Chromatin structure is regulated by post-translational histone modifications, histone variants, nucleosome remodeling, and nucleosome exchange. Histone modifications have been connected to a wide range of biological processes, and function to promote or inhibit protein-DNA interactions by serving as binding sites for regulatory factors or by regulating the state of chromatin folding. The monoubiquitylation of H2B (H2Bub1) has been tightly linked to active transcription, and in particular to transcription elongation. In organisms from yeast to humans, H2Bub1 is established co-transcriptionally through the association of the ubiquitylation machinery with elongating RNA Polymerase II (Pol II) and the mediation of transcription elongation factors. The key issues relating to the function of H2Bub1 are whether it is linked to other biological processes besides transcription, and how its presence in chromatin affects its cellular roles. We hypothesize that H2Bub1 regulates the assembly or activity of multi-protein complexes in a number of different cellular processes because it promotes a stable chromatin environment. We have found that H2Bub1 is present at yeast origins of replication and that it plays a role in progression of the DNA replication fork, a mechanistically similar phenomenon to transcription elongation.
In Specific Aim 1, we will use genetic and molecular approaches to identify the factors that regulate H2Bub1 at origins and define the role of H2Bub1 in replication by examining the effect of an htb-K123R mutation on replication-dependent nucleosome dynamics. The regulation and function of histone modifications have been studied primarily in the context of growing cells. However, most eukaryotic cells spend a large fraction of their life cycle in a non-growing or quiescent state tha is characterized by a general repression of transcription. Yeast stationary phase (SP) has emerged as an excellent model for the study of cellular quiescence in the context of chronological lifespan (CLS). Our studies have revealed that a specific pattern of histone modifications characterize SP quiescent cells. This pattern includes the selective loss of H2Bub1 and its downstream mark of H3K79me2, and the retention of marks associated with transcription initiation (H3K4me3) and elongation (H3K36me3, H3K79me3).
In Specific Aim 2, we will use genetic, molecular, and genomic approaches to investigate the functional significance of these modifications in the development of quiescence and in release of cells from the quiescent state. We will also explore if mutants with shortened or lengthened CLS have perturbed patterns of histone modifications, and if the deubiquitylation of H2B determines the timing of differentiation into quiescent and nonquiescent cells. Together, the studies outlined in the two Aims should provide new mechanistic insights into the role of chromatin in basic cellular processes and reveal how alterations in chromatin structure potentially impact human disease.
H2B ubiquitylation is a histone modification with significant relevance to human health, particularly cancer. A tumor suppressor encodes a key regulator of H2B ubiquitylation, and several downstream histone modifications that are mediated by H2B ubiquitylation are linked to the inappropriate expression of developmentally important genes in leukemia. Understanding the fundamental roles of H2B in chromatin structure and function could lead to the development of new therapies in cancer. Cellular quiescence is a key mechanism for cell and tissue regeneration and has emerged as important factor in the survival of both normal adult and cancer stem cells. Defining the underlying epigenetic mechanisms that contribute to cellular quiescence could eventually lead to the development of new treatments to stimulate cellular regeneration in response to injury or to eliminate cancer stem cells.
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