Post-transcriptional mechanisms underlying age related expression of cytokine mRNAs in hematopoietic stem cells

National Institute On Aging

Investigators

Abstract

In this study we aim to identify novel regulatory mechanisms that may contribute to the chronically elevated inflammatory cytokines in physiological aging, using the newly developed experimental techniques such as direct RNA Sequencing, Assay for Transposase Accessible Chromatin assay (ATAC-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag). Fluorescent reporter mice for the endogenous expression of IFN-gamma have been aging and are now available for isolation of HSCs and microglia. The reporter mice allow for accurate flow sorting of the cytokine-high and cytokine-low cells from each mouse strain. We will compare 9 mice 72 weeks old or older with 9 mice 20-30 weeks old. We have successfully applied direct RNA-Seq, which will be used to simultaneously profile the abundance and decay of RNAs, in human cell-lines and we have established an efficient processing computational pipeline. We are working on further optimizations on the protocol to support sequencing from primary cells. Direct RNA-Seq results will determine key gene regulatory features in the aged HSCs and microglia such as whether RNA degradation increases/decreases during aging and whether RNA degradation varies between the cytokine-high and cytokine-low populations allowing for a selective survival advantage of one population over another. To identify the chromatin mechanisms contributing to RNA differences in the cell populations of interest, we will perform ATAC-seq with a hyperactive Tn5 transposase to make cuts in accessible regions of the genome. ATAC-seq data will be further searched for regions with depletion of cleavage events due to transcription factor binding and underlying motifs will be used to create a list of candidate transcription factors. To maximize the yield of our DNA and RNA extraction experiments, in this fiscal year we have worked towards isolating DNA and RNA from the same cells. Transcription factors that bind the promoter regions of RNAs most altered in RNA expression analysis will be prioritized for further investigation by CUT&Tag.