Suming Huang - US grants

DESCRIPTION (provided by applicant): Aberrant activation of the TAL1/SCL gene has been involved in majority of T cell acute lymphoblastic leukemia (T-ALL). As a hematopoietic-specific basic helix-loop-helix transcription factor, TAL1 is essential for hematopoietic stem cell renewal and lineage development during normal hematopoiesis. Given its relevance to normal hematopoiesis and T-ALL, understanding the transcriptional control of this transcription factor will elucidate the molecular mechanisms underlying the control of normal hematopoiesis and leukemogenesis. Several lines of evidence have established that TAL1 associates with transcriptional coactivators and corepressors which differentially affect TAL1's function in erythroid differentiation. Hence, we hypothesize that histone modifying enzymes regulate TAL1-mediated transcriptional control during hematopoiesis and their misregulations would contribute to the development of T-cell leukemia. In this application, we focus on investigating how the opposite histone modifying enzymes that we have identified as TAL1-associated cofactors regulate TAL1 function in hematopoiesis and leukemogenesis. The specific aims of this research proposal are: 1) Elucidate the biological function of histone demethylase in the control of TAL1-mediated transcription and hematopoiesis; 2) Analyze the role of histone methyltransferase in TAL1-mediated transcriptional regulation during erythroid differentiation; 3) Understand the molecular basis of histone modifying enzymes in TAL1-induced normal hematopoiesis and leukemogenesis. By completing the proposed research, we expect to further understand how TAL1- directed transcriptional activity contributes to normal hematopoiesis and leukemic transformation. We will also gain knowledge on the role of TAL1-associated histone modifying enzymes in these processes. This information will eventually help to design new therapeutic approaches to treat leukemia. Moreover, the study will shed light on the control of transcription factors on hematopoietic stem cell growth and differentiation. Project Narative: Transcription factor TAL1/SCL is critical for the formation of normal blood cells and is frequently associated with a specific form of leukemia, T cell acute lymphoblastic leukemia (T-ALL) which is a malignant disease with very few effective treatments. In this proposal, we will explore how TAL1 regulates the formation of blood cells and the development of leukemia. These studies will provide novel insights into TAL1 function in the regulation of blood cell development and will help to design new therapeutic approaches to treat leukemia.

DESCRIPTION (provided by applicant): The main function of red blood cells, which carry and exchange oxygen, depends on hemoglobin, a heterotetramer composed of two a and two [unreadable]-globin chains and associated iron-binding heme groups. Mutations of globin genes are among the most common inherited diseases and cause mild or severe anemia in the human population. Current treatments of severe anemia are largely unsatisfactory and it is anticipated that knowledge of how the globin genes are regulated will aide in the development of novel therapies. Erythroid-specific expression of the globin genes requires cis-regulatory DNA elements located in gene proximal or distal regions. The [unreadable]-like globin genes are regulated by a locus control region (LCR), which is composed of several DNase I hypersensitive (HS) sites and located far upstream of the genes. HS2 is perhaps the most powerful regulatory element in the LCR. It consists of several binding sites for hematopoietic and ubiquitously expressed transcription factors. One of these sites is an E-box that interacts with the helix-loop-helix protein USF. USF also interacts with E-box elements in the [unreadable]-globin gene promoter and previous work has shown that USF is required for efficient recruitment of RNA polymerase II (Pol II) to LCR element HS2 and to the [unreadable]-globin gene promoter. In addition, we have shown that USF mediates the boundary activity of the chicken 2-globin insulator HS4, which maintains an accessible chromatin conformation over the globin genes in erythroid cells. Our preliminary data demonstrate that USF1 interacts with large co-activator complexes containing two histone methyltransferases PRMT1 and hSET1. We hypothesize that USF recruits histone modifying enzymes to establish and/or maintain an open chromatin structure at boundary elements and at regulatory elements in the [unreadable]-globin locus, which in turn controls erythroid-specific and developmental stage-specific globin expression. We will test the function of USF and associated histone modifying enzymes in establishing and maintaining chromatin barrier function and tissue specific transcriptional regulation of the [unreadable]-globin locus. Finally, we will investigate how the stability of USF is regulated during differentiation of erythroid cells. Our studies on epigenetic alterations in the [unreadable]-globin gene locus are anticipated to provide new insight into the transcriptional control of globin genes and may lead to novel strategies for the molecular therapy of anemia. Furthermore, addressing the role of USF in [unreadable]-globin gene regulation may shed light on the mechanisms involved in enhancer promoter interactions. PUBLIC HEALTH RELEVANCE: Project Narrative: [unreadable]-globin, an important component of hemoglobin, plays a critical function in the transport and exchange of oxygen in red blood cells in which genetic defects of this gene have been implicated in mild to severe anemia. In this proposal, we will investigate the epigenetic mechanisms by which nuclear proteins USF1/2 regulate globin gene expression and chromatin barrier function in the globin loci. The studies will provide a novel insight into USF function in the regulation of developmental stage-specific globin gene expression and will lead to novel strategies for the molecular therapy of anemia.

