The loss of HNRNPA0 alters the selection of myeloid cell fate and replication dynamics at human chromosomal common fragile sites

An allele of HNRNPA0, which encodes an RNA-binding protein that targets AU-rich 3'-UTR elements (AREs) is lost in -5/del(5q) t-MDS/t-AML. I hypothesize that HNRNPA0 is necessary for proper hematopoiesis and that its loss leads to alterations in hematopoietic differentiation due to changes in the expression of its target AU-rich transcripts. Using RNAi technology, I modeled the effects of Hnrnpa0 loss in both primary murine cells and experimental cell systems. I have found that reduced Hnrnpa0 expression leads to a shift from monocytic differentiation towards granulocytic differentiation. Microarray-based global expression profiling revealed that Hnrnpa0 knockdown disproportionally impacts ARE-containing transcripts. This effect includes Egr2, an AU-rich transcript whose expression is decreased almost 2-fold following Hnrnpa0 knockdown. This work implicates Hnrnpa0 in the maintenance of Egr2 transcript stability, without which, myeloid differentiation shifts towards granulopoiesis. Disruption of DNA replication plays a central role in the fragility observed at the common chromosomal fragile sites (CFS). I hypothesized that a deficit of origins in these regions and/or insufficient activity of origins present leads to a local inability to respond to replicative stress, leading ultimately to breakage at the CFS. To test this hypothesis, I have developed a new approach to identify specific replication initiation points (RIPs) using microarray technology to quantify nascent strand DNA abundance. These data indicate that origins located within the fragile region of FRA3B respond differently to replicative stress than origins within non-fragile regions. I have found that both FRA3B and FRA16D have a relative deficit of RIPs compared to non-fragile regions. Treatment with the histone deacetylase inhibitor, TSA, alleviates these differences, suggesting a role for chromatin structure in secondary origin usage. Finally, I have identified at least two sequences that combined appear to represent a novel motif frequently found at RIPs.