Structure And Function Of The Histone Chaperone Rtt106 And Human Brd2

We have solved the structure of the core domain of Rtt106 (Rtt106-M), which is a Saccharomyces cerevisiae histone chaperone with roles in heterochromatin silencing and nucleosome assembly, and have solved the structures of Bromodomains of human Brd2. Rtt106-M adopts an unusual "double pleckstrin homology" domain architecture that represents a novel structural mode for histone chaperones. Aβ-sheet andβ-hairpin structure in Rtt106-M was found out to bind histone H3-H4. A positively-charged and conserved region was identified in one surface of Rtt106-M. This region was then found to bind double-stranded DNA(dsDNA). Mutagenesis studies reveal that the histone and DNA binding activities of Rtt106 are involved in Sir protein-mediated heterochromatin formation.There are an introduction and overview of histone chaperone in chapter 1. The eukaryotic genome DNA is compacted into chromatin which has complex and different levels of structure. The structure of chromatin is dynamicly changed during transcription, replication, DNA repair and epigenetic information inheritance. Many cellular proteins are involved in remodeling the structure of chromatin, including the ATP-dependent chromatin remodeling complex and histone chaperone. The histone chaperones play important roles in DNA replication-dependent and transcription-dependent nucleosome disassembly or assembly. The histone chaperones are also involved in cellular histones storage and transfer. We focus on structure and function of histone chaperones.Chapter 2 introduces the cellular function of Rtt106 and our studies on this important histone chaperone. Rtt106-M adopts a "double PH" domain architecture (PH1-PH2). Rtt106-PH2 domain closely resembles the standard PH domain, while PHI is a derivant of PH domain with insertion sequence in loop regions. The two PH domains are intimately associated with each other through hydrophobic and type-conserved residues. The overall structure of Rtt106-M and Pob3-M is similar with an RMSD of 2.7A. A positively-charged region stretching across one side of the two PH domains is found to bind dsDNA. The PH2 domain could bind H3-H4 and the P-hairpin formed by S12-S13 strands is critical in this interaction. The residues in the loop region of theβ-hairpin are highly conserved, when mutated the interaction between Rtt106-M and H3-H4 is abolished. Rtt106 could bind both dsDNA and H3-H4 indepently. The TPE, immunofluorescence and ChIP experiments reveal that the histone and DNA binding activities of Rtt106 are involved in telomere heterochromatin silencing. In addition, the methods of yeast histone expression and purification, and procedures of tetramer and octamer reconstition in our lab are introduced.Chapter 3 briefly presents our work about the Bromodomains of human Brd2. Brd2 has two Bromodomains-BD1 and BD2. We have solved the solution structure of BD2 and studied its interaction with acetylated H4 tails by NMR chemical shifts perturbation experiments. We have also solved the crystal structures of BD1, BD2 and BD2 in complexed with an acetylated H4 tail.