All of the cells in the peripheral blood are generated from a small population of hematopoietic stem cells (HSC) through a process of proliferation and differentiation known as hematopoiesis. The hematopoietic differentiation program includes well-defined stages at which the progeny of HSC become restricted to specific fates. The goal of this project is to define specific molecular signatures associated with specific stages of hematopoiesis. Project 1. Epigenetic changes, including DNA methylation, histone modifications and transcription factor occupancy is an essential element of normal development and hematopoietic differentiation. Our goal is to comprehensively map these changes in the genomes of primary mouse erythroblasts, megakaryocytes and their common progenitor, the MEP. The ENCODE project has given us a roadmap of changes in the epigenetic code of different cell lines, but to move ENCODE further, we have decided to tackle primary cells. Our previous experiences with genome-wide methylation (Hogart A, Lichtenberg J, Ajay SS, Anderson S, NIH Intramural Sequencing Center, Margulies EH, Bodine DM. Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites. Genome Res. 22 (8) 1407-18, 2012) and the software packages we developed for them (Lichtenberg J, Hogart A, Battle S. Bodine DM. Discovery of motif-based regulatory signatures in whole genome methylation experiments. Bioinformatics Open Source Conference (BOSC), 2012, Long Beach USA) have become the starting point for our more comprehensive studies. We have entered into a consortium with a group at Penn State led by Ross Hardison to take advantage our re respective labs'strengths. Our group has generated methylation and transcription factor profiles for erythroblasts, megakaryocytes and the Megakaryocyte/Erythroid progenitor (MEP;the progenitor that gives rise to both the erythroid and megakaryocytic platelet lineages) and fully differentiated megakaryocytes. We have also begun a genome-wide analysis of histone acetylation in these cells. In addition, we have provided the Hardison lab with cells and chromatin for RNA-Seq analysis of the trancriptome and histone methylation. Project 2. Diamond Blackfan anemia is an inherited bone marrow failure syndrome characterized by a severe deficiency of red blood cells, despite the fact that all other hematopoietic lineages differentiate normally and are present in normal numbers. Mutations in 13 different ribosomal protein genes have been identified in multiple unrelated families, most resulting in haploinsufficiency. However, attempts to generate mouse models of DBA by knocking out the mouse Rps19 gene have been unsuccessful. Preliminary data suggests that the output of the normal mouse Rps19 allele increases to compensate for the loss of the second allele. RPS19's only function appears to be in the assembly of the small ribosomal subunit. We hypothesized that mild deficiencies in the levels of ribosomal proteins that serve additional functions might be better candidates for a mouse model of DBA. Mutations in the RPL11 gene account for 10% of known DBA mutations. In addition to its role in the assembly of the large ribosomal subunit, RPL11 also interacts with MDM2, p53 and the rRNA processing pathways, which may account for the significantly higher frequency of craniofacial and physical abnormalities seen in patients with RPL11 mutations. Our attempts to generate an Rpl11 kockout mouse were unsuccessful, even after screening 1000 potential ES cell clones. We are currently generating an Rpl11 conditional knockout mouse using the CRISPR Cas methodology that we hope will be more efficient. Obviously a realistic mouse model would be useful to to provide an in vivo system to study the mechanism of erythroid failure and to test potentially useful drugs. Project 3. The goal of this project is to provide a molecular diagnosis to each DBA patient. We previously showed that deletions of ribosomal protein genes was responsible for about 10% of all DBA mutations (Farrar JE, Vlachos A, Atsidaftos E, Carlson-Donohoe H, Markello TC, Arceci RJ, Ellis SR, Lipton JM, Bodine DM. Ribosomal protein gene deletions in Diamond-Blackfan Anemia. Blood. 118 (26): 6943-51, 2011). To identify novel DBA mutations, we have initiated whole exome sequencing analysis of 4 unrelated family groups consisting of parents, a DBA proband and either an affected or unaffected sibling. The inclusion of the parents and sibling added a great deal of power to our analysis allowing us to identify 3 strong candidate genes (MCM-2, FLNB and SEMA7A in our first 4 families. We are currently validating these mutations in vitro and we are screening a pool of 96 undiagnosed DBA patients to see if they have any of the newly identified mutation. we plan to enroll three sets of 4 families in the coming year.
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