article: PubMed Central - PubMed

Affiliation: Nuclear Signalling Laboratory, Department of Biochemistry, Oxford University, Oxford, UK.

ABSTRACT
Understanding the function of histone modifications across inducible genes in mammalian cells requires quantitative, comparative analysis of their fate during gene activation and identification of enzymes responsible. We produced high-resolution comparative maps of the distribution and dynamics of H3K4me3, H3K36me3, H3K79me2 and H3K9ac across c-fos and c-jun upon gene induction in murine fibroblasts. In unstimulated cells, continuous turnover of H3K9 acetylation occurs on all K4-trimethylated histone H3 tails; distribution of both modifications coincides across promoter and 5' part of the coding region. In contrast, K36- and K79-methylated H3 tails, which are not dynamically acetylated, are restricted to the coding regions of these genes. Upon stimulation, transcription-dependent increases in H3K4 and H3K36 trimethylation are seen across coding regions, peaking at 5' and 3' ends, respectively. Addressing molecular mechanisms involved, we find that Huntingtin-interacting protein HYPB/Setd2 is responsible for virtually all global and transcription-dependent H3K36 trimethylation, but not H3K36-mono- or dimethylation, in these cells. These studies reveal four distinct layers of histone modification across inducible mammalian genes and show that HYPB/Setd2 is responsible for H3K36 trimethylation throughout the mouse nucleus.

Show MeSH