H4K20me3 and CTCF act reciprocally at TAD boundaries to regulate cell state transitions

Chromatin is organized into topologically associating domains (TADs) that are critical for gene regulation. Transitions between cell states, such as proliferation and quiescence, involve genome-scale chromatin dynamics and transcriptional changes, but the underlying mechanisms are not well-understood. Here we show that the reversible shift between proliferation and quiescence is regulated by the opposing action of CTCF and the histone modification H4K20me3. CTCF is a boundary element that defines TADs, while H4K20me3 has previously been associated with heterochromatin, chromatin compaction, and repetitive elements. Using CUT&Tag, RNA-seq, and functional perturbations, we demonstrate that H4K20me3 and CTCF compete for chromatin, including at TAD boundary elements, with increased H4K20me3 in quiescent cells antagonizing CTCF activity in proliferating cells. Manipulation of H4K20me3 levels shows that in quiescent cells, elevated H4K20me3 alters the chromatin landscape, leading to more compact chromatin architecture, elongated nuclei, and induction of a quiescence gene expression program. Conversely, CTCF binding is associated with open chromatin conformation, rounder nuclei, expression of proliferation-associated genes, lower levels of quiescence marker p27/CDKN1B, and cell division in cultured cells and mice. Fibroblasts deficient for H4K20me3 methyltransferase KMT5C are more proliferative, with reduced expression of quiescence genes, and KMT5C-deficient mice are larger. Our findings reveal a reversible antagonistic interplay between H4K20me3 and CTCF that modulates the functional outcome of chromatin architecture to effect changes in cellular state. This new paradigm for regulation of the proliferation-quiescence transition suggests a molecular basis for common developmental transitions and disorders of proliferation.

H4K20me3, a mark associated with heterochromatin, and CTCF compete for chromatin boundary elementsCTCF and H4K20me3 play opposing roles, with increased CTCF in proliferating cells and elevated H4K20me3 in quiescent cellsModulating H4K20me3 or CTCF reversibly shifts cells between proliferative and quiescent stateMice with reduced H4K20me3 are larger, consistent with hyperproliferation.