Long-term hematopoietic stem cells (HSCs) are capable of self- renewal and differentiation into all mature hematopoietic lineages. Cell specific transcription factors interact with co-factors to orchestrate chromatin structure and facilitate gene expression. To generate a compendium of factors that establish the epigenetic code in HSCs, I completed first large-scale in vivo reverse genetic screen targeting chromatin factors. To accomplish this task, I designed antisense morpholinos for 488 zebrafish orthologs of conserved human chromatin factors and the resultant morphants were analyzed by whole embryo in situ hybridization at 36 hours post fertilization for expression of two HSC specific genes, c- myb and runx1. 25 morpholinos caused near complete knockdown of HSC marker expression and 4 were found to increase HSC marker expression. Of the morpholinos that alter formation, several genes known to be essential for HSC self-renewal and maintenance were identified. For example, knockdown of Mll or Dot1 fail to specify HSCs, as indicated by a reduction in expression of the HSC markers. Reduced expression of six polycomb family members results in a decrease in HSC marker expression. Many of the remaining hits represent factors with no previous function ascribed in hematopoiesis, though some are components of known chromatin remodeling complexes, such as the Hat1 and Hbo1 complexes. The Hbo1 complex binds to target loci through one of the complex members, Ing4, which recognizes H3K4me3 marks. Four members of the Hbo1 complex were hits in this screen and I have found that these factors genetically interact in zebrafish embryos. No role for this complex has been shown previously in HSCs. Ing4 has also been shown to negatively regulate NF-?B, tying the function of this complex to regulation of inflammatory signals in HSCs. This project will connect two important pathways, chromatin remodeling through acetylation and HSC response to inflammatory signals, offering new avenues to pursue in the study of histone modifications in HSCs and for therapeutic alternatives for patients with blood disorders. As a postdoctoral Research Fellow in Hematology/Oncology at the Children's Hospital Boston, Dr. Kathrein will perform her research project in the laboratory of Dr. Leonard Zon, a renowned hematologist, stem cell biologist, and zebrafish researcher. Building on her strong background of studying cell signaling mechanisms, Dr. Kathrein will expand her advanced scientific and technical knowledge to the regulation of hematopoietic cell specification determination using zebrafish as model organism. Under the mentorship of Dr. Zon and a prestigious mentoring committee, Dr. Kathrein has developed an ambitious research and training program that will equip her with highest research skills to ensure her success in the mentored and independent award period. The nurturing environment of the Children's Hospital Boston and the Harvard Medical School will provide the perfect surroundings for Dr. Kathrein's training to become a successful independent scientist.
While transcription factors are well known regulators of hematopoiesis, the molecular mechanisms of how associated chromatin remodeling complexes regulate this process remains largely unknown. The studies outlined in this proposal aim to characterize a novel hematopoietic chromatin complex, the HBO1 complex, how it orchestrates hematopoietic stem cell specification and define the mechanism of HSC regulation by this complex. A component of this complex, ING4, negatively regulates inflammatory signaling pathways, suggesting a role for this complex in HSC response to these signals. My work will broaden our understanding of the components that regulate the histone code in hematopoietic stem cell fate decisions. Furthermore, knowledge of specific genetic defects in these regulators may help identify therapeutic targets that could be useful in the treatment of hematological diseases.
