A screen for epigenetic regulators of hematopoiesis reveals a requirement for the Hbo1 complex during hematopoietic stem cell specification

 描述（由申请人提供）：长期造血干细胞（HSC）能够自我更新并分化为所有成熟的造血谱系。细胞特异性转录因子与辅因子相互作用以协调染色质结构并促进基因表达。为了生成在HSC中建立表观遗传密码的因子的概要，我完成了第一次大规模的针对染色质因子的体内反向遗传筛选。为了完成这一任务，我设计了488个斑马鱼同源的人染色质保守因子的反义吗啉代，并在受精后36小时通过全胚胎原位杂交分析了所得吗啉代的两个HSC特异性基因c-myb和runx 1的表达。25个吗嘟代导致HSC标志物表达的几乎完全敲低，并且发现4个吗嘟代增加HSC标志物表达。在改变形成的吗啉代中，确定了已知对HSC自我更新和维持至关重要的几个基因。例如，MII或Dot 1的敲低不能指定HSC，如HSC标志物表达的减少所示。六个polycomb家族成员的表达减少导致HSC标志物表达减少。许多剩余的命中代表的因素没有以前的功能归因于造血，虽然有些是已知的染色质重塑复合物，如Hat 1和Hbo 1复合物的组成部分。Hbo 1复合物通过复合物成员之一Ing 4与靶基因座结合，Ing 4识别H3 K4 me 3标记。Hbo 1复合物的四个成员在这个屏幕上被击中，我发现这些因子在斑马鱼胚胎中遗传相互作用。以前在HSC中没有显示这种复合物的作用。Ing 4还显示负调节NF-κ B B，将该复合物的功能与HSC中炎症信号的调节联系起来.该项目将连接两个重要的途径，通过乙酰化和HSC对炎症信号的反应进行染色质重塑，为研究HSC中的组蛋白修饰和血液疾病患者的治疗选择提供新的途径。作为波士顿儿童医院血液学/肿瘤学博士后研究员，Kathrein博士将在著名血液学家、干细胞生物学家和斑马鱼研究员伦纳德·佐恩博士的实验室执行她的研究项目。Kathrein博士将在她研究细胞信号传导机制的强大背景的基础上，将她先进的科学和技术知识扩展到以斑马鱼为模式生物的造血细胞规格确定的调节。在Zon博士和一个著名的指导委员会的指导下，Kathrein博士制定了一个雄心勃勃的研究和培训计划，这将使她具备最高的研究技能，以确保她在指导和独立奖励期间取得成功。波士顿儿童医院和哈佛医学院的培养环境将为凯瑟琳博士的培训提供完美的环境，使其成为一名成功的独立科学家。

 DESCRIPTION (provided by applicant): Long-term hematopoietic stem cells (HSCs) are capable of self- renewal and differentiation into all mature hematopoietic lineages. Cell specific transcription factors interact with co-factors to orchestrate chromatin structure and facilitate gene expression. To generate a compendium of factors that establish the epigenetic code in HSCs, I completed first large-scale in vivo reverse genetic screen targeting chromatin factors. To accomplish this task, I designed antisense morpholinos for 488 zebrafish orthologs of conserved human chromatin factors and the resultant morphants were analyzed by whole embryo in situ hybridization at 36 hours post fertilization for expression of two HSC specific genes, c- myb and runx1. 25 morpholinos caused near complete knockdown of HSC marker expression and 4 were found to increase HSC marker expression. Of the morpholinos that alter formation, several genes known to be essential for HSC self-renewal and maintenance were identified. For example, knockdown of Mll or Dot1 fail to specify HSCs, as indicated by a reduction in expression of the HSC markers. Reduced expression of six polycomb family members results in a decrease in HSC marker expression. Many of the remaining hits represent factors with no previous function ascribed in hematopoiesis, though some are components of known chromatin remodeling complexes, such as the Hat1 and Hbo1 complexes. The Hbo1 complex binds to target loci through one of the complex members, Ing4, which recognizes H3K4me3 marks. Four members of the Hbo1 complex were hits in this screen and I have found that these factors genetically interact in zebrafish embryos. No role for this complex has been shown previously in HSCs. Ing4 has also been shown to negatively regulate NF-B, tying the function of this complex to regulation of inflammatory signals in HSCs. This project will connect two important pathways, chromatin remodeling through acetylation and HSC response to inflammatory signals, offering new avenues to pursue in the study of histone modifications in HSCs and for therapeutic alternatives for patients with blood disorders. As a postdoctoral Research Fellow in Hematology/Oncology at the Children's Hospital Boston, Dr. Kathrein will perform her research project in the laboratory of Dr. Leonard Zon, a renowned hematologist, stem cell biologist, and zebrafish researcher. Building on her strong background of studying cell signaling mechanisms, Dr. Kathrein will expand her advanced scientific and technical knowledge to the regulation of hematopoietic cell specification determination using zebrafish as model organism. Under the mentorship of Dr. Zon and a prestigious mentoring committee, Dr. Kathrein has developed an ambitious research and training program that will equip her with highest research skills to ensure her success in the mentored and independent award period. The nurturing environment of the Children's Hospital Boston and the Harvard Medical School will provide the perfect surroundings for Dr. Kathrein's training to become a successful independent scientist.