Poster abstracts

Poster number 105 submitted by Jennifer Pence

Interaction of Son with a heterochromatic transgene array

Jennifer Pence (Biological Sciences, Wright State University), Alok Sharma (Biological Sciences, Wright State University), Paula A. Bubulya (Biological Sciences, Wright State University)

Abstract:
Nuclear speckles are dynamic subnuclear accumulations of pre-mRNA splicing factors and other co-transcriptional processing factors often found nearby active transcription sites (1). The nuclear speckle splicing factor called Son has within its sequence a cluster of unique tandem repeats as well as an arginine-serine-rich (RS) domain, a G-patch and a double-stranded RNA binding domain at its C-terminus. Son’s unique repeats are required for proper nuclear speckle organization (2), and other domains are important for splicing regulation for a subset of human mRNAs (3,4). To investigate Son’s activities at transcription sites in situ, we used the U2OS 2-6-3 cell line containing an integrated transgene array that allows visualization of gene expression at a specific site of chromosome 1 (5). Our lab discovered that unlike other SR proteins that are recruited to this reporter array after transcriptional activation, Son localizes to the inactive reporter gene locus and is absent following transcription induction. Immunofluorescence analysis shows that upon transcriptional activation, enrichment of Son at the U2OS 2-6-3 locus decreases within thirty minutes and leaves the locus within sixty minutes. SR protein alternative splicing factor 2 (ASF/SF2) is not present at the inactive locus and is recruited to the active locus after Son clears the locus. Chromatin immunoprecipitation with anti-Son antibodies (Son-ChIP) indicates that Son enrichment is highest at the promoter region of the U2OS 2-6-3 transgene array. We hypothesize that Son maintains the heterochromatic state of the inactive reporter array, and that Son may be needed during the first steps of gene activation to recruit components required for mRNA synthesis and/or processing. Heterochromatin maintenance would be a novel role in gene expression for a splicing factor. Experiments to determine Son’s specific role in gene expression at this locus as well as endogenous human gene loci are being pursued.

References:
1) Spector, D. L. and Lamond, A. I. (2010). Cold Spring Harb Perspect Biol. doi: 10.1101/ cshperspect.a000646
2) Sharma, A., Takata, H., Shibahara, K., Bubulya, A., and Bubulya, P. A. (2010). Mol. Biol. Cell. 21, 650-663.
3) Sharma, A., Markey, M., Torres-Muñoz, K., Varia, S., Kadakia, M., Bubulya, A., and Bubulya, P. A. (2011). J. Cell Sci. 124, 4268-4298.
4) Ahn, E. Y., Yan, M., Malakhova, O. A., Lo, M. C., Boyapati, A., Ommen, H. B., Hines, R., Hokland, P., and Zhang, D. E. (2011). Molecular Cell. 42, 185–198.
5) Janicki, S. M., Tsukamoto, T., Salghetti, S. E., Tansey, W. P., Sachidanandam, R., Prasanth, K. V., Ried, T., Shav-Tal, R., Bertrand, E., Singer, R. H., and Spector, D. L. (2004). Cell. 116, 683-698.

Keywords: heterochromatin, gene expression, Son