Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
Histone disassembly is one of the mechanisms by which the ATP dependent chromatin remodelers remodel the nucleosomes. However nucleosome remodeling is also regulated by other factors like covalent modifications marks (methylation, acetylation etc. of both histones and chromatin remodelers). and histone chaperones. All these different players act in concert inside the cell, to modulate the chromatin landscape. Histone chaperones can either act independently and or in concert with chromatin remodelers, for nucleosome disassembly. We have previously shown that SWI/SNF has the ability to mediate disassembly of a dinucleosome substrate in a two step process. The first step in this process is the displacement of one H2A-H2B dimer that occurs rapidly, followed by a slower step in which the remaining hexasome is displaced. SWI/SNF mediated disassembly does not require any free DNA as an acceptor or other factors, but does require an adjoining nucleosome. However, little is known about the mechanism by which chaperones act with remodelers in causing disassembly. In this study, we have investigated the role of histone chaperone Nap1 (Nucleosome Assembly Protein1) on the remodeling properties of the yeast remodeler RSC ((Remodels Structure of Chromatin). We focused on the effects of Nap1 when RSC remodels short nucleosomal arrays and compared these to those with mononucleosomes. We found that Nap1 enhances RSC mediated disassembly of nucleosome only on dinucleosome substrates but not on mononucleosomes. The enhanced disassembly is not due to increased ATP hydrolysis as the rate of ATP hydrolysis by RSC does not increase in the presence of Nap1. Enhanced disassembly is also not due to an increase in rate of nucleosome movement. The effects of Nap1 on RSC are limited to dinucleosome substrate, and does not affect its binding or rate of ATP hydrolysis on a free DNA substrate. To further understand the mechanism of Nap1 action, we scanned various mutants on the surface of Nap1, to find out the region of Nap1 that is important for enhancing dinucleosome disassembly by RSC. We found that the same alpha chain and amino acid residues of Nap1 that binds to histone H2A-H2B dimer in solution, is also critical for interaction with RSC during remodelling of nucleosomes, and favours disassembly. This suggests that Nap1 interacts with nucleosomal H2A-H2B during remodeling by RSC, and hence promotes nucleosome disassembly, which the next step in the remodeling process. We have also seen that this enhancement of disassembly is specific to RSC and Nap1, and does not occur with either SWI/SNF, which is a paralog of RSC in yeast or with histone chaperone Vps75, only other member of NAP family in yeast. On the other hand, Nap1 is overall inhibitory for SWI/SNF. Nap1 reduces the rate of nucleosome movement on both mono and di-nucleosomes, by reducing the rate of ATP hydrolysis. Higher concentration of Nap1 inhibits SWI/SNF binding to free DNA and dinucleosomes. Based on an extensive scan using twenty five Nap1 mutants to identify the , we found that the C-terminal region of Nap1 to be inhibitory to SWI/SNF binding to both DNA and dinucleosome while the H2A-H2B binding to inhibitory for SWI/SNF dinucleosome binding. Therefore Nap1 regulates SWI/SNF and RSC complexes in antagonistic ways. This may help explain the activating and occasional repressive roles of RSC and SWI/SNF on gene transcription, by stimulating nucleosome disassembly by RSC and reducing nucleosome movement by SWI/SNF.
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