Project description:H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation (SHM) in AID target loci. H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci, in both S and variable regions, and in non-IgH loci, such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting approximately 5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Spt6 is a unique histone chaperone, capable of regulating the histone epigenetic state of both AID targets and the AID locus. CH12F3-2A cells were transfected with control and Spt6 siRNAs; 24h later, cells were stimulated with CIT to induce CSR. Total RNA was extracted from control and Spt6 siRNA treated cells for mRNA expression profiling.
Project description:H3K4me3 plays a critical role in the activation-induced cytidine deaminase (AID)-induced DNA cleavage of switch (S) regions in the immunoglobulin heavy chain (IgH) locus during class-switch recombination (CSR). The histone chaperone complex facilitates chromatin transcription (FACT) is responsible for forming H3K4me3 at AID target loci. Histone chaperone suppressor of Ty6 (Spt6) also participates in regulating H3K4me3 for CSR and for somatic hypermutation (SHM) in AID target loci. H3K4me3 loss was correlated with defects in AID-induced DNA breakage and reduced mutation frequencies in IgH loci, in both S and variable regions, and in non-IgH loci, such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and small nucleolar RNA host gene 3 (SNHG3). Global gene expression analysis revealed that Spt6 can act as both a positive and negative transcriptional regulator in B cells, affecting approximately 5% of the genes that includes suppressor of Ty4 (Spt4) and AID. Interestingly, Spt6 regulates CSR and AID expression through two distinct histone modification pathways, H3K4me3 and H3K36me3, respectively. Spt6 is a unique histone chaperone, capable of regulating the histone epigenetic state of both AID targets and the AID locus.
Project description:Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the requirements for this critical factor, we have performed genome-wide analyses of transcription, chromatin structure, and histone modifications in an S. pombe spt6 mutant. Our results demonstrate several dramatic changes to transcription and chromatin structure in the spt6 mutant, including an elevation of antisense transcripts at over 70 percent of all genes and general loss of the +1 nucleosome. Furthermore, Spt6 is required for the trimethylation of histone H3 on lysines 4 and 36, marks associated with active transcription. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones. ChIP-seq experiments were performed on wild type and spt6-1 strains on the following proteins: RNA polymerase II (Rpb1), Paf1 Complex (Ctr9), COMPASS (Swd1), Set2, Spt6, histones H2B and H3, histone modifications H3K4me3 and H3K36me3. Experiments were performed in replicates and matching inputs were also sequenced.
Project description:Histone chaperones affect chromatin structure and gene expression through interaction with histones and RNA polymerase II (PolII). Here, we report that the histone chaperone Spt6 counteracts H3K27me3, an epigenetic mark deposited by the Polycomb Repressive Complex 2 (PRC2) and associated with transcriptional repression. We found that Spt6 is required for proper engagement and function of the H3K27 demethylase KDM6A (UTX) on muscle genes and regulates muscle gene expression and cell differentiation. ChIP-Seq experiments revealed an extensive genome-wide overlap of Spt6, PolII and KDM6A at transcribed regions that are devoid of H3K27me3. Mammalian cells and zebrafish embryos with reduced Spt6 display increased H3K27me3 and diminished expression of the master regulator MyoD, resulting in myogenic differentiation defects. As a confirmation for an antagonistic relationship between Spt6 and H3K27me3, inhibition of PRC2 permits MyoD re-expression in myogenic cells with reduced Spt6. Our data indicate that, through cooperation with PolII and KDM6A, Spt6 orchestrates removal of H3K27me3, thus effectively controlling developmental gene expression and cell differentiation. Examination of Spt6 and KDM6A levels in a skeletal muscle cells at various developmental stages
Project description:Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 interacts with elongating RNA polymerase II (RNAPII) via a tandem Src2 homology (tSH2) domain, but it is not known whether this particular interaction is required for the nucleosome reassembly activity of Spt6. Here, we show that Spt6 recruitment to genes and its nucleosome reassembly functions are largely independent of association with RNAPII. Instead, the Spt6-RNAPII association is required for post-transcriptional mRNA turnover. Mechanistically, association of Spt6 with RNAPII couples the Ccr4-Not complex to the transcribed regions of genes, which we show regulates the timely deadenylation and degradation of a broad range of mRNAs including those required for cell cycle progression. Thus, our findings reveal an unexpected control mechanism for mRNA turnover facilitated by a histone chaperone during transcription.
