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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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Effects of H3K36me3 knockdown on gene expression levels: lack of intragenic transcription from IE genes or gapdh. (A) C3H 10T½ cells were untransfected (−), mock transfected (no siRNA, mock) or transfected with Setd2, non-targeting (non-t) or NSD1 siRNA. Cells were quiesced 24 h later, and after a further 24 h left untreated (con) or treated with EGF (50 ng/ml) for 15–120 min. Total RNA was isolated and relative levels of Setd2 and NSD1 mRNA were quantified by qRT–PCR and normalised to 18S rRNA. Average values and s.d. from two independent experiments are plotted. (B) Cells were transfected and stimulated and total RNA isolated as in (A). Kinetics of c-fos, c-jun, CPBP and MKP1 gene expression was assessed by qRT–PCR and normalised to 18S rRNA. Average values and s.d. from two independent experiments are plotted. (C) Cells were transfected as in (A), left unstimulated and levels of cycb, polr3b, glnrs and gapdh mRNA quantified by qRT–PCR, with normalisation to 18S rRNA. Average values and s.d. from two independent experiments are plotted.
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f6a: Effects of H3K36me3 knockdown on gene expression levels: lack of intragenic transcription from IE genes or gapdh. (A) C3H 10T½ cells were untransfected (−), mock transfected (no siRNA, mock) or transfected with Setd2, non-targeting (non-t) or NSD1 siRNA. Cells were quiesced 24 h later, and after a further 24 h left untreated (con) or treated with EGF (50 ng/ml) for 15–120 min. Total RNA was isolated and relative levels of Setd2 and NSD1 mRNA were quantified by qRT–PCR and normalised to 18S rRNA. Average values and s.d. from two independent experiments are plotted. (B) Cells were transfected and stimulated and total RNA isolated as in (A). Kinetics of c-fos, c-jun, CPBP and MKP1 gene expression was assessed by qRT–PCR and normalised to 18S rRNA. Average values and s.d. from two independent experiments are plotted. (C) Cells were transfected as in (A), left unstimulated and levels of cycb, polr3b, glnrs and gapdh mRNA quantified by qRT–PCR, with normalisation to 18S rRNA. Average values and s.d. from two independent experiments are plotted.

Mentions: To investigate the potential role of K36me3 in transcriptional regulation, the effect of K36me3 loss on the transcription of IE and constitutively active genes was assessed by qRT–PCR (Figure 6). As shown previously, knock down of Setd2 mRNA (Figure 6A) led to virtually complete loss of all detectable K36me3 (Figure 6D). The gene induction kinetics of c-fos, c-jun and two other IE genes, core promoter element binding protein (CPBP) and MAP kinase phosphatase 1 (MKP1), was examined (Figure 6B). A recent report in yeast has shown that increased K36me leads to delayed induction of the HIS4 gene, whereas elimination of K36me accelerates HIS4 induction (Nelson et al, 2006). However, loss of K36me3 did not affect the kinetics of induction of any gene tested here (Figure 6B). Second, the steady-state mRNA levels of four constitutively active genes (cycb, polr3b, glnrs and gapdh) were assessed in the absence of K36me3, and no significant changes were observed for any transcript (Figure 6C). Furthermore, we did not observe any intragenic transcription from c-fos, c-jun or gapdh in K36me3-depleted cells (Figure 6E). This is in contrast to the observations in yeast where K36me appears to create a less permissive chromatin structure throughout yeast gene coding regions by recruiting HDAC complexes (Rpd3) via interaction with the chromodomain of Eaf3 (Carrozza et al, 2005; Joshi and Struhl, 2005; Keogh et al, 2005). Loss of K36me leads to increased acetylation, K4me3 and Pol II loading within specific gene coding regions and intragenic transcript initiation from the FLO8 and STE11 genes (Carrozza et al, 2005; Joshi and Struhl, 2005; Keogh et al, 2005; Kizer et al, 2005). Similar to the lack of any intragenic transcription in K36me3-depleted mouse fibroblasts (Figure 6E), we do not detect any increased H3 or H4 acetylation at 3′ regions of IE or housekeeping genes (Supplementary Figure S6), nor any change in Pol II occupancy at these genes (Supplementary Figure S7). These studies do not reveal a clear parallel at c-fos and c-jun in mammalian cells for the relationship between H3K36 methylation and histone deacetylation previously seen in yeast.


