Characterization of the Drosophila Scaffold Attachment Factor B (SAFB)
Alfonso Parra, Catalina
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Gene expression is a process that involves changes in chromatin organization and structure. Chromatin is thought to be organized in a structure consisting of looped domains, which are fixed at their bases to the nuclear matrix or scaffold. SAFB has been identified as a nuclear matrix binding protein in humans. Human SAFBs contain an N-terminal DNA-binding SAP-Box, and an RNA recognition motif (RRM). However it is unknown how the features of SAFB are linked to gene expression and chromatin organization. I have identified a single homologue of SAFB in Drosophila. To understand the role of SAFB in gene expression and nuclear structure, I have begun to characterize Drosophila SAFB. I found two SAFB splice forms, a full length SAFB containing DNA and RNA binding domains, and a smaller splice form lacking the RNA binding domain. I have showed that SAFB is expressed throughout embryogenesis, in adult testis and ovaries, and larval and adult brains. In addition, I made SAFBGFP constructs to characterize the cellular localization of SAFB. In S2 cells, embryos and neuroblasts, GFP-SAFB was found throughout the nucleus and in nuclear speckles and is retained in the matrix after soluble proteins and DNA are removed. Using larval polytene chromosomes, I show that GFP-SAFB binds to specific DNA bands, some of them overlapping with RNA Polymerase II. After heat shock, GFP-SAFB is recruited to the highly expressed heat shock genes. Treatment of polytene chromosomes with RNAse caused the majority of bands to disappear, meaning that the binding of most of SAFB to chromosomes was mostly through RNA. To distinguish binding of SAFB to DNA from protein-protein interaction, I constructed a GFP-tagged version of SAFB lacking the SAP domain, which binds to fewer sites in the genome. RNAse treatment abolished nearly all binding. Together, my data show that Drosophila SAF-B is a component of the nuclear matrix, that localized to specific loci in the chromosomes, and is recruited to actively-transcribed genes.