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Molecular Genetics of Stem Cells

Research Summary

Stuart Orkin's research focuses on stem cell biology, particularly the development and function of the blood system, the relationship between cancer and stem cells, and the mechanisms responsible for self-renewal of stem cells and the switch from fetal to adult hemoglobin.

In this laboratory we study gene regulation as it pertains to the properties and development of stem cells. 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. Mutations of the factor GATA-1 are uniquely associated with M7 acute megakaryoblastic leukemia in individuals with Down syndrome. Previously we demonstrated that truncated GATA-1 in this entity (called GATA-1s) perturbs proliferation and cellular maturation of an embryonic/fetal megakaryocytic progenitor. We have proposed that this cell represents the target for transformation in this leukemia. Currently we seek to determine how GATA-1s acts in concert with other proteins to transform these progenitors and how trisomy for human chromosome 21 synergizes with GATA-1s. In our studies we are employing a variety of approaches, including the use of human ES and iPS (induced pluripotent stem) cells.

Hematopoietic stem cells are rare in the adult bone marrow. Hence, it is not feasible at this time to perform global assessment of gene regulatory networks. To develop a comprehensive view of networks in stem cells, we have turned to mouse ES cells, which are capable of indefinite self-renewal and maintain pluripotency under appropriate culture conditions. Several transcription factors, such as Oct4 and Nanog, were known to be critical for ES cell identity. We began by investigating the protein network surrounding Nanog. Our goal was to define the network in which Nanog operates to sustain pluripotency, and what other proteins participate in these processes. We used affinity chromatography to purify Nanog under native conditions and then employed mass spectrometry to identify associated proteins. After validation of selected associated proteins, we performed affinity purification of associated proteins in an iterative fashion to develop a protein network. This network is remarkable for its concentration of proteins essential for early development, co-downregulation of members of the network during ES cell differentiation, and linkage to multiple repression pathways. In addition, the genes encoding proteins of the network are highly represented among direct targets of Nanog and Oct4. Thus, the network appears to represent a cellular module that functions to maintain ES cell pluripotency. We extended our studies by determining the promoter targets bound by pluripotency factors within the network. We found that target genes fall into two classes: promoters bound by few factors tend to be inactive or repressed, whereas promoters bound by more than four factors (among nine within the network that were studied) are largely active in the pluripotent state and become repressed upon differentiation. We also showed that c-Myc, which is potent in factor-induced cellular reprogramming, functions within a parallel network, presumably facilitating chromatin accessibility. We are continuing to investigate novel transcription factors within the pluripotency network and the role of c-Myc in reprogramming.

Additional regulatory mechanisms in stem cells and development involve "epigenetic" controls, including DNA methylation and histone modification. Our work in ES cells has led to reexamination of the composition and function of the Polycomb repressive complex known as PRC2. This complex, composed of the core components EED, Suz12, and EZH2, is 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. Thus, the nature, and presumably the function, of PRC2 changes dynamically during development and among different cellular lineages. Our ongoing studies are focused on other proteins that associate with PRC2 and on how PRC2 functions during differentiation and contributes to, or opposes, oncogenesis.

Regulatory mechanisms operative in stem cells and development are directly relevant to human cancers. In addition to studies of leukemia, we have developed a powerful mouse model of the most common bone tumor, osteosarcoma. Through inactivation of the tumor suppressors p53 and Rb in bone precursors, we have generated mice that regularly develop osteosarcoma that mimics the human disease very closely. We are using a multidisciplinary approach to focus on the molecular determinants of metastasis and on the role of epigenetic pathways in pathogenesis. In surveys of numerous human tumors (e.g., prostate, breast), it has been noted that the PRC2 complex is overactive and most notably that EZH2 is overexpressed. We are using biochemical and genetics strategies to determine the mechanisms responsible for PRC2 activation and whether PRC2 is essential in tumor formation and/or progression. Such studies may reveal pathways in human cancer that may be exploited in novel therapies.

Rescue of sickle cell disease phenotype by loss of BCL11A.

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 β-like globin is the embryonic ε-globin gene, which is then followed by expression of the fetal γ-globin gene, and ultimately by expression of the adult β-globin gene. Major clinical disorders related to mutation of the coding or regulatory sequences of the β-globin gene, such as sickle cell anemia and the β-thalassemias, would be ameliorated by preventing shutoff of the fetal γ-globin gene or reactivation of the gene in adult life. How γ-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 γ-globin gene expression in adult erythroid cells. Knockdown of BCL11A expression reactivates γ-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 β-globin complex and are exploring how to interfere with BCL11A expression or function as a new approach to therapy for the hemoglobin disorders. Our studies raise the prospect of genome engineering of the BCL11A enhancer as a genetic therapy for hemoglobin disorders.

Partial support for this research is provided by grants from the National Institutes of Health.

As of March 4, 2014

Scientist Profile

Boston Children's Hospital
Medicine and Translational Research, Molecular Biology