KMT2D controls cerebellar granule cell differentiation by temporally activating neuronal transcriptional factor genes [CUT&RUN]

Spatiotemporal gene expression is the fundamental feature for cellular differentiation, including neurogenesis. The epigenetic mechanism underlying spatiotemporal gene regulation during in vivo cellular differentiation remains largely unknown. The cerebellum contains ~80% of the total number of neurons in the human brain. Granule cells (GCs) constitute the vast majority of cerebellar neurons, and GC genesis is spatiotemporally regulated. Here, we show that Atoh1-Cre-mediated knockout (ACKO) of the Kmt2d gene encoding the lysine methyltransferase KMT2D (MLL4 and a COMPASS-like enzyme) in the cerebellar GC lineage in mice inhibits cerebellar GC differentiation while increasing cerebellar cell proliferation. Kmt2d ACKO impaired cerebellum-associated behaviors and caused facial peculiarity, microcephaly, and smaller body size (Kabuki syndrome-like characteristics) in mice. KMT2D temporally activates neuronal differentiation programs in cerebellar GC lineage. KMT2D-mediated activation of the key neuronal transcription factor genes En2, Pax6, and Myt1l is critical for GC differentiation. KMT2D positively programs super-enhancers/enhancers associated with these genes. Single cell RNA-seq analysis showed that Kmt2d ACKO inhibited the transition of GC progenitor to GCs while having a cell-non-autonomous impact on other cerebellar cells. These findings provide a unique epigenetic mechanism in which KMT2D temporally orchestrates gene expression required for cerebellar GC differentiation by programming neuronal enhancers.