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

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

Effects of H3K36me3 knockdown on gene expression levels: lack of intragenic transcription from IE genes or gapdh. (A) C3H 10T½ cells were untransfected (−), mock transfected (no siRNA, mock) or transfected with Setd2, non-targeting (non-t) or NSD1 siRNA. Cells were quiesced 24 h later, and after a further 24 h left untreated (con) or treated with EGF (50 ng/ml) for 15–120 min. Total RNA was isolated and relative levels of Setd2 and NSD1 mRNA were quantified by qRT–PCR and normalised to 18S rRNA. Average values and s.d. from two independent experiments are plotted. (B) Cells were transfected and stimulated and total RNA isolated as in (A). Kinetics of c-fos, c-jun, CPBP and MKP1 gene expression was assessed by qRT–PCR and normalised to 18S rRNA. Average values and s.d. from two independent experiments are plotted. (C) Cells were transfected as in (A), left unstimulated and levels of cycb, polr3b, glnrs and gapdh mRNA quantified by qRT–PCR, with normalisation to 18S rRNA. Average values and s.d. from two independent experiments are plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6a: Effects of H3K36me3 knockdown on gene expression levels: lack of intragenic transcription from IE genes or gapdh. (A) C3H 10T½ cells were untransfected (−), mock transfected (no siRNA, mock) or transfected with Setd2, non-targeting (non-t) or NSD1 siRNA. Cells were quiesced 24 h later, and after a further 24 h left untreated (con) or treated with EGF (50 ng/ml) for 15–120 min. Total RNA was isolated and relative levels of Setd2 and NSD1 mRNA were quantified by qRT–PCR and normalised to 18S rRNA. Average values and s.d. from two independent experiments are plotted. (B) Cells were transfected and stimulated and total RNA isolated as in (A). Kinetics of c-fos, c-jun, CPBP and MKP1 gene expression was assessed by qRT–PCR and normalised to 18S rRNA. Average values and s.d. from two independent experiments are plotted. (C) Cells were transfected as in (A), left unstimulated and levels of cycb, polr3b, glnrs and gapdh mRNA quantified by qRT–PCR, with normalisation to 18S rRNA. Average values and s.d. from two independent experiments are plotted.
Mentions: To investigate the potential role of K36me3 in transcriptional regulation, the effect of K36me3 loss on the transcription of IE and constitutively active genes was assessed by qRT–PCR (Figure 6). As shown previously, knock down of Setd2 mRNA (Figure 6A) led to virtually complete loss of all detectable K36me3 (Figure 6D). The gene induction kinetics of c-fos, c-jun and two other IE genes, core promoter element binding protein (CPBP) and MAP kinase phosphatase 1 (MKP1), was examined (Figure 6B). A recent report in yeast has shown that increased K36me leads to delayed induction of the HIS4 gene, whereas elimination of K36me accelerates HIS4 induction (Nelson et al, 2006). However, loss of K36me3 did not affect the kinetics of induction of any gene tested here (Figure 6B). Second, the steady-state mRNA levels of four constitutively active genes (cycb, polr3b, glnrs and gapdh) were assessed in the absence of K36me3, and no significant changes were observed for any transcript (Figure 6C). Furthermore, we did not observe any intragenic transcription from c-fos, c-jun or gapdh in K36me3-depleted cells (Figure 6E). This is in contrast to the observations in yeast where K36me appears to create a less permissive chromatin structure throughout yeast gene coding regions by recruiting HDAC complexes (Rpd3) via interaction with the chromodomain of Eaf3 (Carrozza et al, 2005; Joshi and Struhl, 2005; Keogh et al, 2005). Loss of K36me leads to increased acetylation, K4me3 and Pol II loading within specific gene coding regions and intragenic transcript initiation from the FLO8 and STE11 genes (Carrozza et al, 2005; Joshi and Struhl, 2005; Keogh et al, 2005; Kizer et al, 2005). Similar to the lack of any intragenic transcription in K36me3-depleted mouse fibroblasts (Figure 6E), we do not detect any increased H3 or H4 acetylation at 3′ regions of IE or housekeeping genes (Supplementary Figure S6), nor any change in Pol II occupancy at these genes (Supplementary Figure S7). These studies do not reveal a clear parallel at c-fos and c-jun in mammalian cells for the relationship between H3K36 methylation and histone deacetylation previously seen in yeast.

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