? DESCRIPTION (provided by applicant): Hox genes are critical for maintaining the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Although ectopic expression of the HoxB4 gene in bone marrow or embryonic stem cells (ESCs) leads to a dramatic expansion and long-term engraftment potential of HSCs, HoxB4 deficient mice exhibit only a mild reduction in progenitors and stem cells in fetal liver and bone marrow. In contrast, mice deficient in both HoxB3 and HoxB4 genes display severe hematopoietic defects with a marked decrease in HSC population indicating that other anterior HoxB genes may cooperate with HoxB4 to specify hematopoietic cell fate. It is important to understand underlying mechanisms by which the anterior HoxB genes are coordinately activated to confer HSC fate. The expression of Hox genes is regulated epigenetically by polycomb (PcG) and trithorax (TrxG) group regulators. We showed that recruitment of hSET1A to the HoxB4 locus governs its transcription activation and promotes HSC fate. Furthermore, we have identified and cloned a HoxB locus associated long intergenic noncoding RNA (lincRNA), HoxBlinc, which controls the development and specification of mesoderm-derived Flk1+ hemangioblasts. HoxBlinc associates with the Setd1a HMT complex to modulate HoxB locus chromatin conformation and anterior HoxB gene activation. Thus, the data suggest that HoxBlinc RNA may play an important role in early hematopoiesis, perhaps by shaping the histone modification landscape and activate hematopoietic lineage specific program of gene expression. However, it remains unknown how HoxBlinc reprograms chromatin state to regulate anterior HoxB genes and hematopoietic specific transcription program and whether HoxBlinc plays a role in targeting histone modifying enzymes to these genes to initiate hematopoietic differentiation. Based on our preliminary data, we hypothesize that selective recruitment of the Setd1a HMT complex to the HoxB locus and coordination of anterior HoxB expression are mediated by HoxBlinc to specify the hematopoietic cell fate. In this proposal, we will examine the role of HoxBlinc in reprograming chromatin state and modulating anterior HoxB gene transcription. We will investigate underlying epigenetic mechanism by which HoxBlinc regulates early hematopoietic lineage commitment and differentiation. By finishing the proposed research, we expect a better understanding of molecular mechanism by which lincRNA and epigenetic regulators control early events of hematopoiesis.

Abstract Hox genes are critical for maintaining the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Although ectopic expression of the HoxB4 gene in bone marrow or embryonic stem cells (ESCs) leads to a dramatic expansion and long-term engraftment potential of HSCs, HoxB4 deficient mice exhibit only a mild reduction in progenitors and stem cells in fetal liver and bone marrow. In contrast, mice deficient in both HoxB3 and HoxB4 genes display severe hematopoietic defects with a marked decrease in HSC population indicating that other anterior HoxB genes may cooperate with HoxB4 to specify hematopoietic cell fate. It is important to understand underlying mechanisms by which the anterior HoxB genes are coordinately activated to confer HSC fate. The expression of Hox genes is regulated epigenetically by polycomb (PcG) and trithorax (TrxG) group regulators. We showed that recruitment of SETD1A to the HoxB4 locus governs its transcription activation and promotes HSC fate. Furthermore, we have identified and cloned a HoxB locus associated long intergenic noncoding RNA (lincRNA), HoxBlinc, which is expressed during early hematopoietic differentiation consistent with H3K4me3 patterns and anterior HoxB gene activation. Furthermore, HoxBlinc associates with the Setd1a HMT complex and controls the specification and differentiation of Flk1+ hemangioblasts. Thus, the data suggest that HoxBlinc RNA may play an important role in early hematopoiesis, at least in part by recruiting Setd1a HMT complexes onto the Hox genes thereby modulating Hox locus chromatin structure and organization. However, it remains unknown how HoxBlinc reprograms chromatin state to regulate anterior HoxB genes and hematopoietic specific transcription program and whether HoxBlinc plays a role in targeting histone modifying enzymes to these genes to initiate hematopoietic differentiation. Based on our preliminary data, we hypothesize that selective recruitment of the Setd1a HMT complex to the HoxB locus and coordination of anterior HoxB expression are mediated by HoxBlinc to specify the hematopoietic cell fate. In this proposal, we will examine the role of HoxBlinc in reprograming chromatin state and modulating anterior HoxB gene transcription. We will investigate underlying epigenetic mechanism by which HoxBlinc regulates early hematopoietic lineage commitment and differentiation. By finishing the proposed research, we expect a better understanding of molecular mechanism by which lincRNA and epigenetic regulators control early events of hematopoiesis.

