Doctor of Philosophy (PhD)
A high degree of chromosome compaction is needed to fit nearly 2 meters of DNA inside a human nucleus of around 10 µm diameter. Correct chromatin folding is crucial to facilitate important nuclear functions such as transcription, DNA replication and DNA repair. Nuclei contain a variety of proteins, many of which help regulate chromatin structure and function. The mechanisms by which these proteins work are diverse and complicated. Here, we study the chromatin interactions of Boundary Element Associated Factor (BEAF) associated sites to gain insight into eukaryotic genome organization. We used circular chromosome conformation capture (4C) technology to detect genome-wide chromatin interactions of four BEAF binding sites (scs’, hts, snf and RpS6), referred to as viewpoints. These viewpoints have characteristics common for BEAF binding sites: multiple CGATA motifs between divergently transcribed, active genes. We verified the interactions detected by 4C using chromosome conformation capture (3C) and fluorescence in situ hybridization (FISH) assays. Our FISH results indicate that BEAF is not essential for mediating the interactions. However, if BEAF is involved, it is (i) functionally redundant with other proteins or (ii) maternally provided BEAF may be sufficient for establishing the detected genomic contacts. In accord with other studies, we find that the most reproducible interactions are within a few hundred kilobases of the viewpoint, which we refer to as cis-common. Lack of reproducibility of cis-unique (cis-interactions unique to replicates) and interchromosomal (referred to as trans-interactions) contacts suggest that at these scales nuclear organization is highly variable and/or dynamic. On the other hand, our 4C data show a consistent enrichment for active chromatin marks and housekeeping genes. Additionally, association with Chromator, GAF, BEAF and M1BP insulator proteins was also noted.
Shrestha, Shraddha, "Analysis of Chromatin Interactions of BEAF-associated Promoters Using 4C" (2018). LSU Doctoral Dissertations. 4578.