Hematopoietic stem cell (HSC) homeostasis is essential for normal hematopoiesis. Consequently, aberrant regulation of this HSC population by overproduction or deficiency is a key contributor to hematologic diseases. RUNX1 is a transcription factor that is master regulator of hematopoiesis and specifically regulates the HSC pool size. There are three isoforms of RUNX1 that differ based on promoter and polyadenylation sequence usage. Most RUNX1 studies focus on RUNX1b/c, which utilize a distal polyadenylation sequence and result in a longer protein product than RUNX1a, which terminates at a proximal polyadenylation site. Interestingly, the shorter RUNX1a isoform is undetectable in committed progenitors and differentiated blood cells. Instead, its expression is restricted to the CD34+ HSC population, where it represents a maximum of 20% of the RUNX1 isoform pool. RUNX1a also exhibits distinct functionality from RUNX1b/c. Overexpression of RUNX1 isoforms in HSCs reveals that RUNX1a promotes HSC self-renewal and expansion whereas RUNX1b/c promote lineage commitment and differentiation. Because of this unique role, RUNX1a is an attractive candidate for therapeutically expanding umbilical cord blood (CB)-derived HSCs for transplantation, a therapy that currently suffers from insufficient numbers of HSCs to effect disease-free survival of treated patients. However, strict overexpression of RUNX1a in CB-derived HSCs is unadvised because unchecked expression has leukemogenic potential. In order to safely manipulate RUNX1a levels for potential therapeutic use, it is necessary to understand how this isoform is generated in vivo. Since RUNX1a expression is developmentally regulated, we hypothesize that the alternative polyadenylation (APA) event that results in its production must be linked to restricted expression patterns of pertinent RNA-binding proteins (RBPs) that facilitate this post- transcriptional modification. In this research proposal, I seek to uncover the novel mechanism of RUNX1 APA. Specifically, I aim to identify the pertinent cis-RNA elements and trans-RBPs that regulate this APA event. Once pertinent cis-elements are determined, antisense morpholino oligonucleotide technology will be utilized to alter APA in CB-derived HSCs, transiently increasing RUNX1a and expanding these HSCs ex vivo. Not only will findings help overcome this hurdle to HSCT, benefitting patients in need, but i will also elucidate upstream regulation of RUNX1, which affects proper HSC homeostasis and subsequent hematopoiesis. Furthermore, this will be a pioneering study of APA in the blood system.
There are three different protein forms of RUNX1, a gene essential for proper blood development. The shortest RUNX1 protein form is specifically important for maintaining and expanding the blood stem cell population, which then gives rise to all other blood cells in the body. This study aims to characterize how the shortest RUNX1 protein form is generated and to manipulate its levels to expand a donor's healthy blood stem cell population for the treatment of patients in need of life-saving blood stem cell transplantatio.
