Transcriptional response to signaling during hematopoiesis

摘要 造血分化包括从祖细胞到前体阶段的进展，以及最终的 成熟。主要转录因子(MTF)，如GATA1和GATA2，激活一个关键的细胞特异性 程序，但其他驱动阶段特异性表达的转录因子仍有待确定。 细胞外信号被传递到细胞核，激活信号转录因子(STF)。我们 研究人CD34细胞向红系分化，并检测其活化和结合情况 针对代表几个信号通路的DNA的特定STF。我们确定了基因组的区域 与MTF和STF共同占据的阶段特定基因相对应。我们把这些称为共用 区域称为转录信号中心(TSCs)，因为它们使相邻的基因可由生长诱导 因子或小分子。BMP信号转录因子Smad1是TSCs活跃的标志， 与GATA因子相邻结合，在分化的每个阶段标记活性基因。Smad1预测 在其他STF结合的地方，如cAMP定向的CREB、WNT定向的TCF7L2和转化生长因子β定向的STF Smad2.每个配体都可以激活(或抑制)TSCs，导致增强子活性和基因表达的改变。 Smad1和GATA因子的共同结合允许BMP诱导靶基因和Smad1位点的突变 在一项研究中，TSC证明了适当的基因表达需要Smad1结合。一次考试 与红系特征相关的单核苷酸多态(SNPs)表明这种富集性 TSCs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。 大多数人红系GWAS基因在TSCs中存在STF结合位点的突变，但只有少数 SNP的数量影响MTF的结合。我们发现TSC内SMAD结合位点的多态 根据凝胶迁移率漂移分析减少Smad1结合，并导致特定的表达减少 人类血细胞中的相关基因。其他信号如PGE2也导致TSCs的激活。我们 研究表明，PGE2可在胚胎发育过程中诱导干细胞诞生，并增强造血干细胞。 (HSC)在鱼、小鼠和人类身上的可移植性。PGE2增强型造血干细胞目前正在进行第四次临床试验 适用于白血病患者。由于PGE2刺激的STF CREB在TSCs中与Smad1相邻结合，我们计划 以检查这两条通路的靶点是否相似，或者特定的基因程序是否根据 配基。我们将评估PGE2和BMP如何改变染色质以导致特定基因表达的变化。我们的 使用微球菌核酸酶敏感性研究的数据表明，在几个小时内，有一种重组 染色质导致更容易接触到由STF结合的区域。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变化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 治疗红系疾病，包括地中海贫血和镰状细胞性贫血。 好了！

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.! !