We have identified and partially characterized several BAT- specific miRNAs and Long Intergenic Noncoding (Linc) RNAs. This grant is focused on understanding their roles in BAT development and function with the long- term goals of determining their mechanisms of action and, their roles in regulatory mechanisms governing BAT development. We have shown that a BAT-enriched microRNA cluster, miR-193b-365, is an essential regulator for brown adipocyte differentiation and lineage determination: knockdown of both miR-193-365 loci in BAT progenitors blocked BAT differentiation and ectopic expression of miR-193b in C2C12 myoblasts induced differentiation into brown adipocytes that undergo adaptive thermogenesis11. At least one other miRNA, miR203 is also highly and specifically enriched in brown fat and our preliminary data indicates that it is essential for development of mature brown adipocytes. We need to determine the mechanisms by which these miRNAs function. Thus we will determine their target mRNAs and the global changes in mRNA levels and translation induced by miR-193- 365 or miR-203 overexpression and knockdown in murine cells;we do this by determining the changes in mRNA composition and by analysis of changes in ribosome occupancy. This will allow us to construct a network of miRNA- mRNA interactions that ultimately modify expression of key human and murine BAT, muscle, and WAT proteins, and also to determine the mechanism by which these miRNAs inhibit synthesis of specific proteins. . We will determine the function of key target mRNAs as targets by reporter gene assays and then by appropriate knockdown or overexpression experiments, with the goal of identifying additional genes that regulate BAT development. To determine the roles of miR-193-365 and miR- 203 during brown fat development in vivo we will generate adipocyte- specific and whole body deletions and then characterize the effects of miR deletion on BAT development, metabolic homeostasis, and thermogenesis. In collaboration with the Broad Institute, we performed RNA-seq to profile the transcriptome of primary brown and white adipocytes, pre-adipocytes and cultured white and brown adipocytes. We identified 481 LincRNAs that are specifically regulated during adipogenesis;RNAi-mediated loss of function screens identified 10 functional LincRNAs required for adipogenesis. Seven other LncRNAs are specifically expressed in BAT cells. Here we will test our hypothesis that these and other BAT- specific LincRNAs are essential for BAT development and we will determine their molecular mechanisms of action. We will focus on nuclear- localized lincRNAs, and determine the effects of their overexpression and knockdown on BAT development and on BAT, WAT, and muscle- specific gene expression. For selected LncRNAs, we will develop RNA pull- down and yeast three- hybrid assays to identify bound proteins as a first step in determining their mechanism of action. In the long- term, we will use Chip experiments to determine how these nuclear LincRNA- protein complexes function to regulate gene expression.

Public Health Relevance

Brown adipose (fat) tissue (BAT) is specialized for thermogenesis (heat generation) and oxidizes (burns) fatty acids, as opposed to storing them as do the more common white fat cells. Increasing the level of brown fat tissue even by a small amount can increase the basal metabolism rate and lessen the chances of developing diet-induced obesity and its attendant consequences such as diabetes and cardiovascular disease. We have identified two groups of noncoding RNAs - micro RNAs (miRs) and long noncoding RNAs - (LncRNAs) that are specifically expressed in brown adipose cells and that when expressed in certain fat or muscle progenitor cells have the potential to increase the amount of brown fat in the body.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK047618-25A1
Application #
8578599
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Haft, Carol R
Project Start
1989-01-01
Project End
2017-04-30
Budget Start
2013-07-01
Budget End
2014-04-30
Support Year
25
Fiscal Year
2013
Total Cost
$679,380
Indirect Cost
$330,980
Name
Whitehead Institute for Biomedical Research
Department
Type
DUNS #
120989983
City
Cambridge
State
MA
Country
United States
Zip Code
02142
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