Characterization of Boundary Element-Associated Factors BEAF-32A and BEAF-32B and Identification of Novel Interaction Partners in Drosophila Melanogaster

Abstract

Regulatory elements are DNA sequences which have specialized activities that coordinate the functions of the genome. Promoters, enhancers, locus control regions, boundary elements (or insulator elements) are examples of DNA sequences that have regulatory properties. In transgenic assays insulator elements have been shown to block communication between regulatory regions, such as enhancers and promoters, when placed between these sequences and also protect genes from position effects when bracketing them, thereby affecting gene expression. Insulator sequences are bound by insulator proteins that direct the function of these sequences. One such insulator protein is the Boundary Element Associated Factor-32 (BEAF-32), a 32 kDa protein which was originally found to bind to the scs’ insulator sequence in the 87A heat shock locus of the Drosophila genome. BEAF-32 has two isoforms: 32A and 32B. BEAF was immunolocalized to numerous binding sites across the Drosophila genome. This was substantiated by various genome-wide mapping experiments, which have identified from 1800 to 6000 BEAF binding sites across the genome. Hence, BEAF-32 likely plays an important role in chromatin organization and gene regulation in combination with other proteins in the nucleus. However, it is not clear how BEAF-32 affects genome organization and gene regulation. We characterized essential domains in the BEAF-32 protein and identified protein partners, some of which include Transcription Factors (TFs). We further mapped the interaction regions inside BEAF and these TFs. We then attempted Fluorescent Recovery After Photobleaching (FRAP) to assess the dynamics of BEAF-32 on polytene chromosomes and also observed banding patterns, with the help of fluorescent protein labels, and evaluated its behavior during mitosis in early embryos. Finally, results obtained with BEAF prompted us to test for physical interactions between various insulator proteins and to check contradictory reported results from the literature to document interactions.