MicroRNAs and hematopoietic differentiation

描述(由申请人提供)：理解和操纵造血分化需要知道在这一过程中协调基因表达程序的调节电路。一类基因调控分子是microRNAs(MiRNAs)-长度约22个核苷酸的微小内源RNAs，被认为利用RNA干扰途径的元件在转录后下调蛋白质编码基因的表达。从miRNAs在造血分化过程中发挥重要调节作用的假设开始，Loish和Bartel实验室合作从小鼠骨髓中克隆了大约100个不同的miRNAs。其中包括五种被称为“造血型miRNAs”的miRNAs，因为它们在造血细胞系中高度或优先表达。这五个基因中的三个也来自与之前与白血病相关的染色体断裂点或异常相关的基因座。初步研究表明，在细胞培养和移植小鼠中，其中一种miRNAs在骨髓前体细胞中的异位表达调节了造血细胞的分化。 这一方案的实验侧重于造血miRNAs，其广泛、长期的目标是了解造血干细胞和祖细胞维持和分化所需的基因调控事件。其具体目的是：1)检测在造血过程中miRNA表达改变的后果。2)确定造血miRNAs的调控靶点，并研究干扰这些靶点的miRNA调控的后果。3)鉴定额外的造血型miRNAs。这些实验包括在造血干细胞和祖细胞中异位表达miRNAs，敲除miRNA基因，体外验证预测的miRNA调控靶点，在体内用不响应miRNA调控的版本替换靶基因，从造血组织克隆更多的miRNAs，以及进一步的表达分析。他们试图将miRNAs放置在特定的基因调控通路中，这是维持造血干细胞以及淋巴和髓系分化所必需的。他们还将解决与miRNA调控有关的更基本的问题，如miRNAs表达的组合控制和脊椎动物mRNAs中功能性miRNA互补位点的特征。因此，这些实验将为理解miRNAs在哺乳动物中的作用以及它们的功能障碍如何导致血液和其他人类疾病提供重要的见解。

DESCRIPTION (provided by applicant): Understanding and manipulating hematopoietic differentiation requires knowing the regulatory circuitry that orchestrates the programs of gene expression during this process. One class of gene regulatory molecules are the microRNAs (miRNAs) - tiny endogenous RNAs, about 22 nucleotides in length, that are thought to use the elements of the RNA-interference pathway to post transcriptionally down-regulate the expression of protein-coding genes. Starting with the hypothesis that miRNAs are playing important regulatory roles during hematopoietic differentiation, the Lodish and Bartel labs have collaborated to clone about 100 different miRNAs from mouse bone marrow. These include five miRNAs referred to as "hematopoietic miRNAs", because they are highly or preferentially expressed in hematopoietic cell lineages. Three of the five also derive from loci associated with chromosomal breakpoints or aberrations previously linked to leukemias. Preliminary studies show that ectopic expression of one of these miRNAs in bone marrow progenitors modulates hematopoietic differentiation both in cell culture and in transplanted mice. The experiments of this proposal focus on the hematopoietic miRNAs with the broad, long-term objective of understanding the gene regulatory events needed for hematopoietic stem cell and progenitor maintenance and differentiation. The specific aims are: 1) To examine the consequences of altered miRNA expression during hematopoiesis. 2) To identify the regulatory targets of hematopoietic miRNAs and examine the consequences of disrupting miRNA regulation of these targets. 3) To identify additional hematopoietic miRNAs. These experiments include the ectopic expression of hematopoietic miRNAs in hematopoietic stem cells and lineage-committed progenitors, knock-outs of miRNA genes, in vitro validation of predicted miRNA regulatory targets, in vivo substitution of target genes with versions unresponsive to miRNA regulation, cloning of additional miRNAs from hematopoietic tissues, and further expression analyses. They seek to place miRNAs within specific gene regulatory pathways needed for hematopoietic stem cell maintenance and lymphoid and myeloid differentiation. They will also address more fundamental issues regarding miRNA regulation, such as the combinatorial control of expression by miRNAs and the features of functional miRNA complementary sites within vertebrate mRNAs. Thus, these experiments will provide important insights for understanding the action of miRNAs in mammals and how their dysfunction might contribute to both hematological and other human diseases.