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

 描述(申请人提供)：长期造血干细胞(HSCs)能够自我更新并分化为所有成熟的造血系。细胞特异性转录因子与辅因子相互作用，协调染色质结构，促进基因表达。为了生成在造血干细胞中建立表观遗传密码的因素概要，我完成了第一次针对染色质因素的大规模体内反向遗传筛选。为了完成这一任务，我为488个人类保守的染色质因子的斑马鱼同源基因设计了反义吗啉，并在受精后36小时通过全胚胎原位杂交分析了两个HSC特异性基因c-myb和RUNX1的表达。25例吗啡使HSC标志物表达几乎完全下调，4例使HSC标志物表达增强。在改变形成的吗啡肽中，已确定了几个已知对HSC自我更新和维持至关重要的基因。例如，MLL或Dot1的敲除不能指定HSC，如HSC标志物的表达减少所示。6个多梳家族成员的表达减少会导致HSC标志物表达的降低。尽管有些是已知的染色质重塑复合体的成分，如Hat1和Hbo1复合体，但许多剩余的Hit代表了以前在造血中没有功能的因子。Hbo1复合体通过复合体成员之一Ing4与靶基因结合，Ing4识别H3K4me3标记。Hbo1复合体的四个成员在这个屏幕上被点击，我发现这些因素在斑马鱼胚胎中存在基因上的相互作用。这个复合体以前在HSCs中没有表现出任何作用。ING4还被证明可以负向调节NF-B，将这种复合体的功能与HSC中的炎症信号的调节联系在一起。该项目将连接两条重要的途径，即通过乙酰化的染色质重塑和HSC对炎症信号的反应，为HSC组蛋白修饰的研究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/肿瘤学的博士后研究员，卡苏林博士将在著名的血液学家、干细胞生物学家和斑马鱼研究员伦纳德·宗博士的实验室里进行她的研究项目。基于她研究细胞信号机制的深厚背景，凯特林博士将把她的先进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测定的调节方面。在宗博士和一个享有盛誉的指导委员会的指导下，卡特林博士制定了一项雄心勃勃的研究和培训计划，将使她具备最高的研究技能，以确保她在指导和独立获奖期间取得成功。波士顿儿童医院和哈佛医学院的培育环境将为凯特林博士培养成为一名成功的独立科学家提供完美的环境。

 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.