Regulation of hematopoiesis is complex, involving several different cell types and their gene products functioning together in the context of the marrow microenvironment (ME);there is little understanding currently of how the coordinated expression of these genes is regulated. Regulation of gene expression by microRNAs (miRs) and other non-coding RNAs has been invoked to explain how large vertebrates have highly specialized cellular and tissue functions despite having limited number of protein coding transcripts. We hypothesize that miRs contribute in a significant way to hematopoietic regulation and form part of a functional network of trans- acting regulators. Preliminary data generated from the analysis of 2 functionally distinct stromal cell lines reveal differential expression of several miRs that may target transcripts of factors which contribute to their distinct phenotypes. For example, one of these cell lines designated HS5 expresses >40 fold more miR-886-3p than a second one, called HS27a. This miR was found to directly down-regulate the expression of CXCL12, a chemokine known to be critical to the homing of the hematopoietic stem cell (HSC) to its niche. Although important information can be gained from such conventional approaches to defining miR-mRNA interactions (expression profiling, bio-informatic prediction algorithms and functional studies based on over-expression or knock-down), they fall short of demonstrating direct miR-mRNA interactions in vivo. Hence we have optimized a recently described biochemical technique that identifies those miRs and mRNAs interacting with each other in the context of the RISC (RNA-Induced Silencing Complex). This technique, termed HITS-CLIP (High Through-put Sequencing following Cross-Linked Immuno Precipitation) utilizes ultraviolet (UV) radiation to cross-link miRs and mRNAs to the protein Argonaute, a component of the RISC followed by its immune precipitation and high throughput sequencing of the RNA. This application proposes a systems-biology approach based on HITS-CLIP to define the role of miRs in the hematopoietic ME.
In Aim 1, we will define the murine homologues of miR-886-3p and develop a novel in vivo model to test the function of these miRs in the ME.
In Aim 2, we will utilize the HITS-CLIP methodology to systematically define the miR-mRNA interactions in 4 distinct cellular populations of the ME: two stromal cell types (as defined by HS5 and HS27a), macrophages and endothelial cells. Specific miRs that target 3 genes critical to maintaining the hematopoietic stem cell (HSC) niche (CXCL12, Angiopoietin1 and Jagged1) will be identified and functionally validated.
In Aim 3, we will use a systems-biology approach to organize the HITS-CLIP data generated to predict and test specific functional networks that regulate groups of genes that may direct the HSC fate decisions. The results will provide a better understanding of how the hematopoietic ME is regulated, which is critical to devising new therapies for hematologic disorders where the ME is dysregulated.

Public Health Relevance

The goal of this project is to define the mechanisms by which different cells in the bone marrow microenvironment interact to regulate blood cell production while retaining a stem cell population. Defining these mechanisms will inform the development of better therapies for diverse blood disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL104070-04
Application #
8616779
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Thomas, John
Project Start
2011-04-08
Project End
2016-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
4
Fiscal Year
2014
Total Cost
$957,841
Indirect Cost
$382,561
Name
Yale University
Department
None
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Balakrishnan, Ilango; Yang, Xiaodong; Brown, Joseph et al. (2014) Genome-wide analysis of miRNA-mRNA interactions in marrow stromal cells. Stem Cells 32:662-73
Pillai, Manoj M; Gillen, Austin E; Yamamoto, Tomomi M et al. (2014) HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer. Breast Cancer Res Treat 146:85-97
Ramakrishnan, Aravind; Torok-Storb, Beverly; Pillai, Manoj M (2013) Primary marrow-derived stromal cells: isolation and manipulation. Methods Mol Biol 1035:75-101