Chromatin Structure & Dynamics
|研究人員 / 簡介|
My laboratory has focused on functional analyses of histone modifications, centered on the regulation and function of histone H2B ubiquitylation (H2Bub). In the past years, my research group provided new insight into the biological functions of chromatin dynamics regulated by H2Bub. In collaboration with Dr. Didier Devys at the IGBMC, France, we analyzed the H2Bub deubiquitylation activity of SAGA and observed that SAGA acts on the whole transcribed genome in a rapid manner and this highly dynamic association of the complex with chromatin is required for RNA polymerase II transcription (Bonnet et al., 2014). In addition to transcriptional regulation, it has also been shown that H2Bub promotes recovery from replication stress; however, the underlying molecular mechanism remains unclear. We employed a genome-wide approach to investigate how H2Bub affects DNA replication under stress. We observed that coordination of chromatin assembly and DNA damage checkpoint activity by H2Bub is critical for the cell response to replication stress. The replication proteins on replicating DNA are akin to a train on the tracks, and movement of this train is carefully controlled. Our data indicate that H2Bub helps organize DNA in the nuclei during DNA replication; this process plays a similar role to the brakes on a train, serving to slow down replication, and thus maintaining stable progression of replication under environmental stress (Lin et al., 2014). In addition to the molecular function of histone modifications, we recently demonstrate that the expression of Snf1, a member of the AMP-activated protein kinase family is influenced by ubiquitin specific protease Ubp8 and Ubp10, whose best known target is histone H2B (Hsu et al., 2015). In this study we provide evidence the level of Snf1 protein is dramatically decreased in an UBP8 and UBP10 deleted yeast mutant (ubp8D ubp10D), and this is independent of transcriptional regulation and proteasome-mediated degradation. We identified that a novel feedback loop connects Snf1 protein level and its phosphorylation in a manner independent of its ubiquitin-mediated degradation, and this dynamic regulation of Snf1 activity is crucial for adaptation to environmental stress.
H2B ubiquitylation and the Asf1 histone chaperone mediate the formation and maintenance of heterochromatin architecture
Heterochromatin is a heritable form of gene repression, with critical roles in development and cell identity. Understanding how chromatin structure results in such repression is a fundamental question. Chromatin is assembled and disassembled during transcription, replication, and repair by Anti-silencing function 1 (Asf1), a highly conserved histone chaperone. Transcription and DNA replication are also affected by histone modifications, such as H2B ubiquitylation (H2Bub), which exerts effects on nucleosome dynamics. We report here that H2Bub and Asf1 synergistically maintain transcriptional silencing at yeast telomeres and mating loci. By monitoring silencing establishment at the HML locus in real time, we found that the initiation of transcriptional repression was attenuated and never fully established in a mutant lacking both Asf1 and H2Bub. These findings are consistent with a significant reduction in both the binding of the Sir2 and Sir3 repressor proteins at HML silencers, and spreading of these proteins. In addition, mutants lacking H2Bub and Asf1 show defects in both nucleosome assembly and higher-order heterochromatin organization at the HML locus. Our findings reveal a novel role for H2Bub and Asf1 in epigenetic silencing through promoting nucleosome formation. We propose that nucleosome assembly is an ongoing requirement for the formation and maintenance of heterochromatin.
H2B mono-ubiquitylation promotes error-free DNA damage tolerance pathways by fine-tuning chromatin structure around replication forks
DNA lesions bypass is mediated by DNA damage tolerance (DDT) pathways activated by the ubiquitylation of the replication clamp PCNA. However, it is largely unknown how these pathways take action within the context of chromatin. Histone H2B mono-ubiquitylation (H2Bub) has been previously shown to promote nucleosome dynamics during transcription elongation and DNA double strand breaks in yeast or human cells. In our study, we provide evidence that suggests that H2Bub modulates chromatin dynamics and contributes to error-free DDT processes during and after replication. Several lines of evidence support this conclusion. We find that:
(1) In the absence of H2Bub, MMS-damaged forks are resistant to restart and accompanied with the accumulation of unrepaired ssDNA gaps.
(2) Both template switch and salvage HR pathways are impaired in a manner that is downstream of the post-modification of PCNA in cells lack of H2Bub.
(3) Our genetic data support that DNA synthesis during gap repair is dependent on H2Bub.
(4) H2Bub is epistatic to RAD51 and promotes Rad51 recruitment to DNA damage sites.
(5) H2Bub is required for promoting chromatin accessibility around damaged forks.
Taken together, those results point out the importance of chromatin dynamics around replication forks for efficient recruitment of factors to facilitate the DNA damage tolerance pathways and uphold the genome integrity. Our findings are the first to indicate that H2Bub promotes DNA damage tolerance, through the regulation of chromatin configuration. As such, this project will provides a major advance in our understanding of DNA damage tolerance pathways in relationship with chromatin structure.
Bonnet, J., Wang, C.Y., Baptista, T., Vincent, S.D., Hsiao, W.C., Stierle, M., Kao, C.F., Tora, L., and Devys, D. (2014). The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription. Genes Dev 28, 1999-2012.
Hsu, H.E., Liu, T.N., Yeh, C.S., Chang, T.H., Lo, Y.C., and Kao, C.F. (2015). Feedback Control of Snf1 Protein and Its Phosphorylation Is Necessary for Adaptation to Environmental Stress. J Biol Chem 290, 16786-16796.
Lin, C.Y., Wu, M.Y., Gay, S., Marjavaara, L., Lai, M.S., Hsiao, W.C., Hung, S.H., Tseng, H.Y., Wright, D.E., Wang, C.Y., et al. (2014). H2B Mono-ubiquitylation Facilitates Fork Stalling and Recovery during Replication Stress by Coordinating Rad53 Activation and Chromatin Assembly. PLoS Genet 10, e1004667.
Publications in peer-reviewed journals in recent 5 years
(Total citation; 1653 /H-index:15 )