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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2010-08-8581 |
| Date | 2010 August 1900 |
| Creators | Alfonso Parra, Catalina |
| Contributors | Maggert, Keith |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | application/pdf |
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