Date of Degree

2-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Shireen Saleque

Linda Spatz

Committee Members

Linda Spatz

Mark Emerson

Laurel A. Eckhardt

E. Richard Stanley

Subject Categories

Cell and Developmental Biology

Abstract

The molecular programs that govern the specification of erythroid and megakaryocytic lineages remain incompletely defined. Gene targeting experiments have shown the transcriptional repressor Gfi (Growth factor independence) 1b to be essential for the generation of both erythroid and megakaryocyte cells. Transcriptional repression of Gfi1b target genes is mediated mainly by the cofactors LSD (lysine demethylase) 1 and CoREST/Rcor1 (REST corepressor 1) or other Rcor factors. To understand the mechanism of Gfi1b action, its target genes were identified by chromatin immunoprecipitation (ChIP on Chip) screens. In this study we present the role of Rgs18 (Regulator of G protein signaling 18), a GTPase activating protein (GAP) and a transcriptional target of Gfi1b, in mediating erythro-megakaryocytic lineage specification in murine and human contexts.

Following identification of Rgs18 as a potential Gfi1b and LSD1 target, its regulation by these factors was ascertained in erythro-megakaryocytic cells. Subsequently, the role of Rgs18 in erythro-megakaryocyte differentiation was interrogated by cDNA and shRNA mediated manipulation of expression in primary hematopoietic progenitors and cell lines. To determine the role of Rgs18 in vivo rgs18-/- mice have been generated and their phenotypes will be analyzed shortly. In parallel, to trace the underlying mechanistic alterations responsible for the phenotypes obtained by the above manipulations, the status of two branches of the MAPK (mitogen activated protein kinase) pathway and gene expression patterns of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1/ Klf1 (Kruppel like factor 1) were determined in Rgs18 manipulated cells. Also Rgs18 interacting proteins were identified in megakaryocytic cells. Rgs18 expression was found to be low in immature megakaryoblasts in keeping with strong Gfi1b and LSD1 expression, but was reciprocally upregulated in mature megakaryocytes following declining Gfi1b and LSD1 levels in cells and on the rgs18 promoter. In contrast, expression of Gfi1b was strong in immature erythroid cells and increased further in mature cells. Manipulation of Rgs18 expression in murine hematopoietic progenitors and a multi-potential human hematopoietic cell line produced opposite outcomes in the two lineages, with expression augmenting megakaryocytic, and potently suppressing erythroid differentiation and vice versa. These phenotypes resulted from differential impact of Rgs18 expression on the p38 and ERK branches of MAPK signaling in the erythroid and megakaryocytic lineages. Repercussions of these signaling changes impacted relative expression of the mutually antagonistic transcription factors Fli1 and Klf1 and were compensated by ectopic Fli1 expression demonstrating activity of this transcription factor downstream of Rgs18.

These results identify Rgs18 as a critical downstream effector of Gfi1b and an upstream regulator of MAPK signaling and Klf1/Fli1 gene expression. Sustained Gfi1b expression during erythroid differentiation represses Rgs18 and limits megakaryocytic gene expression in these cells. However during the progress of megakaryocytic differentiation, declining Gfi1b levels result in robust expression of Rgs18 and lineage progression. Preliminary analysis of Rgs18 interactors in megakaryocytes indicates that Rgs18 likely modulates the MAPK pathway by impacting Gαi, cAMP, Ras signaling and certain cytoskeletal proteins. These results will be further extended and confirmed by phenotypic analysis of rgs18-/- mice, and by characterization of novel Rgs18 interacting proteins.

 
 

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