Identification of enhancers in notochordal cell lineages

Abstract

The nucleus pulposus (NP) of the intervertebral disc is derived from the notochord, which is a flexible rod-like structure that serves as the embryonic axial skeleton. Embryonic notochord produces signals for the induction and patterning of the surrounding sclerotome, which later gives rise to the vertebral body of the spine. The NP is a gelatinous tissue core in the center of the intervertebral disc. It is surrounded by tough, ring-like fibrous laminar structure, called the annulus fibrosus (AF), and lies between a pair of cartilaginous endplates of adjacent vertebral bodies that joins together to form the spine. As the vertebrae forms, the notochord segments and becomes NP tissue in the future discs. In human and chondrodystrophic species, notochord-like cells remain in the NP after birth, but they gradually disappear with age, and this correlates with the onset of disc degeneration, known as disc degenerative disorder. Whereas in non-chondrodystrophic species, the notochordal cells remain in the NP throughout their lives, and they do not develop disc degeneration. Currently, little is known about the gene expression changes that control the development and maturation of NP. Gene enhancers are found crucial in controlling the variation in gene expression during the progression from notochordal cells (NCCs) towards mature NP cells (NPCs), and establishing cell identity. It is also known that epigenetic modifications play important role in the regulation of gene expression in early developmental and during cell lineage commitment. To identify the enhancer elements that are involved in the gene regulation during the development and maturation of NP. Genome-wide characterisation of the active enhancers were performed in NCCs from E8.5-9.5 stage embryos, and NPCs from newborn (P2-P3) and adult (8-12 weeks) NP harvested from 〖Foxa2〗^mNE -Cre/Z/EG mice. Histone modification H3K27ac showed specific enrichment at the enhancer regions of Sox9, Agc1 and Cd24 in SV40-immortalised notochordal cell line (SV40-NCC) and was selected for annotating active enhancers in the primary cells. ChIP-seq was successfully performed from small numbers of NCCs and NPCs, by coupling with the linear DNA amplification (LinDA) protocol. ChIP-seq results showed the H3K27ac-enriched regions represented putative active enhancers in NCCs and NPCs. The putative enhancers of NCCs showed association with upregulation of genes involved in embryonic development, such as Celsr1 and Axin2. Similarly, the putative enhancer for Col1a2 and Adam12 in NPCs also associated with the upregulation of gene expression in the NPCs, which is related to the chondrocyte phenotype. Enrichment of the Adam12 enhancer in the adult NPCs also suggests possible correlation to the onset of disc degeneration in aging NP. Overall, the putative enhancers identified in NCCs and NPCs by annotation of epigenetic mark would be useful for the understanding of gene regulation in the development of NP.