Transcriptional response to signaling during hematopoiesis

造血过程中对信号传导的转录反应

基本信息

批准号：
10312777
负责人：
LEONARD Ira ZON
金额：
$ 52.38万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-15 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10312777
关键词：
ATAC-seq Acute Adopted Affect Binding Binding Sites Biological Assay Birth Blood Blood Cells CD34 gene CREB1 gene CRISPR/Cas technology Cell Differentiation process Cell Nucleus Cell Size Cells ChIP-seq Chromatin Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Complex Cyclic AMP DNA DNA Binding Data Development Dinoprostone Disease Embryonic Development Enhancers Erythrocytes Erythroid Erythroid Cells Erythropoiesis Fishes GATA1 gene Gel Gene Expression Gene Mutation Genes Genetic Genetic Diseases Genetic Polymorphism Genetic Transcription Genetic Variation Genome Goals Grant Growth Factor Hematological Disease Hematopoiesis Hematopoietic Hematopoietic Stem Cell Transplantation Hematopoietic stem cells High-Throughput Nucleotide Sequencing Hour Human Human Genetics Human Genome Knowledge Lead Ligands MADH2 gene Maintenance Micrococcal Nuclease Minority Mus Mutate Mutation Nucleic Acid Regulatory Sequences Outcome Pathway interactions Patients Phosphorylation Process Protein Biochemistry Role Sickle Cell Anemia Signal Pathway Signal Transduction Single Nucleotide Polymorphism Site Specificity TCF7L2 gene Thalassemia Therapeutic Transcriptional Activation Transforming Growth Factor beta Transposase Variant Work base erythroid differentiation extracellular genetic manipulation genome editing genome wide association study genome-wide hematopoietic differentiation hematopoietic stem cell differentiation leukemia novel therapeutics peripheral blood progenitor programs protein complex recruit response small molecule stem cells trait transcription factor

项目摘要

ABSTRACT Hematopoietic differentiation involves progression from the progenitor to precursor stages, and final maturation. Master Transcription factors (MTFs) such as GATA1 and GATA2 activate a critical cell-specific program, but additional transcription factors that drive stage-specific expression remain to be defined. Extracellular signals are transmitted to the nucleus, which activate signaling transcription factors (STFs). We studied human CD34 cells differentiated to the erythroid lineage, and examined the activation and binding of specific STFs to DNA representing several signaling pathways. We identified regions of the genome corresponding to stage-specific genes that are co-occupied by MTFs and STFs. We called these co-occupied regions “transcriptional signaling centers” (TSCs) because they render the adjacent genes inducible by growth factors or small molecules. The BMP-signaling transcription factor SMAD1 is a marker of active TSCs and binds adjacent to GATA-factors to mark active genes at each stage of differentiation. SMAD1 is predictive of where other STFs bind, such as the cAMP-directed CREB, WNT-directed TCF7L2, and TGFβ-directed SMAD2. Each ligand can activate (or repress) TSCs, leading to altered enhancer activity and gene expression. Co-binding of SMAD1 and GATA factors allows BMP induction of target genes, and mutation of a SMAD1-site in one TSC demonstrated a requirement of SMAD1-binding for appropriate gene expression. An examination of single nucleotide polymorphisms (SNPs) associated with erythroid traits demonstrates enrichment of such variations at TSCs, where many mutations occur at SMAD or other STF binding sites within the local region. The majority of human erythroid GWAS genes have mutations in STF binding sites in TSCs, but only a minority of SNPs affect the binding of MTFs. We showed that a polymorphism in a SMAD binding site within a TSC reduces SMAD1 binding based on gel mobility shift analysis and causes a specific reduction of expression of the associated gene in human blood cells. Other signals such as PGE2 also lead to activation of TSCs. We have shown that PGE2 induces stem cell birth during embryogenesis, and enhances hematopoietic stem cell (HSC) transplantability in fish, mice and humans. PGE2 enhanced HSCs are currently in a fourth clinical trial for patients with leukemia. Since the PGE2-stimulated STF CREB binds adjacent to SMAD1 in TSCs, we plan to examine if targets of both pathways are similar, or if specific gene programs are activated according to ligands. We will evaluate how PGE2 and BMP alter chromatin to lead to specific gene expression changes. Our data using micrococcal nuclease sensitivity studies suggest that within a few hours, there is a reorganization of chromatin resulting in greater accessibility of regions bound by the STFs. We plan to utilize ChIP-seq, ATAC- seq and protein biochemistry to examine how these chromatin alterations lead to gene expression changes. Understanding the specificity of signaling pathways and their impact on gene expression may lead to novel therapies for erythroid disorders including thalassemia and sickle cell anemia.! !

抽象的造血分化涉及从祖细胞到前体阶段以及最终阶段的进展 GATA1 和 GATA2 等主转录因子 (MTF) 激活关键的细胞特异性。程序，但驱动阶段特异性表达的其他转录因子仍有待定义。细胞外信号传输到细胞核，细胞核激活信号转录因子（STF）。研究了分化为红系谱系的人类 CD34 细胞，并检查了我们确定了代表多种信号通路的 DNA 的特定 STF。对应于由 MTF 和 STF 共同占据的阶段特异性基因，我们将其称为“共同占据”。区域“转录信号中心”（TSC），因为它们使相邻基因可被生长诱导 BMP 信号转录因子 SMAD1 是活性 TSC 的标记物和 SMAD1 与邻近的 GATA 因子结合以标记每个分化阶段的活性基因，从而预测分化。其他 STF 结合的地方，例如 cAMP 导向的 CREB、WNT 导向的 TCF7L2 和 TGFβ 导向的 SMAD2。每种配体都可以激活（或抑制）TSC，从而改变增强子活性和基因表达。 SMAD1 和 GATA 因子的共同结合允许 BMP 诱导靶基因以及 SMAD1 位点的突变在一个 TSC 中，证明了适当的基因表达需要 SMAD1 结合。与红系性状相关的单核苷酸多态性（SNP）的研究表明，这些特征的富集 TSC 的变异，其中许多突变发生在局部区域内的 SMAD 或其他 STF 结合位点。大多数人红系 GWAS 基因在 TSC 中的 STF 结合位点有突变，但只有少数我们发现 TSC 内 SMAD 结合位点的多态性会影响 MTF 的结合。基于凝胶迁移率变化分析减少 SMAD1 结合，并导致特定的表达减少人类血细胞中的相关基因（例如 PGE2）也会导致 TSC 的激活。研究表明，PGE2 在胚胎发生过程中诱导干细胞诞生，并增强造血干细胞 (HSC) 鱼、小鼠和人类中 PGE2 增强的 HSC 的可移植性目前正在进行第四项临床试验。对于白血病患者，由于 PGE2 刺激的 STF CREB 与 TSC 中的 SMAD1 相邻，我们计划检查两条途径的目标是否相似，或者特定的基因程序是否根据我们将评估 PGE2 和 BMP 如何改变染色质以导致特定的基因表达变化。使用微球菌核酸酶敏感性研究的数据表明，在几个小时内，就会发生重组染色质导致 STF 结合区域的可及性更高，我们计划利用 ChIP-seq、ATAC-。 seq 和蛋白质生物化学来检查这些染色质改变如何导致基因表达变化。了解信号通路的特异性及其对基因表达的影响可能会带来新的发现治疗红细胞疾病，包括地中海贫血和镰状细胞性贫血。！！