Reconstitution and structural dissection of transcription-coupled histone H3.3 deposition

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Project Summary DNA-histone interactions are at least transiently lost during replication and transcription, which causes profound change in chromatin structure, and accompanies aging. Thus, efficient maintenance of chromatin structure during replication and transcription is known to be critical for cell survival and normal aging. Recently, the H3.3 histone variant has garnered particular interest in the context of aging because the H3.3 variant accumulates with age and impacts gene expression. In metazoans, the histone variant H3.3 along with H4 is deposited throughout transcribed genes by the HUC (HIRA/UBN1/CABIN1) complex, while in ascomycetes, such as yeast, only one form of histone H3, which shares the same evolutionary ancestry with H3.3 in metazoans, is deposited by the Hir (Hir1/Hir2/Hir3/Hpc2) complex. Understanding how the Hir complex functions in yeast will ultimately facilitate the development of therapeutic pathways to treat the onset of age-associated disease in humans. As my thesis project, I sought a possible cofactor of the Hir complex in yeast whole cell extract using mass spectrometry, and identified Spt4/5, a highly-conserved pol II elongation factor required for transcription of nearly all mRNA genes, which provided first direct evidence of physical interaction between pol II and histone chaperones for histone deposition. This proposal aims to uncover the molecular basis of Spt4/5 in facilitating histone deposition after passage of transcribing pol II by recruiting the Hir complex. I expect this study to provide the first molecular evidence of the interplay between histone deposition and transcription, as well as a first-of-its-kind structural view of its structural basis with following aims: (1) Investigate the functional roles of Spt4/5 in transcription coupled H3/H4 deposition in vitro transcription system. (2) Determine the molecular basis of the Hir complex in the transcription elongation complex via crosslinking mass spectrometry and cryo-EM.