As primitive hematopoietic stem and progenitor cells multiply and differentiate, their progeny become restricted to specific hematopoietic lineages, eventually becoming committed to one single type of cell that ultimately enters the blood circulation. The Hematopoiesis Section's research focuses on erythropoiesis, the regenerative process in which undifferentiated hematopoietic cells differentiate into red blood cells. Perturbations of this process cause a variety of disorders ranging from hematologic malignancy to anemia.
One of the Hematopoiesis Section's research objectives is to understand the epigenetic changes that accompany erythroid differentiation, including how they differ from the epigenetic profiles of cells from other lineages. To accomplish this goal, the Hematopoiesis Section participates in a consortium designed to extend the ENCyclopedia Of DNA Elements (ENCODE) Project into the study of primary hematopoietic cells. ENCODE was developed to identify all functional elements in the human genome, including epigenetic marks and sites occupied by transcription factors, primarily focusing on cell lines or cultured cells. The hematopoietic system provides a unique opportunity for studybecause primary hematopoietic cells can be sorted into nearly homogeneous populations representing distinct lineages and stages of differentiation using flow cytometry. This allows the determination of the epigenetic state of the genome in freshly isolated, specific cell types. These data can be correlated with transcriptional profiles of both protein encoding genes (mRNA), as well as long noncoding RNAs (lncRNA), to determine the regulatory signature that accompanies differentiation into a specific lineage (i.e. red cells).
The goal of these epigenetic studies is to determine how the genome of mouse hematopoietic progenitor cells programs them to become red blood cells. By working with the mouse system, hypotheses derived from observations can be tested directly in the myriad mouse models available to study erythroid differentiation. The Hematopoiesis Section is developing a comprehensive map of the chromosomal binding locations of erythroid transcription factors (i.e. GATA1 and GATA2, EKLF, NFE2, SCL/TAL), the histone code and the methylation signature in primary mouse erythroblasts, megakaryocytes (which generate platelets) and the common progenitor of these two cell types, the megakaryocyte/erythroid progenitor (MEP). The collective chromatin signatures emerging from the analysis of these cell populations are compared to find correlations between the RNASeq expression profiles of mRNA and lncRNA levels in each cell type. The computational integration of the ChIPSeq, MethySeq and RNASeq data uses a new NextGen analysis software platform developed in the Hematopoiesis Section. Hematopoiesis Section researchers anticipate that activation and repression signatures for coordinately regulated genes will be identified. The long-term goal is to identify the critical regulatory pathways that promote erythroid proliferation and differentiation and to develop small molecules or compounds that could be used to treat anemia.
The Hematopoiesis Section also studies erythropoiesis using the model of a genetically-determined form of anemia that affects humans, called Diamond-Blackfan anemia. DBA is an inherited autosomal dominant disorder associated with failed erythropoiesis, congenital malformations and a predisposition to cancer. Approximately 55 percent of DBA patients have mutations in genes encoding one of 13 ribosomal protein subunits, which result in haploinsufficiency. Because DBA mutations are incompletely penetrant, the lack of a molecular diagnosis in 45 percent of patients prevents the use of sibling donors for hematopoietic stem cell transplantation, the only curative therapy for DBA. The lack of a molecular diagnosis also complicates genotype/phenotype analysis of DBA patients and the possibility of family planning for parents and siblings of affected individuals. Dr. Bodine's group hypothesized that deletions of ribosomal protein genes were responsible for a significant number of the unknown molecular defects in DBA patients, and established a clinical research protocol with the Diamond Blackfan Anemia Registry (DBAR) to perform copy number analysis on DBA patients with unknown molecular defects. The results showed that about 25 percent of such patients had deletions of a known DBA gene, leaving about 30 percent of DBA patients without a molecular diagnosis. A large-scale targeted sequence analysis of all 80 ribosomal protein genes in the remaining DBA patients revealed no mutations, leading to the hypothesis that mutations in non-ribosomal protein genes are the cause of DBA in these patients.
To test this hypothesis, Hematopoiesis Section researchers have undertaken a comprehensive approach to identify the complete spectrum of mutations in DBA patients. Within the DBAR cohort of more than 650 patients, representing a rich clinical database, there are multiple DBA families having two or more affected siblings without known DBA gene deletions, and normal sequence of all 80 ribosomal protein genes. In order to identify candidate mutations, the Hematopoiesis Section uses whole exome sequencing (WES) to analyze DNA from the DBA proband, the parents, and an unaffected sibling in each family. The involvement of candidate mutations in the differentiation of erythroid cells is validated using shRNA knockdown or overexpression experiments in vitro. In addition, in order to establish the frequency of newly discovered mutations in the DBA patient population, targeted sequencing of candidate genes is used to screen a panel of 100 additional DBA patients that lack a molecular diagnosis. A fully genotyped set of DBAR patients (combined with patients in the UK and Japanese DBA registries) will uncover correlations between the genotypes and clinical phenotypes, including steroid responsiveness and spontaneous remission. These studies should be valuable immediately in selecting the best therapeutic options for DBA patients.
Hematopoiesis Section Members
Nancy E. Seidel, Biologist
Amanda P. Cline, Chemist
Jens Lichtenberg, Ph.D., Postdoctoral Fellow
Elisabeth F. Heuston, Ph.D., Postdoctoral Fellow
Nathan A. Krump, Post Baccalaureate Student
Last Updated: November 18, 2015