Genome-wide identification of active enhancers in the developing mouse nucleus pulposus

Abstract

Notochord cells (NCCs) and their derivative nucleus pulposus cells (NPCs) are major cell types in nucleus pulposus (the central part of intervertebral discs) at different development stages. Progressive differentiation of NCCs to NPCs is reported to correlate with the onset and development of disc degeneration. To genome-wide identify active enhancers in NCCs and NPCs, to help unveil the molecular mechanisms underpin development and maintenance of intervertebral discs, NCCs, Neonatal NPCs and Adult NPCs were harvested from Foxa2mNE-Cre x Z/EG mice for ChIP-Seq. NCCs, Neonatal NPCs were also collected for RNA-Seq. H3K27ac was selected to be the active enhancer marker for ChIP. By comparing the enhancer profiles at three development stages, we observed higher degree of overlapping of enhancer regions between Neonatal and Adult NPCs, which indicates they share common regulatory mechanisms in development. Functional annotations on active enhancers revealed that TGF-beta signaling pathway is significantly enriched in NCC/NPC enhancer nearby genes, corresponds to findings in mouse RNA-seq analysis. In addition, Smad family member motifs are significantly enriched in NCC and NPC enhancers. Mice with Smad4 mutant are with twisted tails and deformed intervertebral discs, which supports the importance of Smad4 in intervertebral disc development. With integrative analysis of ChIP-Seq and RNA-Seq data we have associated highly expressed nearby genes with putative enhancers especially those with Smad motifs. Further experimental validations of putative enhancers are undergoing in zebrafish and mouse model.