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Forkhead transcription factor Fkh1: insights into functional regulatory domains crucial for recruitment of Sin3 histone deacetylase complex.

ABSTRACT: Transcription factors are inextricably linked with histone deacetylases leading to compact chromatin. The Forkhead transcription factor Fkh1 is mainly a negative transcriptional regulator which affects cell cycle control, silencing of mating-type cassettes and induction of pseudohyphal growth in the yeast Saccharomyces cerevisiae. Markedly, Fkh1 impinges chromatin architecture by recruiting large regulatory complexes. Implication of Fkh1 with transcriptional corepressor complexes remains largely unexplored. In this work we show that Fkh1 directly recruits corepressors Sin3 and Tup1 (but not Cyc8), providing evidence for its influence on epigenetic regulation. We also identified the specific domain of Fkh1 mediating Sin3 recruitment and substantiated that amino acids 51-125 of Fkh1 bind PAH2 of Sin3. Importantly, this part of Fkh1 overlaps with its Forkhead-associated domain (FHA). To analyse this domain in more detail, selected amino acids were replaced by alanine, revealing that hydrophobic amino acids L74 and I78 are important for Fkh1-Sin3 binding. In addition, we could prove Fkh1 recruitment to promoters of cell cycle genes CLB2 and SWI5. Notably, Sin3 is also recruited to these promoters but only in the presence of functional Fkh1. Our results disclose that recruitment of Sin3 to Fkh1 requires precisely positioned Fkh1/Sin3 binding sites which provide an extended view on the genetic control of cell cycle genes CLB2 and SWI5 and the mechanism of transcriptional repression by modulation of chromatin architecture at the G2/M transition.

SUBMITTER: Aref R

PROVIDER: S-EPMC8139909 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Regulation of gene expression by histone-modifying enzymes is essential to control cell fate decisions and developmental processes. Two histone-modifying enzymes, RPD3, a deacetylase, and dKDM5/LID, a demethylase, are present in a single complex, coordinated through the SIN3 scaffold protein. While the SIN3 complex has been demonstrated to have functional histone deacetylase activity, the role of the demethylase dKDM5/LID as part of the complex has not been investigated.Here, we analyzed the developmental and transcriptional activities of dKDM5/LID in relation to SIN3. Knockdown of either Sin3A or lid resulted in decreased cell proliferation in S2 cells and wing imaginal discs. Conditional knockdown of either Sin3A or lid resulted in flies that displayed wing developmental defects. Interestingly, overexpression of dKDM5/LID rescued the wing developmental defect due to reduced levels of SIN3 in female flies, indicating a major role for dKDM5/LID in cooperation with SIN3 during development. Together, these observed phenotypes strongly suggest that dKDM5/LID as part of the SIN3 complex can impact previously uncharacterized transcriptional networks. Transcriptome analysis revealed that SIN3 and dKDM5/LID regulate many common genes. While several genes implicated in cell cycle and wing developmental pathways were affected upon altering the level of these chromatin factors, a significant affect was also observed on genes required to mount an effective stress response. Further, under conditions of induced oxidative stress, reduction of SIN3 and/or dKDM5/LID altered the expression of a greater number of genes involved in cell cycle-related processes relative to normal conditions. This highlights an important role for SIN3 and dKDM5/LID proteins to maintain proper progression through the cell cycle in environments of cellular stress. Further, we find that target genes are bound by both SIN3 and dKDM5/LID, however, histone acetylation, not methylation, plays a predominant role in gene regulation by the SIN3 complex.We have provided genetic evidence to demonstrate functional cooperation between the histone demethylase dKDM5/LID and SIN3. Biochemical and transcriptome data further support functional links between these proteins. Together, the data provide a solid framework for analyzing the gene regulatory pathways through which SIN3 and dKDM5/LID control diverse biological processes in the organism.

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Forkhead transcription factor Fkh1: insights into functional regulatory domains crucial for recruitment of Sin3 histone deacetylase complex.

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