UC Berkeley

In all eukaryotic organisms, DNA is packaged into chromatin, which controls gene expression and genomic stability. The chromatin landscape is dynamic and responds to environmental signals to direct different cellular programs. Chromatin architecture is regulated by a network of structural and enzymatic proteins that interact with histones to alter nucleosome composition. In my dissertation, I examined 1) the interaction between histone chaperones and newly synthesized histones during nucleosome assembly and 2) the interplay between canonical histone isotypes, histone variants, and histone modifications. My work established how the unusual domain architecture of a newly discovered histone chaperone, Rtt106, provides specificity for acetylated histone cargo. Additionally, I discovered several new histone modifications and examined the relationship between the histone variant H2A.Z and two canonical histone H2B isotypes (Htb1 and Htb2). My dissertation establishes a framework for understanding the additional levels of genomic regulation achieved by histone chaperones, variants, isotypes, and modifications.

In chapter 2, I examine the structural basis for the specificity of the histone chaperone Rtt106 for H3 molecules modified by an acetylation at lysine 56 (H3K56ac). The X ray crystal structure, determined by our collaborators Andy Antczak and James Berger, revealed that Rtt106 contains a double pleckstrin homology (PH) motif. A targeted mutational screen identified two regions on Rtt106 that, when mutated individually, each disrupted Rtt106-H3 binding. One region was a basic surface on the N-terminal PH domain and the other was a loop within the C-terminal PH domain. Although binding experiments did not directly identify an H3K56ac binding pocket on Rtt106, a comparative analysis with the chromatin remodeling protein Pob3 implicated the C-terminal loop as the source of H3K56ac-specificity in the Rtt106-H3 interaction. This work establishes new domain architecture for acetyl-lysine recognition and expands our understanding of how chaperone-histone binding is regulated.

Armed with Rtt106 mutants that reduced H3 binding activity, in chapter 3 I examine the role of Rtt106-mediated nucleosome assembly during replication, transcription, and silencing. Although Rtt106 mutant proteins localized to origins of replication and silent chromatin, without the ability to bind H3, these mutants could not deliver H3K56ac into chromatin. Reduced H3K56ac occupancy was detrimental to replication whereas excess unincorporated H3K56ac was antagonistic to silencing. In contrast, H3K56ac binding was required to recruit Rtt106 to histone gene promoters. Without recruitment of Rtt106 to these loci, we observed defects in H3K56ac incorporation and histone gene repression. Our work demonstrates that Rtt106-H3 binding is necessary for all known branches Rtt106-mediated nucleosome assembly, however these different branches rely on distinct genomic localization cues to target Rtt106 to chromatin.

To analyze the relationships between canonical histones, modifications, and variants, in chapter 4 I explore the unique functions of two histone H2B isotypes, Htb1 and Htb2. In collaboration with the Freitas lab at the Ohio State University, we discovered three new modifications on H2B, one of which was isotype-specific. Although the dimeric association of H2A.Z with H2B did not reveal an isotype-specific interaction, the interplay between canonical histone isotypes and variants remains an intriguing paradigm for chromatin regulation. In collaboration with the Giaever lab at the University of Toronto, we used chemical genomic profiling to define unique functions associated with the Htb1 and Htb2 isotypes. However, all chemical sensitivities identified resulted from changes in histone expression rather than Htb1 and Htb2 protein activity. Although we are still searching for a functional distinction between the two H2B isotypes, mass spectrometry analyses coupled with chemical-genomic profiling represents a promising strategy for discovering these relationships and defining their functional impact.