Concerted neuron-specific splicing events restrict nucleosome engagement of the LSD1 histone demethylase complex

How cell-type-specific chromatin landscapes emerge and progress during metazoan ontogenesis remains an important question. Transcription factors are expressed in a cell type-specific manner and recruit chromatin-regulatory machinery to specific genomic loci. In contrast, most chromatin-regulatory proteins are expressed broadly and are assumed to exert the same intrinsic function across cell types. However, human genetics studies have revealed an unexpected vulnerability of neurodevelopment to chromatin factor mutations with unknown mechanisms. Here, we report that 13 chromatin regulators undergo evolutionary-conserved neuron-specific splicing events involving microexons. Two of these 13 factors are integral components of a histone H3K4 demethylase complex; the catalytic subunit LSD1 and an H3K4me0-reader protein PHF21A. We found that canonical PHF21A (PHF21A-c) binds to DNA by AT-hook motif, and the neuronal counterpart PHF21A-n lacks this DNA-binding function. PHF21A-n showed significantly weaker affinity to the histone H3 tail compared to PHF21A-c. Furthermore, neuronal LSD1 splicing led to reduced affinity of LSD1 to the nucleosome. In-vitro reconstitution of the canonical and neuronal PHF21A-LSD1 complexes, combined with in-vivo methylation mapping, identified the neuronal complex as a hypomorphic H3K4 demethylating machinery with reduced nucleosome engagement. The neuronal PHF21A, albeit its weaker nucleosome binding, is necessary for normal gene expression and H3K4 landscape in developing neurons. Thus, ubiquitously expressed chromatin regulatory complexes can exert neuron-specific functions via alternative splicing of their subunits. Overall design: CUT&RUN was performed using H3K4me2, H3K9me2, H4K20me2 and IgG antibodies on mouse embryonic day 16.5 cortex. 2 biological replicates per genotype (both wild-type and Phf21a homozygous knockout). RNA-seq was performed using RNAs from E16 cortices, 3 embryos per genotype (WT or Phf21a-/-, the sex was balanced between genotypes),