Transcriptional response to signaling during hematopoiesis

摘要 造血分化涉及从祖细胞到前体阶段的进展，以及最终的分化。 成熟主转录因子（MTF）如GATA 1和GATA 2激活一个关键的细胞特异性转录因子。 程序，但驱动阶段特异性表达的其他转录因子仍有待确定。 细胞外信号被传递到细胞核，细胞核激活信号转录因子（STFs）。我们 研究了分化为红系的人CD 34细胞，并检查了CD 34细胞的激活和结合。 特异性STF与代表几种信号通路的DNA结合。我们确定了基因组的区域 对应于由MTF和STF共同占据的阶段特异性基因。我们称之为共同占有 区域“转录信号中心”（TSCs），因为它们使相邻的基因可被生长诱导 因子或小分子。BMP信号传导转录因子SMAD 1是活性TSC的标志物， 与GATA因子相邻结合，以标记每个分化阶段的活性基因。SMAD 1预测 在其他STF结合的地方，如cAMP导向的CREB、WNT导向的TCF 7 L2和TGFβ导向的 SMAD2.每种配体都可以激活（或抑制）TSC，导致增强子活性和基因表达的改变。 SMAD 1和加塔因子的共结合允许BMP诱导靶基因和SMAD 1位点的突变 在一个TSC中，证实了SMAD 1结合对于适当的基因表达的需要。检查 与红系性状相关的单核苷酸多态性（SNP）的富集表明， 在TSC处的变异，其中许多突变发生在SMAD或局部区域内的其他STF结合位点处。 大多数人红系GWAS基因在TSCs中的STF结合位点有突变，但只有少数人GWAS基因在TSCs中的STF结合位点有突变。 SNPs影响MTF的结合。我们发现TSC内SMAD结合位点的多态性 基于凝胶迁移率变动分析减少SMAD 1结合，并导致 人类血细胞中的相关基因。其他信号如PGE 2也会导致TSC的激活。我们 已经表明，PGE 2在胚胎发生过程中诱导干细胞产生，并增强造血干细胞 (HSC)在鱼、小鼠和人类中的可移植性。PGE 2增强的HSC目前正在进行第四次临床试验 治疗白血病的方法由于PGE 2刺激的STF CREB在TSC中与SMAD 1相邻结合，我们计划 以检查两种途径的靶点是否相似，或者是否根据特定的基因程序被激活， 配体。我们将评估PGE 2和BMP如何改变染色质，导致特定的基因表达变化。我们 使用微球菌核酸酶敏感性研究的数据表明，在几个小时内， 染色质，导致STF结合的区域的更大可及性。我们计划利用ChIP-seq，ATAC- seq和蛋白质生物化学来研究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导致新的 治疗红细胞疾病，包括地中海贫血和镰状细胞贫血。 !

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.! !