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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-induced increases in H3K4me3 and H3K36me3 within c-fos and c-jun are transcription-dependent. (A) Quiescent cells were pretreated (10 min) with DRB (25 μg/ml) or untreated (con) prior to stimulation with EGF (50 ng/ml) for 15, 30 or 60 min or no-stimulation (−). Quiescent cells were also treated with DRB alone for 25, 40 or 70 min as controls, respectively. Total mRNA was isolated and relative levels of c-fos and c-jun mRNA were quantified by qRT–PCR with normalisation to gapdh mRNA. A representative experiment is shown. Error bars represent the s.d. from triplicate PCRs. (B) Formaldehyde crosslinked mononucleosomes were prepared from quiescent cells treated as in (A) and used for ChIPs with an anti-RNA-polymerase-II (Pol II) antibody. Recovery of c-fos and c-jun coding DNA sequences were quantified by real-time PCR. Average % input recoveries and s.d. from 3–4 independent experiments are plotted. (C) ChIPs and real-time PCR were performed as described in (B) using H3K4me3- (top panel) and H3K36me3- (bottom panel) specific antibodies. Amplicons analysed are indicated above each graph. Average % input recoveries and s.d. from 3–4 independent experiments are plotted.
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f3: EGF-induced increases in H3K4me3 and H3K36me3 within c-fos and c-jun are transcription-dependent. (A) Quiescent cells were pretreated (10 min) with DRB (25 μg/ml) or untreated (con) prior to stimulation with EGF (50 ng/ml) for 15, 30 or 60 min or no-stimulation (−). Quiescent cells were also treated with DRB alone for 25, 40 or 70 min as controls, respectively. Total mRNA was isolated and relative levels of c-fos and c-jun mRNA were quantified by qRT–PCR with normalisation to gapdh mRNA. A representative experiment is shown. Error bars represent the s.d. from triplicate PCRs. (B) Formaldehyde crosslinked mononucleosomes were prepared from quiescent cells treated as in (A) and used for ChIPs with an anti-RNA-polymerase-II (Pol II) antibody. Recovery of c-fos and c-jun coding DNA sequences were quantified by real-time PCR. Average % input recoveries and s.d. from 3–4 independent experiments are plotted. (C) ChIPs and real-time PCR were performed as described in (B) using H3K4me3- (top panel) and H3K36me3- (bottom panel) specific antibodies. Amplicons analysed are indicated above each graph. Average % input recoveries and s.d. from 3–4 independent experiments are plotted.

Mentions: As shown in yeast (reviewed in Gerber and Shilatifard, 2003; Hampsey and Reinberg, 2003; Sims et al, 2004), stimulation-dependent increases in K4me3 and K36me3 across coding regions suggest a link with elongating Pol II, a possibility we tested with the transcriptional inhibitor DRB. Quantitative reverse transcription PCR (qRT–PCR) shows that DRB ablates induction of c-fos and c-jun (Figure 3A). This correlates with loss of Pol II across the coding region of both genes under these conditions, shown by ChIPs using anti-Pol II antibodies raised against its N terminus (Figure 3B). Similar results were obtained at several different positions within the coding regions of c-fos and c-jun (Supplementary Figure S4A), except for +444 of c-fos, which showed increased Pol II loading in response to DRB at later time points. This may be explained by the transcriptional pause site reported at this region (Mechti et al, 1991; Coulon et al, 1999).


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

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

EGF-induced increases in H3K4me3 and H3K36me3 within c-fos and c-jun are transcription-dependent. (A) Quiescent cells were pretreated (10 min) with DRB (25 μg/ml) or untreated (con) prior to stimulation with EGF (50 ng/ml) for 15, 30 or 60 min or no-stimulation (−). Quiescent cells were also treated with DRB alone for 25, 40 or 70 min as controls, respectively. Total mRNA was isolated and relative levels of c-fos and c-jun mRNA were quantified by qRT–PCR with normalisation to gapdh mRNA. A representative experiment is shown. Error bars represent the s.d. from triplicate PCRs. (B) Formaldehyde crosslinked mononucleosomes were prepared from quiescent cells treated as in (A) and used for ChIPs with an anti-RNA-polymerase-II (Pol II) antibody. Recovery of c-fos and c-jun coding DNA sequences were quantified by real-time PCR. Average % input recoveries and s.d. from 3–4 independent experiments are plotted. (C) ChIPs and real-time PCR were performed as described in (B) using H3K4me3- (top panel) and H3K36me3- (bottom panel) specific antibodies. Amplicons analysed are indicated above each graph. Average % input recoveries and s.d. from 3–4 independent experiments are plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168397&req=5

f3: EGF-induced increases in H3K4me3 and H3K36me3 within c-fos and c-jun are transcription-dependent. (A) Quiescent cells were pretreated (10 min) with DRB (25 μg/ml) or untreated (con) prior to stimulation with EGF (50 ng/ml) for 15, 30 or 60 min or no-stimulation (−). Quiescent cells were also treated with DRB alone for 25, 40 or 70 min as controls, respectively. Total mRNA was isolated and relative levels of c-fos and c-jun mRNA were quantified by qRT–PCR with normalisation to gapdh mRNA. A representative experiment is shown. Error bars represent the s.d. from triplicate PCRs. (B) Formaldehyde crosslinked mononucleosomes were prepared from quiescent cells treated as in (A) and used for ChIPs with an anti-RNA-polymerase-II (Pol II) antibody. Recovery of c-fos and c-jun coding DNA sequences were quantified by real-time PCR. Average % input recoveries and s.d. from 3–4 independent experiments are plotted. (C) ChIPs and real-time PCR were performed as described in (B) using H3K4me3- (top panel) and H3K36me3- (bottom panel) specific antibodies. Amplicons analysed are indicated above each graph. Average % input recoveries and s.d. from 3–4 independent experiments are plotted.
Mentions: As shown in yeast (reviewed in Gerber and Shilatifard, 2003; Hampsey and Reinberg, 2003; Sims et al, 2004), stimulation-dependent increases in K4me3 and K36me3 across coding regions suggest a link with elongating Pol II, a possibility we tested with the transcriptional inhibitor DRB. Quantitative reverse transcription PCR (qRT–PCR) shows that DRB ablates induction of c-fos and c-jun (Figure 3A). This correlates with loss of Pol II across the coding region of both genes under these conditions, shown by ChIPs using anti-Pol II antibodies raised against its N terminus (Figure 3B). Similar results were obtained at several different positions within the coding regions of c-fos and c-jun (Supplementary Figure S4A), except for +444 of c-fos, which showed increased Pol II loading in response to DRB at later time points. This may be explained by the transcriptional pause site reported at this region (Mechti et al, 1991; Coulon et al, 1999).

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