Role of the ultraconserved genomic regions (UCRs) in hematopoiesis

描述（由申请人提供）：造血系统是最复杂的组织之一，包括许多生存所必需的不同类型的细胞。这一过程受到造血细胞中存在的信号通路和转录因子的复杂相互作用的高度调节。在人类白血病中异常表达的一类新的转录超保守基因组区（ucr）的发现，促使我们寻求这些ucr在特定的造血谱系中是否存在差异表达，以及ucr是否在造血干细胞（HPCs）向特定谱系，特别是红系和巨核细胞谱系的分化中发挥作用。利用一种新型的ucr微阵列平台，我们表征了造血分化不同阶段的ucr表达，并确定了与特定谱系相关的独特特征。本提案的总体目标是证明转录的ucr在造血过程中具有功能相关性。为了实现这一目标，我们提出了以下具体目标：1)特异性目标#1：通过形态学、免疫表型、集落测定和骨髓移植实验，研究异位过表达或抑制UCRs表达对红细胞和巨核细胞“体外”和“体内”谱系分化的影响；2)特异性目标#2：研究GATA-1是否通过以下方式调节UCRs表达：1)利用条件GATA-1细胞系统分析GATA-1激活后UCRs的表达；2)利用cDNA末端快速扩增技术（RACE）鉴定UCRs转录起始位点；3)利用chip测序技术鉴定GATA-1结合位点；4)开展启动子功能研究。这项研究有可能确定新的造血调节因子，并可能为基因表达和细胞命运决定的基础生物学提供见解。如果成功，这项研究将揭示干细胞分化中超保守的非编码和编码rna的无与伦比的分子信息库，这将对该领域具有非凡的价值。

DESCRIPTION (provided by applicant): The hematopoietic system is one of the most complex tissues and includes a number of different cell types essential for survival. This process is highly regulated by the complex interplay of signaling pathways and changing sets of transcription factors that are present in hematopoietic cells. The discovery of a new class of transcribed ultraconserved genomic regions (UCRs) that are aberrantly expressed in human leukemias, prompt us to seek whether these UCRs are differentially expressed among the specific hematopoietic lineages and whether UCRs play a role in the differentiation of hematopoietic stem cells (HPCs) into specific lineages, in particular the erythroid and megakaryocytic lineages. Using a novel UCRs microarray platform we have characterized UCRs expression at different stages of hematopoietic differentiation and identified distinctive signatures associated with particular lineages. The overall goal of this proposal is to demonstrate that transcribed UCRs are functionally relevant during hematopoiesis. To achieve this goal we propose the following specific aims: 1) Specific Aim #1: To investigate the effects of ectopic over-expression or inhibition of UCRs expression on erythrocyte and megakaryocyte lineage differentiation "in vitro" and "in vivo" by using morphology, immunophenotype, colony assays and bone marrow transplant assay experiments and 2) Specific Aim #2: To investigate whether GATA-1 regulates UCRs expression by: 1) analyzing UCRs expression after GATA-1 activation using a conditional GATA-1 cell system; 2) identifying UCRs transcription starting sites using Rapid Amplification of cDNA ends (RACE); 3) identifying GATA-1 binding sites using Chip-sequencing and 4) performing promoter functional studies. This research has the potential to identify novel regulators of hematopoiesis and may give insights into basic biology of gene expression and cell fate determination. If successful, this research will uncover an unparalleled reservoir of molecular information on ultraconserved non-coding and coding RNAs in stem cell differentiation that will be extraordinarily valuable for the field. PUBLIC HEALTH RELEVANCE: This research has the potential to give insights into the basic mechanisms of blood formation and to identify novel regulators in this process. This new information will be extraordinarily valuable for the field. Potential benefits include: 1) direct insight into normal hematopoietic regulation will also be important to understand malfunction in leukemias and other hematologic disorders and 2) the pharmacological modulation of hematopoietic stem cells by targeting critical regulators could be used to treat a wide variety of blood cell disorders that arise from abnormalities in the red or platelet family.