Investigating higher-order chromatin structure and SAGA cooperativity using existing and modified in vitro chromatin systems

Sinha, Divya
Major Professor
Michael A. Shogren-Knaak
Committee Member
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Biochemistry, Biophysics and Molecular Biology

The organization of genomic DNA with histones and other proteins ensures the proper storage, utilization and segregation of DNA's encoded information in a healthy eukaryotic cell. These components comprise chromatin, and knowledge of its structure, mechanisms of its formation, and the dynamic changes it undergoes is essential to gain insights into chromatin's role in controlling gene regulation and the defects associated with misregulation. Here we describe our efforts to understand the different aspects of chromatin by using existing and modified in vitro chromatin model systems.

To better understand histone-histone interactions involved in the formation of higher-order chromatin structure, we employed a disulfide cross-linking strategy previously used to study short-range nucleosomal interactions. Using in vitro assembled nucleosomal arrays, we show that histones H4 and H2A, belonging to nucleosomes on different arrays, directly interact with each other under conditions that promote array-array associations. Additionally, prior intra-array cross-linking of nucleosomal arrays has an antagonistic effect on inter-array self-association. Together, our data show the role of H4-H2A contacts in the interplay between short-range nucleosomal compaction and higher-order chromatin structure.

Nucleosomal arrays used above, generated by assembling histone octamers on DNA templates, provide an excellent model system due to their homogeneity and reproducibility in assembly. However, there is still a need for improved and novel systems to further expand the scope of in vitro chromatin studies. We have generated new DNA templates for nucleosome assembly and have improved our native chemical ligation technique for generating post-translationally modified histones, reducing protein racemization. Acetylated histones generated by the modified histone ligation method were successfully used for the study of ATP-dependent chromatin remodeling by SWI/SNF and RSC complexes.

Finally, previous studies from our group using nucleosomal arrays showed that SAGA, a histone acetyltransferase complex, acetylated nucleosomes cooperatively. Preliminary results indicated that this cooperativity requires functional Gcn5 bromodomain and acetylation of Ada3 subunit. Our follow-up experiments to dissect the role of individual lysine acetylation on Ada3 show that Ada3 lysine 8 pre-acetylated peptide binds tighter to the Gcn5 bromodomain and is a better substrate for acetylation by the Gcn5/Ada2/Ada3 subcomplex as compared to the Ada3 lysine 14 pre-acetylated peptide.