Biochemical Characterization of the Domain Architecture of Chromatin Assembly Motor Proteins Human CHD1 and CHD2
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CitationLiu, Jessica Chishow. 2015. Biochemical Characterization of the Domain Architecture of Chromatin Assembly Motor Proteins Human CHD1 and CHD2. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractThe sites where the basic unit of chromatin, the nucleosome, is assembled greatly affects the dynamic compaction/decompaction of eukaryotic genetic material and how the DNA is accessed, read, and interpreted. The nucleosome, which consists of ~147 base pairs of DNA wrapped in a left-handed superhelix around an octameric core made up of histone proteins, is the targeted substrate for ATP-dependent protein machineries called chromatin remodelers. Remodelers are essential regulators of DNA accessibility and are often grouped into four families: SWI/SNF, INO80/SWR1, ISWI, and CHD. Though remodelers can act as large multi-subunit complexes, all have a unique core SNF2-like ATPase that utilizes the energy from ATP hydrolysis to translocate along DNA. This DNA translocase activity of the catalytic ATPase domain acts in coordination with auxiliary domains or accessory subunits to disrupt histone-DNA contacts, resulting in distinct remodeling outcomes. Furthermore, the assembly of DNA into nucleosomal arrays is a specialized activity catalyzed by a subset of remodelers. Identifying remodeler proteins responsible for nucleosome assembly and delineating the mechanisms through which remodelers assemble and remodel nucleosomes are key goals in the field of chromatin biology.
CHD proteins have important roles in regulating gene expression through their remodeling activities. While yeast cells only have one CHD protein (CHD1), mammalians possess nine proteins (CHD1-9) that are further categorized into subfamilies on the basis of additional sequences flanking the central ATPase domain. CHD2 is in the same subfamily as CHD1 and has been linked to developmental regulation but the enzymatic activity of CHD2 has not been well characterized. Given the homology between human CHD2 and CHD1, which is an important assembly protein in other species (S. cerevisiae and D. melanogaster), we set out to delineate the biochemical properties of human CHD2 and the CHD1 human counterpart.
In this dissertation work, we examined the biochemical activities of recombinant human CHD1 and CHD2. We used in vitro chromatin assembly and remodeling assays and showed CHD2 assembles nucleosomal arrays and remodels nucleosomes while CHD1 exhibits less robust activity by comparison. We used radiometric ATPase and electrophoretic mobility gel shift assays to measure the ATPase and DNA-binding activities of human CHD1 and CHD2 and assessed the contribution from conserved accessory domains using systematic protein truncations. We found the N-terminal chromodomains are inhibitory for the ATPase and DNA-binding activities of both CHD1 and CHD2 while providing substrate specificity for the latter. Moreover, we showed the DNA-binding domain of CHD2 enhances its ATPase and remodeling activities. The distinct in vitro activities exhibited by human CHD1 and CHD2 suggest they have non-redundant roles in vivo with important mechanistic implications for remodeling by CHD proteins. In a broader sense, our findings have added to the number of known assembly motor proteins and aids in our understanding of how remodelers have evolved auxiliary domains to carry out specific functions such as chromatin assembly.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:14226060
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