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Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique
Published on: January 14, 2016
Dynamic changes in the genomic localization of DNA replication-related element binding factor during the cell cycle
B V Gurudatta1, Jingping Yang, Kevin Van Bortle
1Department of Biology, Emory University, Atlanta, GA, USA.
Cell Cycle (Georgetown, Tex.)
|April 30, 2013
Summary
DNA replication factor DREF interacts with Drosophila insulator proteins, revealing a role in chromosome organization and cell cycle regulation. These interactions are crucial for maintaining genome structure and marking sites for DNA replication initiation.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- DNA Replication Element (DREF) is involved in regulating DNA replication genes.
- Insulators are DNA-protein complexes mediating chromosome interactions, with known Drosophila examples like BEAF-32, dCTCF, and Su(Hw).
Purpose of the Study:
- To investigate the genomic localization and functional interactions of DREF with Drosophila insulator proteins.
- To elucidate the role of DREF and insulator proteins in chromosome organization and cell cycle progression.
Main Methods:
- Co-localization studies of DREF with insulator proteins (BEAF-32, dCTCF, CP190) at genomic sites.
- Analysis of DREF enrichment patterns and its correlation with insulator proteins.
- Investigation of mutations in insulator proteins on DREF-induced cell proliferation.
- Chromatin binding analysis of DREF during mitosis.
- Integration of Hi-C data to define topological domain borders.
Main Results:
- DREF and BEAF-32 co-localize at genomic sites with an inverse enrichment correlation.
- DREF also co-localizes with other insulator proteins (dCTCF, CP190), suggesting functional interdependence.
- Mutations in insulator proteins affect DREF-mediated cell proliferation.
- DREF remains bound to chromatin during mitosis at specific sites, co-localizing with key insulator proteins and replication factors (Orc2, Mcm2).
- These sites align with topological domain borders identified by Hi-C.
Conclusions:
- DREF and insulator proteins cooperate to maintain chromosome organization throughout the cell cycle.
- They mark specific genomic sites for pre-replication complex assembly and gene bookmarking during the M/G1 transition.
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