All ETDs from UAB

Advisory Committee Chair

Peter D Burrows

Advisory Committee Members

David M Bedwell

Christopher A Klug

Chander Raman

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Pax5 is an essential regulator for B lineage cell development and controls hundreds of positive and negative target genes. The nuclear matrix (NM) has long been proposed to provide a dynamic structural support for biological reactions inside of the nucleus, including DNA replication, RNA transcription, and RNA splicing. Although Pax5 binding sites have been identified in numerous Pax5 target genes, it is not clear how Pax5 controls so many target genes. In this dissertation, I demonstrate that Pax5 association with the NM is essential for regulation of the B lineage specific gene expression program. In part I, I found that the majority of the endogenous Pax5 protein in human and murine B lineage cells is associated with the NM, where it is distributed closely with the NM bound RNA polymerase II and the TATA box binding protein (TBP) at discrete foci. Lysine 67, 87, and 89 to Alanine mutation within Pax5 diminished its NM association and compromised Pax5-mediated global gene expression. Chromatin immunoprecipitation and NM precipitation assay results further showed that association with the NM is required for Pax5 to recruit the Cd19 promoter to the NM bound RNA polymerase complex. Based on these results, we propose a model that Pax5 activates B lineage specific target gene expression through recruiting its target genes to the NM-bound transcription centers. In part II, I performed global proteomic analyses to compare NM associated proteins in wild type verses Pax5-/- pro B cells. Interestingly, our results showed that wild type pro B cells have established a B lineage specific NM infrastructure with the enrichment of important transcription factors for lymphocyte or B lineage development. By contrast, different groups of non-B lineage transcription factors are enriched in the NM of Pax5-/- pro B cells. Moreover, we characterized that Bcl11a-XL is a specific Pax5 target gene. Forced expression of Bcl11a-XL induces a subset of Pax5 target genes. These results indicate that Pax5 can reorganize the NM to fully control B lineage specific gene expression program. Taken together, the results provide the first evidence that Pax5 controls B lineage specific gene expression through association with the NM and reorganization of the NM infrastructure.

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