Project description:The disassembly and reassembly of nucleosomes by histone chaperones is an essential activity during eukaryotic transcription elongation. This highly conserved process maintains chromatin integrity by transiently removing nucleosomes as barriers and then restoring them in the wake of transcription. While transcription elongation requires multiple histone chaperones, there is little understanding of how most of them function and why so many are required. Here, we show that the histone chaperone Spt6 acts through its acidic, intrinsically disordered N-terminal domain (NTD) to bind histones and control chromatin structure. The Spt6 NTD is essential for viability and its histone binding activity is conserved between yeast and humans. The essential nature of the Spt6 NTD can be bypassed by changes in another histone chaperone, FACT, revealing a close functional connection between the two. Our results have led to a mechanistic model for dynamic cooperation between multiple histone chaperones during transcription elongation.
Project description:The disassembly and reassembly of nucleosomes by histone chaperones is an essential activity during eukaryotic transcription elongation. This highly conserved process maintains chromatin integrity by transiently removing nucleosomes as barriers and then restoring them in the wake of transcription. While transcription elongation requires multiple histone chaperones, there is little understanding of how most of them function and why so many are required. Here, we show that the histone chaperone Spt6 acts through its acidic, intrinsically disordered N-terminal domain (NTD) to bind histones and control chromatin structure. The Spt6 NTD is essential for viability and its histone binding activity is conserved between yeast and humans. The essential nature of the Spt6 NTD can be bypassed by changes in another histone chaperone, FACT, revealing a close functional connection between the two. Our results have led to a mechanistic model for dynamic cooperation between multiple histone chaperones during transcription elongation.
Project description:Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the requirements for this critical factor, we have performed genome-wide analyses of transcription, chromatin structure, and histone modifications in an S. pombe spt6 mutant. Our results demonstrate several dramatic changes to transcription and chromatin structure in the spt6 mutant, including an elevation of antisense transcripts at over 70 percent of all genes and general loss of the +1 nucleosome. Furthermore, Spt6 is required for the trimethylation of histone H3 on lysines 4 and 36, marks associated with active transcription. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones. RNA-seq experiments were performed on wild type and spt6-1 strains in replicate
Project description:The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. In all, eight samples were processed - four genotypes (1. SPT6, RPB3-untagged; 2. SPT6, RPB3-tagged; 3. spt6-1004, RPB3-untagged; 4. spt6-1004; RPB3-tagged) in biological duplicates.
Project description:Histone chaperones affect chromatin structure and gene expression through interaction with histones and RNA polymerase II (PolII). Here, we report that the histone chaperone Spt6 counteracts H3K27me3, an epigenetic mark deposited by the Polycomb Repressive Complex 2 (PRC2) and associated with transcriptional repression. We found that Spt6 is required for proper engagement and function of the H3K27 demethylase KDM6A (UTX) on muscle genes and regulates muscle gene expression and cell differentiation. ChIP-Seq experiments revealed an extensive genome-wide overlap of Spt6, PolII and KDM6A at transcribed regions that are devoid of H3K27me3. Mammalian cells and zebrafish embryos with reduced Spt6 display increased H3K27me3 and diminished expression of the master regulator MyoD, resulting in myogenic differentiation defects. As a confirmation for an antagonistic relationship between Spt6 and H3K27me3, inhibition of PRC2 permits MyoD re-expression in myogenic cells with reduced Spt6. Our data indicate that, through cooperation with PolII and KDM6A, Spt6 orchestrates removal of H3K27me3, thus effectively controlling developmental gene expression and cell differentiation.