Our Science – Keller Website
Jonathan R. Keller, Ph.D.
Molecular Regulation of Hematopoietic Cell Growth/Differentiation
Hematopoiesis is a multi-stage developmental process that is maintained throughout life by a limited number of hematopoietic stem cells (HSC), which proliferate, self renew, and differentiate into mature blood cells of all lineages. Central questions about the molecular events that regulate HSC quiescence, survival, self-renewal, and lineage commitment remain to be answered. A significant effort in this field is focused on learning to direct these processes to expand and maintain hematopoietic stem cells (HSC) for the treatment of leukemia and other hematological disorders. Transcription factors are the ultimate downstream mediators of extrinsic signals received by the micro environment and cell intrinsic signals. Our current focus is to define how transcriptional regulators promote HSC self renewal and cell specification to multiple hematopoietic cell lineages using stem cell line models; knock-out mice and normal hematopoietic cells. Since transcription factors are frequently deregulated or mutated in leukemia, we will also evaluate if specific transcriptional regulators represent potential therapeutic targets. We believe that knowledge of the molecular and cellular regulation of HSC will contribute to 1) an improved understanding of the mechanism(s) that regulate normal HSC development, 2) if deregulation of these processes contributes to hematopoietic malignancies, 3) the development of biopharmaceuticals to treat leukemia, 4) improved methods of bone marrow transplantation, regenerative medicine, and gene therapy.
Function of the Inhibitors of DNA Binding (Id) Proteins in Normal Hematopoietic Development
Id proteins are members of the helix loop helix (HLH) family of proteins that function as dominant negative regulators of other basic HLH proteins, which regulate the differentiation and proliferation of many cell lineages. We found in our initial studies that Id1 expression is induced by hematopoietic growth factors (HGF) that promote myeloid cell differentiation in HSC and their progeny, and that overexpression of Id1 in normal HSC promotes myeloid, and inhibits B cell development in vitro and in vivo. Thus, we hypothesized that IL-3 may direct HSC fate toward myeloid versus lymphoid cells fate via the induction of Id1 protein. To define the physiological role of Id1 in normal hematopoietic development, we examined hematopoiesis in Id1-/- and Id2-/- mice. Id1-/- mice are viable, reproduce, and have normal life spans, however, we observed novel hematopoietic phenotypes in these mice including 1) increased numbers of neutrophils and monocytes, decreased numbers of B cells, and increased total WBC in the PB, 2) decreased BM cellularity, and 3) increased HSPC cell cycling in vivo. We found that mice transplanted with HSC/HPC that over express Id1 have a similar phenotype, including increased HPC cycling and decreased B cell development. This suggested that the Id1-/- hematopoietic phenotype may not be due to the loss of Id1 in hematopoietic cells, but likely due to the loss of Id1 in cells that constitute the microenvironment. We discovered that Id1 is required for the proper functioning of the hematopoietic microenvironment in vivo, and that loss of Id1 disrupts normal cytokine production. The results of these studies leave open a number of questions including: Does Id1 regulate the development and/or function of stromal cell lineages? Are stromal cell precursors, including mesenchymal stem/progenitor cells (MSC/MPC), or endothelial cell (EC) progenitors, present in normal numbers in Id1-/- mice? Is their differentiation affected by loss of Id1? What are the molecular mechanism(s) by which Id genes regulate microenvironmental function?
Since Id2 was highly expressed in normal HSC, we hypothesized that Id2 may regulate normal HSC biology. We found that Id2 is an intrinsic negative regulator of B cell development. Id2-/- mice showed enhanced B cell development in the BM and spleen, while overexpression of Id2 inhibited B cell development by regulating E2A proteins. We also discovered that Id2 is a positive regulator of erythroid cell development. We demonstrated that Id2 regulates erythroid development by competing with GATA-1 for PU.1 binding, which relieves PU.1 mediated repression of GATA-1 activity, leading to enhanced expression of genes required for erythroid development. Based on these findings, we concluded that Id2 functions distinctly from Id1, and intrinsically regulates lymphoid and erythroid cell fate via interaction with different target proteins, E2A and PU.1. These studies raised questions about the role of Id2 in the growth and development of HSC and MPP cells. For example: Is Id2 expressed in LT-HSC, ST-HSC and MPP? Does Id2 regulate the self-renewal, survival and proliferation of HSC? Future studies are planned to evaluate Id gene loss in specific stromal and hematopoietic cells using novel Id1 and Id2 conditional knock out mouse models. Finally, we discovered that Id2 is a direct target of Gfi-1, and that Gfi-1 regulates B cell development by repressing Id2 gene expression. Future studies are planned to further evaluate the regulation of Id1, 1d2 and Id3 gene expression in HSPC.
Id Genes and Leukemia, and Novel Transcriptional Regulators
Id genes are overexpressed in a broad range of cancers, suggesting that these proteins may represent therapeutic targets to treat cancer. We discovered that deregulated Id1 expression in HSC immortalizes BMC and promotes a myeloid proliferative disease (MPD) in vivo. We found that Id1 and Id2 genes are overexpressed in AML cell lines and human AML patient samples, suggesting that deregulated expression of Id genes may contribute to the initiation and progression of AML. To test this, we plan to investigate if deregulated expression of Id2 and Id3 also contribute to MPD, lymphomas or AML, and investigate how Id genes immortalize murine HPC and contribute to MPD. We will also determine if Id genes are required for the survival and growth of human AML cell lines, and AML patient samples.
In an effort to identify novel transcriptional regulators of myeloid cell growth and differentiation, we have compared the global gene expression profile of undifferentiated and differentiating hematopoietic progenitor cells. We have identified a novel zinc finger transcription factor of unknown function, POGZ, which is down regulated during the early stages of myeloid development. We have generated a mouse strain with a targeted deletion of POGZ. POGZ-/- mice do not survive beyond the first few hours of life, and die at birth of unknown causes, suggesting that POGZ is essential for mouse survival. We have discovered that fetal liver hematopoietic cell development is impaired in POGZ-/- mice, which includes a dramatic decrease in cellularity. In addition, fetal thymic development is arrested at a very early stage of development suggesting that POGZ is required for thymic development. We have discovered that POGZ binds DNA through a consensus sequence found in numerous promoters, including those promoters that regulate genes that are required for T cell development. We have developed a mouse strain to conditionally remove POGZ in specific tissues and hematopoietic lineages. POGZ will be deleted in adult hematopoietic cells using the mx1-cre and vav-cre transgenic mice, in T cells using Lck-cre and B cells using CD-19-cre. Future studies are focused on the role of POGZ in hematopoietic development and more specifically the development of lymphoid lineage cells.
This page was last updated on 9/30/2013.