DNA accessibility determines the level of Notch signal strength necessary for lineage specific target gene expression (RNA-Seq)

Using the Notch signaling pathway, hematopoietic stem and progenitor cells (HSPC) sense and respond to the surrounding microenvironment to regulate cell fate outcome. Our previous studies have indicated that quantitative differences in Notch signal strength mediate cell-fate decisions during hematopoiesis. Low signal strength favors HSPC self-renewal, along with inhibition of myelopoiesis, whereas high signal strength promotes T cell differentiation. The basis for target gene selectivity in response to quantitative differences in Notch signal strength is unknown. Focusing on the high dose dependent induction of the T cell lineage, we found that targets essential for T cell commitment, including IL2ra, CD3 and Rag1, establish promoter DNA accessibility only upon exposure to high levels of Notch signaling.  We further find that low promoter CpG content is a feature capable of maintaining the ground state DNA inaccessibility of these lineage determining genes. Our results suggest that DNA inaccessibility at the promoters of essential T cell commitment genes, mediated in part through low CpG content, provides robust protection against stochastic activation in inappropriate Notch signaling contexts, ensuring the maintenance of HSPC developmental plasticity. Total RNA was extracted with the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. RNA was quantified by Nanodrop. RNA quality was assessed using either the Agilent Bioanalyzer or Tapestation. Samples with RIN values of > 8 were submitted to the High Throughput Genomics Center (www.htseq.org) or the Fred Hutchinson Cancer Research Center Genomic Shared Resource for Illumina library preparation and sequencing on a Hi-Seq2000 machine