Toshimi Yoshida, PhD, Instructor in Dermatology
Taku Naito, PhD, Instructor in Dermatology
John Seavitt, PhD, Research Fellow
Mariko Kashiwagi, PhD, Research Fellow
Idit Hazan, PhD, Research Fellow
Ila Joshi, PhD, Research Fellow
Samuel Ng, Graduate Student, Program in Immunology HMS
Jiangwen Zhang, PhD, Senior Computational Biologist FAS Center for Systems Biology, Harvard University
Bruce Morgan, PhD, Associate Professor of Dermatology, CBRC
In many developmental systems, nuclear regulators have been implicated in coupling key events in gene expression with specific cell fate and lineage decisions. Focusing on this topic in the hemo-lymphoid system, we have identified the Ikaros gene family of zinc finger DNA binding proteins and shown in a series of genetic studies that they function as key regulators of lymphocyte specification working from the level of the hemopoietic stem cell (HSC). These fundamental studies continue to contribute insight into the earliest steps of the B and T cell differentiation pathways and the molecular network that supports cell fate decisions in the hemo-lymphoid system.
The molecular mechanisms by which Ikaros and its family members exert their range of effects in the hemo-lymphoid system have been under investigation using molecular and biochemical approaches. Importantly, purification of Ikaros and its family member Aiolos from both resting and activated lymphocytes has shown that they exist as part of two functionally distinct chromatin remodeling machineries; an Ikaros-NURD complex, which contains the DNA-dependent ATPase Mi-2 and histone deacetylases, and an Ikaros-SWI/SNF complex which contains the DNA-dependent ATPase Brg-1. Essentially all of the Ikaros found in lymphocytes is in these two higher order molecular entities. This provides an unprecedented example of a factor with demonstrated function as an essential regulator of mammalian development that acts possibly by guiding functionally distinct chromatin remodeling machineries. This combination of attributes makes Ikaros an ideal entry point to study epigenetics in stem cell commitment and differentiation. Our current work focuses on the ability of Ikaros proteins to partition in these functionally distinct higher order molecular complexes and the way that these distinct complexes alter chromatin structure and expression of lineage-specific genes. The genetic interactions between Ikaros and its chromatin remodeling associates are being explored in a series of conditional inactivation studies in mice. These studies are providing evidence of a regulatory network that is comprised of positive and negative regulatory factors whose intimate interactions are responsible for developmental decisions and homeostasis in the hemopoietic system.
Studies on Ikaros gene regulation continue to target our original goal of determining the combination of transcriptional inputs required for induction of a HSC gene expression program. Ikaros regulatory elements coupled with genes encoding fluorescent markers are proving invaluable for the effective separation of rare stem cell and precursor activities and are allowing an in vivo real time analysis of their development. Achieving these goals will dramatically increase the ability to analyze and manipulate development of the HSC in vitro with potential therapeutic implications.
Areas of research:
1. Ontogeny of the hemo-lymphoid system.
Critical insight into the regulation of the early steps of the hemo-lymphoid pathways was obtained by studying mice with Ikaros mutations. Ikaros expressed in the HSC is required for the production and maintenance but also the differentiation of HSC into the lymphoid pathways. Studies on the hemo-lymphoid hierarchy in the Ikaros mutant mice have identified gene targets responsible for some of the cellular defects and have generated a platform on which to study the molecular mechanisms employed by these factors. Studies on Ikaros regulatory elements, which are active in the HSC and progenitor compartments, are also providing us with powerful molecular tools to study transcriptional regulation at the very first steps in the hemopoietic pathway. These Ikaros regulation studies have increased our ability to manipulate gene expression in the early hemo-lymphoid populations and further dissect the HSC compartment and lymphoid progenitors and precursors. (Select publications: 1, 2, 8, 10)
