Study Of Rest As A Negative Regulator Of P16Ink4A

Author

Monica B. Gireud, The University of Texas Graduate School of Bionedical Sciences at HoustonFollow

Date of Graduation

8-2011

Document Type

Thesis (MS)

Program Affiliation

Biomedical Sciences

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Vidya Gopalakrishnan, Ph.D.

Committee Member

Dennis Hughes, M.D., Ph.D.

Committee Member

George Calin, M.D., Ph.D.

Committee Member

Jill Schumacher, Ph.D.

Committee Member

Dean Lee, M.D., Ph.D.

Committee Member

Kenneth Aldape, M.D.

Abstract

STUDY OF REST AS A NEGATIVE REGULATOR OF P16^INK4A

Monica Gireud, B.S.

Thesis Advisor: Vidya Gopalakrishnan, Ph.D.

The RE1 Silencing Transcription Factor (REST) is a negative regulator of neuronal differentiation. It is expressed ubiquitously in early embryos, but downregulated in neural progenitors concomitant with onset of neuronal differentiation in these cells. REST has been widely studied as a negative regulator of neuronal differentiation genes. Our recent work identified a novel role for REST in control of cell proliferation. However, the underlying molecular mechanism(s) are not known and is a focus of the current thesis project. Here, we provide evidence that REST signaling controls the expression of the cyclin-dependent kinase inhibitor, p16^Ink4a, a negative regulator of the cell cycle and passage through G1. We determined that REST expression in the proliferating granule progenitors of the cerebellum and its lack of expression in the differentiated neurons is reciprocally correlated with that of p16^Ink4a. Decline in REST levels in differentiating primary and neural stem cells immortalized with v-myc (NSC-M) granule progenitors in vitro was also associated with upregulation of p16^Ink4a expression. Conversely, constitutive human REST transgene expression in NSC-M cells (NSC-MRs) blocked p16^Ink4 upregulation, even under neuronal differentiation conditions. However, the lack of a consensus REST DNA binding RE1 element in the regulatory regions of p16^Ink4a locus suggested an indirect regulation of p16^Ink4a by REST. Based on work from other groups that showed repression of p16^Ink4a transcription by the polycomb protein Bmi-1, and its negative regulation by microRNA-203 (miR-203) and our identification of a RE1 element in the downstream regulatory region of miR-203, we asked if the p16^Ink4a expression was controlled by REST through a series of negative regulatory events involving miR-203 and Bmi-1. We observed that Bmi1 -expression mirrored that of REST and inversely correlated with that of miR-203 in the postnatal cerebellum and in vitro differentiated granule and NSC-M progenitors. In contrast, forced REST transgene expression in NSC-MR cells abrogated the decrease in Bmi-1 levels and elevation in miR-203 expression. Significant REST binding to the miR-203 RE1 element was also observed in NSC-M cells, indicating that REST had the potential to directly regulate miR-203 expression. In conclusion, our studies suggest a role for REST in control of cell cycle transit in neural progenitors through negative regulation of p16^Ink4a. Further validation of these results in REST knockout mice is needed, and is ongoing.

Keywords

REST, p16, miR-203, Bmi-1, Cell proliferation