The Transcriptional Corepressor SIN3 Directly Regulates Genes Involved in Methionine Catabolism and Affects Histone Methylation, Linking Epigenetics and Metabolism

Abstract

Chromatin modification and cellular metabolism are tightly connected. Chromatin modifiers regulate the expression of genes involved in metabolism and, in turn, the levels of metabolites. The generated metabolites are utilized by chromatin modifiers to affect epigenetic modification. The mechanism for this cross-talk, however, remains incompletely understood. The corepressor SIN3 controls histone acetylation through association with the histone deacetylase RPD3. The SIN3 complex is known to regulate genes involved in a number of metabolic processes. Here, we find that Drosophila SIN3 binds to the promoter region of genes involved in methionine catabolism and that this binding affects histone modification, which in turn influences gene expression. Specifically, we observe that reduced expression of SIN3 leads to an increase in S-adenosylmethionine (SAM), which is the major cellular donor of methyl groups for protein modification. Additionally, Sin3A knockdown results in an increase in global histone H3K4me3 levels. Furthermore, decreased H3K4me3 caused by knockdown of either SAM synthetase (Sam-S) or the histone methyltransferase Set1 is restored to near normal levels when SIN3 is also reduced. Taken together, these results indicate that knockdown of Sin3A directly alters the expression of methionine metabolic genes to increase SAM, which in turn leads to an increase in global H3K4me3. Our study reveals that SIN3 is an important epigenetic regulator directly connecting methionine metabolism and histone modification.

Keywords: Drosophila; S-adenosylmethionine (SAM); SIN3; gene transcription; histone acetylation; histone methylation; methionine.

FIGURE 1.

Methionine metabolism in Drosophila. The methionine cycle generates the major methyl donor SAM. The key Drosophila genes encoding SAM synthetase, adenosylhomocysteinase, and cystathionine-β-synthase are listed. Set1 encodes one of methyltransferases. CG10623 encodes a putative methionine synthase.

FIGURE 2.

Transcription of methionine metabolic genes is regulated by SIN3. A, expression of methionine metabolic genes as determined in an RNA-Seq profile (16). B, verification of Sin3A knockdown. Whole cell extracts from RNAi-treated cells were subjected to Western blotting analysis using the indicated antibodies. α-Tubulin acted as the loading control. Protein size markers are indicated on the right. C, real time qRT-PCR analysis of transcription of methionine metabolic genes and histone methyltransferase genes. The results are the averages of three independent biological replicates. The error bars represent standard error of the mean. Statistically significant results comparing individual knockdown samples to the control are indicated on knockdown samples. GFP RNAi cells are the control cells. **, p < 0.01.

FIGURE 3.

H3K9ac and H3K4me3 levels at the promoters of methionine metabolic genes are regulated by SIN3. A, SIN3 ChIP-seq signals at methionine metabolic genes. B, ChIP-qPCR analysis of H3K9ac and H3K4me3 levels at the promoters of methionine metabolic genes. The results are the average of three independent biological replicates. The error bars represent standard error of the mean. Statistically significant results comparing individual knockdown samples to the control are indicated on knockdown samples. GFP RNAi cells are the control cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, reduction of SIN3 does not affect the protein level of RPD3. Whole cell extracts from RNAi-treated cells were subjected to Western blotting analysis using the indicated antibodies. β-Actin acted as the loading control. Protein size markers are indicated on the right.

FIGURE 4.

Levels of SAM and global H3K4me3 are regulated by SIN3. A, effects of SIN3 on the cellular concentration of the metabolites involved in methionine metabolism. B, whole cell extracts from GFP RNAi control and Sin3A knockdown S2 cells were subjected to Western blotting analysis using the indicated antibodies. Protein size markers are indicated on the right. C, Western blots as shown in B were repeated with protein extracts prepared from at least three independent cultures, and the results were quantified after normalization to histone H4. The error bars represent standard error of the mean. Statistically significant results comparing individual knockdown samples to the control are indicated on knockdown samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Decreased global H3K4me3 levels caused by reduction of SAM-S or SET1 is restored to near control levels upon Sin3A knockdown. A, verification of Sin3A knockdown. Whole cell extracts from RNAi-treated cells were subjected to Western blotting analysis using the indicated antibodies. α-Tubulin acted as the loading control. Protein size markers are indicated on the right. B, real time qRT-PCR analysis of Sam-S and Set1 transcript level. C, whole cell extracts from RNAi-treated cells were subjected to Western blotting analysis using the indicated antibodies. Protein size markers are indicated on the right. D, Western blots as shown in C were repeated with protein extracts prepared from at least three independent cultures, and the results were quantified after normalization to histone H4. Set1-1 KD and Set1-2 KD used two different dsRNA targeting different regions of Set1 mRNA, but these regions overlap. Therefore, we used Set1-1 oligonucleotides for the rest of the study and referred to it as Set1 KD. The error bars represent standard error of the mean. Statistically significant results comparing individual knockdown samples to the control are indicated on knockdown samples. p values were also calculated between the double knockdown samples and each single knockdown sample for the two tested genes, e.g. Sin3A + Sam-S KD to Sin3A KD or Sin3A + Sam-S KD to Sam-S KD. Statistically significant results are indicated with the bars. GFP RNAi cells are the control cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001.