Taiping Chen, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Intestinal stem cells (ISCs) are critical drivers of epithelial homeostasis and regeneration. There are major gaps in knowledge concerning the identities of distinct ISC populations and the roles of epigenetic regulators in ISC self-renewal and multipotency. Lack of such knowledge has significantly hindered the understanding of the functions of ISCs, as well as the regulatory networks involved, in intestinal homeostasis and regeneration. The long-term goal of the applicant's research is to harness the reversibility of epigenetic modifications, including post-translational modifications of histones, to promote tissue regeneration and repair. The objective of this application is to determine the role of Eset (also known as Setdb1 and KMT1E), a histone H3K9 methyltransferase expressed in a fraction of ISCs, in ISC functions such as intestinal homeostasis and regeneration. The central hypothesis is that Eset specifically marks active ISCs and acts as a key corepressor of transcription factors in regulating a gene program that is essential for the maintenance or functioning of these cells. This hypothesis has been formulated on the basis of preliminary data generated in the applicant's laboratory. The rationale for the proposed research is that defining the identity and functions of active ISCs and understanding how they are regulated have the potential to deliver preventive and therapeutic value for intestinal damage, which is frequently associated with chemo- and radiotherapy, drug-mediated toxicity, and various inflammatory disorders. Guided by strong preliminary data, this hypothesis will be tested in mouse models by pursuing three specific aims: 1) Validate Eset as a specific marker of active intestinal stem cells; 2) Determine the role of Eset in intestinal stem cell functions; and 3) Elucidate the molecular mechanisms by which Eset exerts its function in intestinal stem cells. Specifically, the applicant proposes to compare the proliferation rates of Eset-positive and -negative ISCs and identify other key properties (radiosensitivity, transcriptional profile) of Eset-positive ISCs (aim 1), to determine the effects of Eset deficiency and overexpression on intestinal homeostasis and regeneration in vivo and on ISC viability, self-renewal, and differentiation ex vivo (aim 2), and to identify Eset upstream regulators and downstream effectors and to elucidate the mechanistic interactions between Eset and key transcription factors in the regulation of gene expression in ISCs (aim 3). The proposed research is innovative, in the applicant's opinion, because the concept that Eset specifically marks active ISCs is novel and innovative approaches (e.g. in vivo lineage tracing, ex vivo ISC culture, histone peptide arrays) will be used. The project is significant, because results from the proposed studies are expected to vertically advance the fields of intestinal stem cells and epithelial biology by further defining the identity and functions of active ISCs and providing novel insights into epigenetic regulation of ISC functions. These results also have potential translational impact. The restricted expression of Eset in ISCs makes it a promising target for preventive and therapeutic interventions for intestinal damage.

The Immunodeficiency, Centromeric instability, and Facial anomalies (ICF) syndrome is a rare autosomal recessive disorder, with the vast majority of cases carrying mutations in either the DNA methyltransferase gene DNMT3B (ICF1) or the zinc finger protein gene ZBTB24 (ICF2). A hallmark of ICF syndrome is loss of DNA methylation in specific genomic regions, which is believed to be the primary defect underlying other phenotypic abnormalities, including antibody deficiency (hypogammaglobulinemia), facial dysmorphism, and mental retardation. Patients with ICF syndrome usually die of recurrent infections in early childhood. Although ICF syndrome was first reported nearly four decades ago, little progress has been made in understanding the pathogenesis of the disease, largely because of the lack of appropriate in vitro and in vivo models. Modeling ICF syndrome using Dnmt3b mutant mice has been a challenge, because complete inactivation of Dnmt3b leads to embryonic lethality and mice carrying ICF-like point mutations fail to recapitulate antibody deficiency. Preliminary data from the applicant's laboratory revealed that Zbtb24 depletion in mouse embryonic stem cells (ESCs) results in substantial Dnmt3b downregulation and DNA methylation alterations characteristic of ICF syndrome and that conditional Zbtb24 deletion in the hematopoietic lineage leads to severe hypogammaglobulinemia in mice, apparently due to defects in plasma cell differentiation or survival. The objective of this application is to determine the role of Zbtb24 in the regulation of DNA methylation and antibody production. The central hypothesis is that Zbtb24, via regulating Dnmt3b expression, controls DNA methylation, gene expression, and chromatin structure in lymphocyte populations that are important for antibody production. The applicant proposes to use Zbtb24-deficient ESCs to elucidate the molecular mechanism by which Zbtb24 regulates Dnmt3b expression and determine the impacts of Zbtb24 deficiency on DNA methylation, gene expression, and chromatin structure (aim 1). The applicant also proposes to use the Zbtb24-mutant ICF mouse model to determine the cellular defects involved in antibody deficiency and elucidate the links between aberrant DNA methylation and the defects in gene expression, chromatin structure and molecular signaling that contribute to antibody deficiency (aim 2). The project is significant, because results from the proposed work will not only fundamentally advance mechanistic understanding of ICF syndrome, but could also shed light on the etiology and defects underlying other antibody deficiency diseases, including common variable immunodeficiency (CVID). These results will also have general implications in understanding the role of epigenetic mechanisms in immunity and immunological disorders. The project has potential translational impact as well, because results from the proposed work could lead to identification of novel therapeutic strategies for ICF syndrome and other immunodeficiency diseases, and the innovative ICF mouse model could be valuable for preclinical testing of therapeutics in the future.

PROJECT SUMMARY Platelets are critical for hemostasis, thrombosis and inflammatory responses. Billions of platelets circulate in mammalian blood to prevent blood loss in case of tissue injury. The lifespan of platelets is short (5-9 days in humans); as a consequence, several million platelets have to be produced every hour to maintain their physiological blood counts and to avoid the risk of bleeding. Platelets are generated in the bone marrow from megakaryocytes that, in turn, develop from hematopoietic stem and progenitor cells (HSPCs). Although important signaling pathways and transcription factors have been shown to play important roles in platelet biogenesis (thrombopoiesis), little is known about the epigenetic regulation of this process and the key epigenetic regulators involved. Because epigenetic regulators are essential components of regulatory networks that act collaboratively with transcription factors during cellular differentiation, lack of such knowledge significantly hinders the elucidation of the molecular networks controlling platelet generation and function. We have published significant work on the MLL3/4 (mixed lineage leukemia 3&4; KMT2C/KMT2D) histone methyltransferase (KMT) complex, including uncovering an important role for MLL4 in myeloid leukemia, a role for the MLL3/4 complex adaptor subunit PTIP (Pax interaction with transcription-activation domain protein-1) in lymphocyte class switch recombination and, recently, in maintaining normal and leukemic hematopoietic stem cell niches. For the latter studies, we have generated a mouse model of conditional inactivation of PTIP in HSPCs and consistently observed that PTIP deficiency led to thrombocytopenia. We recently generated a MK- specific mouse model and established that the observed reduction in platelets is an intrinsic effect of PTIP deletion in the MK lineage. Preliminary analysis performed on MLL4-deficient mice revealed mild, but significant, thrombocytopenia. Based on our preliminary data and published work, we hypothesize that PTIP is required for platelet generation by functioning together with MLL3 and MLL4 to direct a thrombopoiesis-specific gene expression program. The objective of this project is to determine the role of PTIP and its associated KMTs MLL3/4 in platelet generation. Results from the proposed studies are expected to fundamentally advance the fields of platelet biology by further defining the epigenetic mechanisms that regulate platelet generation. Finally, results from our work will likely contribute to the development of novel in vivo therapies aimed at enhancing platelet production or ex vivo expansion of platelets. Our long-term goal is to harness the reversibility of post-translation modifications of histones to promote human health. !