In this laboratory we study gene regulation as it pertains to the properties and development of stem cells. We are particularly interested in the blood system and how either genetic or somatic mutations ultimately influence the differentiation or function of the cells generated from hematopoietic stem cells (HSCs).  Stem cells, whether of embryonic or adult origins, give rise to additional stem cells (the process of self-renewal) and generate cells representative of specific lineages (the process of differentiation). Our efforts are directed toward understanding the nature and function of genes that control these processes and how disturbances in gene networks may lead to cancer. In addition to studying adult stem cells or progenitors for the blood system, we also have investigated the nuclear factors that control self-renewal of mouse embryonic stem (ES) cells. Our goal is to identify basic mechanisms that may be employed by different types of stem cells, and perhaps reveal molecular pathways shared among stem cells.

We have been interested in how nuclear regulatory proteins control formation and function of hematopoietic stem cells. Previously, we identified transcription factors that specify hematopoietic stem cells and individual lineages. These factors are often the targets of somatic mutation or chromosomal translocation in human leukemias. We have identified the critical regulators of hematopoietic stem cells and specific lineages (notably, the erythroid lineage) and defined the regulatory network within which they function by combining genome-wide occupancy and gene expression analyses. To validate functional requirements in cells or in vivo we have used classical gene targeting, and more recently CRISPR/Cas9 editing.

Ongoing projects include the following:

  1. Epigenetic regulation in stem cells and hematopoiesis: Besides the central transcription factors, gene expression is controlled by additional protein complexes that remodel chromatin or modify histones. Our group has a long-standing interest in the Polycomb repressive complex known as PRC2 complex, which is composed of the core components EED, Suz12, and EZH2, and responsible for the repressive chromatin mark H3K27me3, which is believed to recruit another Polycomb complex, PRC1, to achieve permanent gene silencing. Through protein purification and sequencing, we have shown that the PRC2 complex is more complex than previously believed. Complexes contain alternatively EZH2 or a related protein EZH1, but not both. In addition, the composition and function of the complex changes during differentiation and is subject to control by the master lineage transcription factors. Our recent work is directed toward an understanding of the various roles of PRC2 and its non-canonical functions, both in normal development and in oncogenesis.
  1. Genetic vulnerabilities in cancers: Regulatory mechanisms operative in stem cells and development are directly relevant to human cancers. Although the primary drivers of oncogenesis may not be targeted by small molecules, cancer cells acquire vulnerabilities that distinguish them from normal cells. In specific instances, we are using genome-wide vulnerability screens to identify new targets for potential therapy in cancer. For example, we have found that malignant rhabdoid tumors due to loss of Swi/Snf components and chronic myelogenous leukemia stem cells are vulnerable to loss of Ezh2, whereas osteosarcoma cells are sensitive to loss of the protein methytransferase Prmt1. We are exploring additional cancer dependencies and the mechanistic bases in each instance.
  1. Regulation of the switch from fetal (HbF) to adult (HbA) hemoglobin: The regulation of globin genes has been an important paradigm since the beginning of the recombinant DNA era. During normal human development, globin genes are expressed successively: in early fetal life the predominant epsilon-like globin is the embryonic beta-globin gene, which is then followed by expression of the fetal gamma-globin gene, and ultimately by expression of the adult beta-globin gene. Major clinical disorders related to mutation of the coding or regulatory sequences of the beta-globin gene, such as sickle cell anemia and the beta-thalassemias, would be ameliorated by preventing shutoff of the fetal -globin gene or reactivation of the gene in adult life. How gamma-globin gene silencing is achieved and maintained has been the focus of the field for more than two decades. Recently, taking advantage of a genome-wide association study (GWAS) that identified single-nucleotide polymorphisms (SNPs) correlating with the level of fetal hemoglobin in patients, we showed that the zinc finger protein BCL11A functions as a silencer of gamma-globin gene expression in adult erythroid cells. Knockdown of BCL11Aexpression reactivates gamma-globin gene expression. We have demonstrated that loss of BCL11A alone in erythroid cells is sufficient to reverse the phenotype of sickle cell disease through the reactivation of fetal hemoglobin. Moreover, the quantitative level of BCL11A expression, and therefore the level of fetal hemoglobin, is controlled through an adult, erythroid-specific enhancer within the BCL11A gene. We are examining the mechanism by which BCL11A functions in the beta-globin complex and are exploring how to interfere with BCL11A expression or function as a new approach to therapy for the hemoglobin disorders. We are also interested in defining how BCL11A functions in concert with the NuRD chromatin complex and also with another fetal-globin repressor, LRF. Our current work suggests that the major components involved in silencing of HbF are now in hand. The mechanisms by which these components act are critical to development of targeted approaches to HbF manipulation for therapy.
  1. Gene editing for therapy of hemoglobin disorders: We have identified a potent erythroid-specific enhancer in the BCL11A which is the site of genetic variation seen in GWAS. Through a novel approach of saturating mutagenesis by CRISPR/Cas9 and functional read-out, we have discovered a discrete “Achilles heel” in the enhancer that can be targeted by a single cleavage and NHEJ to disrupt the majority of enhancer activity and BCL11A expression, specifically in the erythroid cell compartment. HbF silencing is then partially relieved. This discovery forms the basis for gene editing as an improved form of gene therapy for the hemoglobin disorders, which is now moving to clinical trials. We are interested in strategies for maximizing the effectiveness of editing in the enhancer and also comparing the consequences of editing in the enhancer with editing other potential targets for upregulation of HbF.
  1. Chemical approaches to reactivation of HbF: Our long-term goal is to identify small molecules that modulate BCL11A and/or gamma-globin gene silencing as an approach that can be applied globally for patients with hemoglobin disorders. As a transcription factor, BCL11A has traditionally been viewed as a member of an “undruggable” class of proteins. New chemical approaches offer the possibility of identifying small molecules that modulate transcription factor function. We are engaged in biochemical and biophysical strategies that should lead to small molecules that bind BCL11A and may affect its function either directly or indirectly. This project requires close collaboration between molecular biologists, chemists, and structural biologists. Identification of small molecules that could be developed further into drugs for HbF reactivation would transform the management of sickle cell and thalassemia patients worldwide.