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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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Setd2 is responsible for H3K36 trimethylation at IE and housekeeping genes. (A) Schematic representation of the gapdh, polr3b, glnrs and cycb housekeeping genes showing regions amplified by primers used for real-time PCR. Primer positions shown indicate 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. Transcription termination sites are shown as filled circles. (B) Cells were untransfected (−) or transfected with Setd2 or non-targeting (non-t) siRNA. Cells were quiesced 24 h later, and after a further 24 h were unstimulated (−) or stimulated with EGF (50 ng/ml) for 15–60 min. Formaldehyde crosslinked mononucleosomes were prepared and aliquots of each sample were heated to reverse the crosslinks, separated by 15% SDS–PAGE, transferred to PVDF and immunoblotted with an H3K36me3-specific antibody. Membranes were stained with Ponceau S before immunoblotting to verify even loading. (D) Aliquots of formaldehyde crosslinked mononucleosomes from unstimulated cells prepared as in (B) were used in ChIP assays with H3K36me3- (i), H3K36me2- (ii) and H3K36me1- (iii) specific antibodies. Recovery of gapdh, cycb, polr3b and glnrs coding region sequences were quantified by real-time PCR. Average % input recoveries and s.d. from two independent experiments are plotted.
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f5a: Setd2 is responsible for H3K36 trimethylation at IE and housekeeping genes. (A) Schematic representation of the gapdh, polr3b, glnrs and cycb housekeeping genes showing regions amplified by primers used for real-time PCR. Primer positions shown indicate 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. Transcription termination sites are shown as filled circles. (B) Cells were untransfected (−) or transfected with Setd2 or non-targeting (non-t) siRNA. Cells were quiesced 24 h later, and after a further 24 h were unstimulated (−) or stimulated with EGF (50 ng/ml) for 15–60 min. Formaldehyde crosslinked mononucleosomes were prepared and aliquots of each sample were heated to reverse the crosslinks, separated by 15% SDS–PAGE, transferred to PVDF and immunoblotted with an H3K36me3-specific antibody. Membranes were stained with Ponceau S before immunoblotting to verify even loading. (D) Aliquots of formaldehyde crosslinked mononucleosomes from unstimulated cells prepared as in (B) were used in ChIP assays with H3K36me3- (i), H3K36me2- (ii) and H3K36me1- (iii) specific antibodies. Recovery of gapdh, cycb, polr3b and glnrs coding region sequences were quantified by real-time PCR. Average % input recoveries and s.d. from two independent experiments are plotted.

Mentions: Next, we used ChIP to analyse all states of K36 methylation at c-fos- and c-jun coding regions (mid and 3′ end) after Setd2 knockdown. Four housekeeping genes were also analysed; glyceraldehyde-3-phosphate-dehydrogenase (gapdh), RNA-polymerase III subunit b (polr3b), glutaminyl-tRNA synthetase (glnrs) and cyclophilin b (cycb) (shown schematically in Figure 5A).


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

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

Setd2 is responsible for H3K36 trimethylation at IE and housekeeping genes. (A) Schematic representation of the gapdh, polr3b, glnrs and cycb housekeeping genes showing regions amplified by primers used for real-time PCR. Primer positions shown indicate 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. Transcription termination sites are shown as filled circles. (B) Cells were untransfected (−) or transfected with Setd2 or non-targeting (non-t) siRNA. Cells were quiesced 24 h later, and after a further 24 h were unstimulated (−) or stimulated with EGF (50 ng/ml) for 15–60 min. Formaldehyde crosslinked mononucleosomes were prepared and aliquots of each sample were heated to reverse the crosslinks, separated by 15% SDS–PAGE, transferred to PVDF and immunoblotted with an H3K36me3-specific antibody. Membranes were stained with Ponceau S before immunoblotting to verify even loading. (D) Aliquots of formaldehyde crosslinked mononucleosomes from unstimulated cells prepared as in (B) were used in ChIP assays with H3K36me3- (i), H3K36me2- (ii) and H3K36me1- (iii) specific antibodies. Recovery of gapdh, cycb, polr3b and glnrs coding region sequences were quantified by real-time PCR. Average % input recoveries and s.d. from two independent experiments are plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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f5a: Setd2 is responsible for H3K36 trimethylation at IE and housekeeping genes. (A) Schematic representation of the gapdh, polr3b, glnrs and cycb housekeeping genes showing regions amplified by primers used for real-time PCR. Primer positions shown indicate 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. Transcription termination sites are shown as filled circles. (B) Cells were untransfected (−) or transfected with Setd2 or non-targeting (non-t) siRNA. Cells were quiesced 24 h later, and after a further 24 h were unstimulated (−) or stimulated with EGF (50 ng/ml) for 15–60 min. Formaldehyde crosslinked mononucleosomes were prepared and aliquots of each sample were heated to reverse the crosslinks, separated by 15% SDS–PAGE, transferred to PVDF and immunoblotted with an H3K36me3-specific antibody. Membranes were stained with Ponceau S before immunoblotting to verify even loading. (D) Aliquots of formaldehyde crosslinked mononucleosomes from unstimulated cells prepared as in (B) were used in ChIP assays with H3K36me3- (i), H3K36me2- (ii) and H3K36me1- (iii) specific antibodies. Recovery of gapdh, cycb, polr3b and glnrs coding region sequences were quantified by real-time PCR. Average % input recoveries and s.d. from two independent experiments are plotted.
Mentions: Next, we used ChIP to analyse all states of K36 methylation at c-fos- and c-jun coding regions (mid and 3′ end) after Setd2 knockdown. Four housekeeping genes were also analysed; glyceraldehyde-3-phosphate-dehydrogenase (gapdh), RNA-polymerase III subunit b (polr3b), glutaminyl-tRNA synthetase (glnrs) and cyclophilin b (cycb) (shown schematically in Figure 5A).

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