We will determine the effects of epigenetic modifications on replication origin activation and silencing within the immunoglobulin heavy chain (Igh) locus. We have established the location of many replication initiation sites throughout the Igh locus and have determined how they change during B cell development. We will now focus on modifying the Igh locus in murine ES and proB cells to understand how chromatin modifications associated with active and inactive chromatin domains affect the activation of initiation domains of DNA replication within the locus. For these studies, we have engineered ES cells to contain an exchangeable cassette at two sites within the Igh locus. Using these ES cells, we propose to target to these sites, specific proteins that modify chromatin structure and have histone modifying activities. We already have demonstrated that we can tether a transcriptional activator to Gal4 target sites that we have inserted in the Igh region, resulting in histone modifications and the activation of a previously silent gene. We will also modify the locus by inserting much larger targeting sequences containing frequent recognition sites. We will determine the effects of epigenetic modifications on replication initiation, the direction and rate of replication fork progression and on the time in S phase during which the Igh and flanking regions replicate. We will use two novel approaches that we have recently developed: single molecule analysis of replicated DNA (SMARD) and high-resolution timing array (HRTA) analysis. The role of higher order chromosome structure in regulating replication will also be investigated. We will use the ES cells with the Igh locus, modified by cassette exchange, to produce chimeric mice and homozygous knock-in mice to determine the effect on B cell development of epigenetic changes in the Igh locus. We will characterize these changes using the detailed information we have already obtained about replication initiation and fork direction, and nuclear organization of the Igh locus at different stages of normal development in the B lineage.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Santangelo, George M
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Albert Einstein College of Medicine
Anatomy/Cell Biology
Schools of Medicine
United States
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Pan, Xiaolei; Drosopoulos, William C; Sethi, Louisa et al. (2017) FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres. Proc Natl Acad Sci U S A 114:E5940-E5949
Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P et al. (2016) G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV. Nucleic Acids Res 44:3675-94
Madireddy, Advaitha; Kosiyatrakul, Settapong T; Boisvert, Rebecca A et al. (2016) FANCD2 Facilitates Replication through Common Fragile Sites. Mol Cell 64:388-404
Drosopoulos, William C; Kosiyatrakul, Settapong T; Schildkraut, Carl L (2015) BLM helicase facilitates telomere replication during leading strand synthesis of telomeres. J Cell Biol 210:191-208
Calderano, Simone Guedes; Drosopoulos, William C; Quaresma, Marina Mônaco et al. (2015) Single molecule analysis of Trypanosoma brucei DNA replication dynamics. Nucleic Acids Res 43:2655-65
Gerhardt, Jeannine; Zaninovic, Nikica; Zhan, Qiansheng et al. (2014) Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation. J Cell Biol 206:599-607
Gerhardt, Jeannine; Tomishima, Mark J; Zaninovic, Nikica et al. (2014) The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells. Mol Cell 53:19-31
Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa et al. (2014) Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery. J Cell Biol 206:493-507
Jeong, Yeon-Tae; Rossi, Mario; Cermak, Lukas et al. (2013) FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress. J Cell Biol 200:141-9
Drosopoulos, William C; Kosiyatrakul, Settapong T; Yan, Zi et al. (2012) Human telomeres replicate using chromosome-specific, rather than universal, replication programs. J Cell Biol 197:253-66

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