2. Immune response/lymphocyte function.
Studies on the mature T and B cell populations in mice with deficits in Ikaros, Aiolos and more recently in their associate Mi-2 have identified a nuclear network that controls a lymphocyte's response to stimulation and its subsequent maturation to an effector state. Studies on Aiolos deficient mice in particular have provided us with some of the mechanisms that control the fate of naive mature B cell upon antigenic encounter. Importantly, the decision of a naive B cell to assume a marginal zone or a follicular B cell fate, a B cell's ability to enter a Germinal Center (GC) reaction, as well as selection of GC B cells for high antigen specificity are all processes that are dependent on Aiolos. Furthermore, the high affinity long-lived plasma cell population that resides in the bone marrow and is responsible for long-term humoral immunity but not other plasma cells requires Aiolos for its generation. (Select publications: 5, 6, 12, 13)
3. Lymphoid cancers.
Proliferative responses in T and B cells depend on levels of the DNA binding isoforms of Ikaros and Aiolos or their activity. Progressive reduction in Ikaros and Aiolos DNA binding factors in respective T and B cell populations results in a progressive increase in proliferative responses and cause rapid neoplastic transformations. (Select publications 3, 5, 6, 12)
4. Chromatin regulation of hemo-lymphopoiesis.
The Ikaros family proteins represent an important group of developmental regulators. Their association with chromatin remodeling machineries (NURD and SWI/SNF) has provided evidence for a critical role of chromatin regulation in lineage-specification. Studies on the CD8 gene have provided us insight on how Ikaros promotes accessibility at the CD8 enhancers and allows for activation of these enhancers during T cell development. The wealth of markers for lineage progression, and the ability to analyze this process in mouse mutants and in vitro, make the hemo-lymphoid system an ideal model to study the role of directed chromatin remodeling in lineage progression. The Ikaros family and its associates are uniquely suited representatives for these studies. The insights gained by studying Ikaros and its associates, their gene targets and effects on chromatin structure serve as a paradigm for research in this field. They also provide us with the ability to study how hemopoiesis and the immune response are regulated at this molecular level. (Select publications: 7, 9 and 13)
5. Development of complex epithelia.
Development, morphogenesis and self-renewal of skin appendages share some intriguing parallels to hemopoiesis. The accessibility, and self-renewal features, coupled with its highly organized and fascinating architecture, make the skin an attractive system for exploring the molecular mechanisms that underlie stem cell properties as well as complex differentiation programs. The Ikaros homologues Helios and Dedalos and their associated chromatin remodelers are highly expressed in the developing epithelia of the skin. We are now utilizing epithelial-specific conditional inactivation and biochemical approaches to address the role of these factors during skin development and morphogenesis. We hope that our past expertise on the hemopoietic system combined with the local expertise on skin differentiation will in the near future provide a breakthrough in the mechanisms that control epithelial development.
Transgenic and Gene Targeting Facility
nt breakthroughs in lineage transcriptional priming
Table.1 Lineage-specific signatures deduced by K-mean clustering (Ng. et. al., Immunity 2009; 30: 1-15)
|Signature||Definition||Expression||Gene probe #|
|stem||Contains self-renewing genes.||HSC (LT- + ST-)||483|
|s-mpp||No significant expression of lineage specific genes .||HSC/MPP, LMPP||315|
|s-ery||Eerythroid lineage specific genes primed in HSC. 1st wave of erythroid lineage specific expression program.||HSC/MPP, MEP||373|
|s-myly||Lymphoid and myeloid lineage specific genes primed in HSC. 1st wave of lymphoid and myeloid lineage specific expression program.||HSC/MPP, LMPP, GMP, ProB||1340|
|r-myly||Lymphoid and myeloid progenitors-primed lineage specific genes. 2nd wave of lymphoid and myeloid lineage specific expression program.||LMPP, GMP, ProB||92|
|diff||No expression with lineage specific genes. demarcating a progenitor-restricted state.||GMP, MEP, ProB||761|
|d-ery||Erythroid progenitor-specific. 2rd wave of erythroid lineage specific expression program.||MEP||888|
|d-my||Myeloid progenitor-specific. 3rd wave of myeloid lineage program.||GMP||151|
|d-ly||Lymphoid progenitor-specific. 3rd wave of lymphoid lineage program.||LMPP, proB||23|