This proposal addresses the mechanism for assembling the specialized chromatin found at the centromeres and telomeres of eukaryotic chromosomes. Because this chromatin differs from most in not becoming decondensed during interphase, the term, heterochromatin has been used to describe it. The roles of heterochromatin are multi-faceted and range from structural functions in chromosome mechanics and nuclear architecture to gene regulation. Abnormalities in chromosome segregation and gene regulation are often the root causes of human genetic disease. The array of functions for heterochromatin in the nucleus make it pertinent both to understanding and treating these diseases. This proposal describes experiments we are using to dissect the process of heterochromatin assembly in Drosophila. Through biochemistry and cytogenetics, we have uncovered a role for the origin recognition complex (ORC) in recruiting a highly conserved heterochromatin-associated protein (HP1) to heterochromatin. We have also identified a novel protein that is a component of a putative HP1 recruitment complex containing HP1 and ORC protein (HP1/ORC-associated protein, HOAP). Preliminary biochemical and genetic data indicate that it is also required for heterochromatin assembly. The function of HOAP in this process will be further examined. We are using chromatin immunoprecipitations to retrieve the binding sequences for ORC and HOAP in heterochromatin to which HP1 is being recruited. The authenticity of these sequences as binding sites for these proteins will be tested by in situ hybridizations to mitotic chromosomes coupled with immunostaining. Studies to characterize differentially phosphorylated populations of HP1 in interphase nuclei are also planned. One of these populations is specifically associated with ORC and HOAP and has been proposed to have a specific function in nucleating heterochromatin assembly, whereas the other populations may be extended from these hypothetical nucleation sites. We will be preparing immunospecific reagents and mutant HP1 transgenes to address the functions of these populations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM059765-01
Application #
2885122
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1999-08-01
Project End
2003-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Li, Hui; Rodriguez, Janel; Yoo, Youngdong et al. (2011) Cooperative and antagonistic contributions of two heterochromatin proteins to transcriptional regulation of the Drosophila sex determination decision. PLoS Genet 7:e1002122
Bi, Xiaolin; Srikanta, Deepa; Fanti, Laura et al. (2005) Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance. Proc Natl Acad Sci U S A 102:15167-72
Badugu, Ramakrishna; Yoo, Youngdong; Singh, Prim B et al. (2005) Mutations in the heterochromatin protein 1 (HP1) hinge domain affect HP1 protein interactions and chromosomal distribution. Chromosoma 113:370-84
Badugu, RamaKrishna; Shareef, Mohammed Momin; Kellum, Rebecca (2003) Novel Drosophila heterochromatin protein 1 (HP1)/origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations. J Biol Chem 278:34491-8
Shareef, Mohammed M; Badugu, RamaKrishna; Kellum, Rebecca (2003) HP1/ORC complex and heterochromatin assembly. Genetica 117:127-34
Rashkova, Svetlana; Karam, Sarah E; Kellum, Rebecca et al. (2002) Gag proteins of the two Drosophila telomeric retrotransposons are targeted to chromosome ends. J Cell Biol 159:397-402