UCLA

Quiescence, reversible exit from the cell cycle, is a conserved process that is important for homeostasis and tissue repair across biological systems. Yet, epigenetic and chromatin changes associated with quiescence remain poorly understood.We found that nuclei in primary human dermal fibroblasts induced into quiescence by serum starvation or contact inhibition are smaller and more elliptical than nuclei in proliferating fibroblasts. Quiescent fibroblasts exhibit a reduction in the levels of multiple chromatin-associated structural proteins, including CTCF. Surprisingly, CTCF knockdown was sufficient to induce fibroblasts into a state with smaller and more elliptical nuclei and reduce proliferation rates. CTCF overexpression resulted in larger and more circular nuclei and fibroblasts that continued to cycle when challenged with quiescence signals. RNA-seq revealed that genes downregulated with CTCF knockdown, and genes inducedwith CTCF overexpression, were enriched for cell cycle GO terms. The findings suggest that when fibroblasts are stimulated to proliferate, CTCF is recruited to chromatin where it induces expression of cell-cycle-related genes. Consistent with this model, proliferating fibroblasts in healing wounds in mice contain higher levels of CTCF than quiescent fibroblasts far from wounds. H4K20me3 is upregulated in quiescent fibroblasts and knockdown of its associated methyltransferase, Suv4-20h2, results in an increased fraction of cells in S phase. CUT&TAG revealed that CTCF and H4K20me3 are both present at TAD boundaries. Super-resolution microscopy showed that CTCF and H4K20me3 are spatially anticorrelated within the nucleus, a finding confirmed by CUT&TAG. Overexpression of CTCF results in lower levels of H4K20me3, while CTCF knockdown causes elevated H4K20me3. Further, inhibiting Suv4-20h methyltransferase activity with small molecule A196 results in lower levels of H4K20me3, elevated chromatin-bound CTCF, larger and rounder nuclei, and upregulation of genes enriched for cell cycle. At specific TAD boundaries, CUT&TAG data confirms higher levels of chromatin-bound CTCF and lower levels of H4K20me3 at the same genomic location in proliferating compared with quiescent cells. Taken together, our data support a new paradigm for the regulation of the proliferation- quiescence transition. In the proliferating state, cells recruit CTCF and form TADs, which result in enhancer-promoter interactions, larger and more circular nuclei, upregulation of cell cycle genes, and cell cycle progression. In the quiescent state, these same genomic loci are blocked by H4K20me3 deposition; less CTCF is bound, the nuclei are smaller and elliptical, and cell-cycle-associated genes are repressed.