Abstract: Aberrant activation of TAL1 oncogene is associated with up to 60% of T-cell acute lymphoblastic leukemia (T-ALL) patients. Its ectopic expression also led to development of leukemia or lymphoma in mice. In contrast, deletion of TAL1 in T-ALL cells lost leukemic phenotype and induced apoptosis. Furthermore, the TAL1 expressing T-ALL subtype is associated with poor prognosis and high rate of relapse. These data suggest that dysregulation of TAL1 oncogene plays an important role in T-ALL leukemogenesis. However, in normal hematopoiesis, TAL1 is a hematopoietic-specific member of the basic helix-loop-helix family of transcription factors required for self-renewal of hematopoietic stem cells and the development of all hematopoietic lineages. Because of its relevance to normal hematopoietic differentiation and T-cell leukemia, it is critical to know how TAL1 oncogene is differentially activated in normal hematopoietic cells and T-cell acute leukemia. Understanding of the epigenetic mechanisms governing TAL1 transcriptional regulation will provide a new insight into epigenetic control of hematopoiesis as well as pathogenesis of T-ALL diseases which may lead to new strategies for leukemia diagnosis and therapeutic approaches. We recently found that TAL1 transcription is regulated by different intra- and interchromosomal loops in normal hematopoietic and leukemia cells, respectively. These intra- and interchromosomal loops alter the cell- type specific enhancers that interact with the TAL1 promoter. Based on these data, we hypothesize that repositioning of the TAL1 gene in a close proximity with T-cell specific transcriptionally active loci within nucleus is essential for aberrantly activating TAL1 oncogene in T-ALL. In this proposal, we will investigate the underlying molecular mechanisms that connect chromatin loops with transcriptional activation decision of the TAL1 oncogene as well as elucidate the role of CTCF and enhancer regulatory elements mediated chromatin interactions in regulation of TAL1 gene during normal hematopoiesis and leukemogenesis. The specific aims are: 1) Evaluate the role of CTCF mediated genome organization in regulation of enhancer/ promoter interaction and TAL1 transcription during hematopoiesis and T cell leukemogenesis; 2) Study the molecular mechanisms by which interchromosomal loops result in aberrant activation of TAL1 oncogene in T-ALL.

Abstract Hox genes, especially HOXA and HOXB genes, are critical for maintaining the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Dysregulation of HOXA and/or HOXB genes is a dominant mechanism of leukemic transformation. Aberrant HOX gene expression is associated with fusion genes involving MLL1 and NUP98, and mutations in NPM1 and CEBPA. However, the epigenetic mechanisms that regulate HOX gene transcription to control HSC function remain to be explored. Furthermore, it is critical to elucidate how HOX genes are aberrantly activated during leukemogenesis. Better understanding of these critical questions will assist in the development of highly effective and selective targeted therapies. We recently identified a HoxB locus associated long intergenic noncoding RNA (lncRNA), HoxBlinc, which controls hematopoietic lineage commitment and differentiation by organizing CTCF mediated active chromatin domain to facilitate anterior HoxB gene activation. HoxBlinc recruits the Setd1a/MLL1 complexes to activate HoxB genes. We found that HoxBlinc lncRNA is overexpressed in significant portions of AML patients, and AML patients with high HoxBlinc expression had significantly shortened survival compare to patients with low HOCBLINC expression. Furthermore, transgenic expression of HoxBlinc in mice leads to increased pools of LT-HSCs and ST-HSCs, and development of lethal AML-like disease. We hypothesize that HoxBlinc lncRNA is a critical epigenetic regulator of HSCs, by controlling the activation of Hox and other key HSC-regulating genes through modulation of chromatin dynamics. In addition, up-regulation of HoxBlinc may represent a potent oncogenic event in leukemogenesis. To test these hypothesis, we will: 1) determine the role of HoxBlinc lncRNA in HSC biology and behavior by performing serial transplantation and paired daughter cell assays using purified HoxBlinc-Tg HSCs; 2) determine whether transgenic HoxBlinc expression is sufficient to perturb hematopoiesis and cause myeloid malignancies in mice; 3) investigate the mechanism(s) by which HoxBlinc lncRNA regulates behaviors of different stages of HSCs by examining global changes in HoxBlinc chromatin binding, 3D chromatin organization, histone modifications, chromatin accessibility, as well as transcription profiles in HSCs purified from WT and HoxBlinc-Tg mice; 4) examine if the function exerted by HoxBlinc lncRNA in HSCs is dependent on the Setd1a/MLL1 complexes; 5) explore whether HOXBLINC lncRNA can serve as an effective therapeutic target for AMLs by examining whether HoxBlinc loss is capable of mitigating NPM1C+- or other mutation-driven myeloid malignancies via abrogating the signature aberrant HOX gene expression. Success of the proposed studies will result in fundamental knowledge regarding the regulation of HSCs by lncRNAs. Our studies could establish HoxBlinc as a powerful oncogenic lncRNAs during leukemogenesis. HOXBLINC lncRNA might represent a novel therapeutic target for AMLs with high HOXBLINC expression.