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的发展，最终成熟。诸如GATA1和GATA2之类的主转录因子（MTF）激活了关键细胞特异性程序，但是驱动特定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，该细胞核激活信号转录因子（STF）。我们研究了与红细胞谱系区分开的人CD34细胞，并检查了激活和结合特定的STF到代表几个信号通路的DNA。我们确定了基因组的区域对应于由MTF和STF共同占领的阶段特异性基因。我们称这些同时称为区域“转录信号传导中心”（TSC），因为它们使相邻的基因由生长诱导因素或小分子。 BMP信号转录因子SMAD1是活动TSC的标记，结合与GATA因子相邻的，以在分化的每个阶段标记活性基因。 Smad1是预测的在其他STF结合的地方，例如CAMP定向的Creb，Wnt定向的TCF7L2和TGFβ指导 Smad2。每个配体可以激活（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，并突变Smad1位置在一个TSC中，SMAD1结合对适当的基因表达的要求。考试与红细胞性状相关的单个核苷酸多态性（SNP）证明了这种富集 TSC的变化，其中许多突变发生在局部区域内的SMAD或其他STF结合位点。大多数人红细胞GWA基因在TSC中的STF结合位点中具有突变，但只有少数 SNP会影响MTF的结合。我们表明TSC中SMAD结合位点的多态性根据凝胶迁移率分析降低SMAD1结合，并导致表达的特定降低人类血细胞中的相关基因。其他信号（例如PGE2）也导致TSC的激活。我们已经表明，PGE2在胚胎发生过程中诱导干细胞出生，并增强造血干细胞（HSC）在鱼，小鼠和人类中的移植性。 PGE2增强的HSC目前正在第四次临床试验中对于白血病患者。由于PGE2刺激的STF CREB在TSC中与SMAD1相邻结合，我们计划检查两种途径的目标是否相似，或者是否根据特定的基因程序被激活配体。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。我们的使用微局局核酸酶敏感性研究的数据表明，在几个小时内，重组染色质导致由STF约束的区域更大的可及性。我们计划利用chip-seq，atac- SEQ和蛋白质生物化学检查这些染色质的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导致新颖促红色疾病在内的疗法，包括丘脑贫血和镰状细胞贫血。呢