Several multiprotein complexes are implicated in the ATP-dependent remodeling of nucleosomes for transcription, including the evolutionarily conserved S. cerevisiae Snf/Swi complex. Other essential processes also require the temporal restructuring of chromatin as cells progress through the cell division cycle or undergo differentiation, any of which might require Snf/Swi or related factors. A novel sixteen- protein complex capable of remodeling the structure of chromatin (RSC) has been purified from yeast. Despite a shared ATP-dependent nucleosome remodeling activity, the functions of RSC and Snf/Swi are distinct: Rsc polypeptides are indispensable, and thermolabile mutations in two RSC genes, STH1 (SNF2 homolog) and SFH1 (SNF5 homolog), arrest cells at the G2/M transition of the cell cycle. A study of sth1 and sfh1 conditional mutants therefore presents an excellent opportunity to investigate the mechanisms that link chromatin remodeling to progression through the cell division cycle. To identify targets of RSC function, screens for extragenic suppressors of an sth1-ts mutation, including multicopy suppressors and suppressor mutations, and for second-site mutations that are lethal in combination with this ts allele will be completed. In the second aim, new sfh1-ts alleles, identified in a genetic screen and from a targeted mutagenesis based on the corresponding snf5-ts mutations, will be characterized functionally and biochemically. Conditional alleles can lead to partial or complete loss of SFH1 function by impairing distinct activities, including cell cycle progression, G1- specific phosphorylation, transcriptional activation, and assembly into RSC. The premise that the abundance, phosphorylation state, or activities of Sth1p or Sfh1p fluctuate in the cell cycle, will be tested in aim three. sth1 and sfh1 mutants will be assayed for the ability to carry out specific functions implicated in the dynamic reorganization of chromatin during the cell cycle, including those required for the completion of DNA replication or chromosome condensation and segregation. In the fourth objective, mechanism(s) by which RSC restructures nucleosomes will be studied in vivo using a novel probe of the chromatin structure of mini-chromosomes. The long-term goal of this project is to define the physiological function of RCS, including the transduction pathway(s) that links chromatin remodeling to cell cycle progression. Elucidation of this connection will impact directly on our understanding of mechanisms that control differentiation and development in multicellular organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056700-04
Application #
6386783
Study Section
Genetics Study Section (GEN)
Program Officer
Carter, Anthony D
Project Start
1998-08-01
Project End
2003-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
4
Fiscal Year
2001
Total Cost
$250,330
Indirect Cost
Name
Suny Downstate Medical Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
068552207
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
Liang, Bing; Qiu, Jiajing; Ratnakumar, Kajan et al. (2007) RSC functions as an early double-strand-break sensor in the cell's response to DNA damage. Curr Biol 17:1432-7
Chai, Bob; Huang, Jian; Cairns, Bradley R et al. (2005) Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair. Genes Dev 19:1656-61
Huang, Jian; Liang, Bing; Qiu, Jiajing et al. (2005) ATP-dependent chromatin-remodeling complexes in DNA double-strand break repair: remodeling, pairing and (re)pairing. Cell Cycle 4:1713-5
Huang, Jian; Laurent, Brehon C (2004) A Role for the RSC chromatin remodeler in regulating cohesion of sister chromatid arms. Cell Cycle 3:973-5
Geng, Fuqiang; Laurent, Brehon C (2004) Roles of SWI/SNF and HATs throughout the dynamic transcription of a yeast glucose-repressible gene. EMBO J 23:127-37
Huang, Jian; Hsu, Jing-Mei; Laurent, Brehon C (2004) The RSC nucleosome-remodeling complex is required for Cohesin's association with chromosome arms. Mol Cell 13:739-50
Hsu, Jing-Mei; Huang, Jian; Meluh, Pamela B et al. (2003) The yeast RSC chromatin-remodeling complex is required for kinetochore function in chromosome segregation. Mol Cell Biol 23:3202-15
Chai, Bob; Hsu, Jing-mei; Du, Jian et al. (2002) Yeast RSC function is required for organization of the cellular cytoskeleton via an alternative PKC1 pathway. Genetics 161:575-84
Geng, F; Cao, Y; Laurent, B C (2001) Essential roles of Snf5p in Snf-Swi chromatin remodeling in vivo. Mol Cell Biol 21:4311-20