Division of labor among H3K4 Methyltransferases Define Distinct Facets of Homeostatic Plasticity
Histone H3 lysine 4 methylaton (H3K4me), a hallmark of transcriptionally engaged chromatin, is installed by the lysine methyltransferases 2 (KMT2) family of six enzymes in mammals. The six enzymes appear to have arisen from gene duplication of the ancient three enzymes found in the fruit fly. Heterozygous mutations in any of the six enzymes result in monogenic neurodevelopmental disorders, indicating nonredundant yet poorly understood roles of KMT2 family enzymes in neurodevelopment. Recent evidence suggests that histone methyltransferase activity may not be central to KMT2 functions; however, the enzymatic activity is evolutionary conserved, implicating the presence of selective pressure to maintain the catalytic activity. Here, we show that H3K4 methylation is dynamically regulated during prolonged alteration of neuronal activity. The perturbation of H3K4me by the H3.3K4M mutant blocks synaptic scaling, a form of homeostatic plasticity that buffers the impact of prolonged reductions or increases in network activity. Unexpectedly, we found that the six individual enzymes are all necessary for synaptic scaling and that the roles of KMT2 enzymes segregate into evolutionary-defined subfamilies; KMT2A and KMT2B (fly-Trx homologs) for synaptic downscaling, KMT2C and KMT2D (Trr homologs) for upscaling, and KMT2F and KMT2G (dSet homologs) for both directions. Selective blocking of KMT2A enzymatic activity by a small molecule and targeted disruption of the enzymatic domain both blocked the synaptic downscaling and interfered with the activity-dependent transcriptional program. Furthermore, our study revealed specific phases of synaptic downscaling, i.e., induction and maintenance, in which KMT2A and KMT2B play distinct roles. These results suggest that mammalian brains have co-opted intricate H3K4me installation to achieve stability of the expanding neuronal circuits. Overall design: Bromouridine sequencing (BrU-seq) was conducted as previously described with modification (Garay et al., 2020). In brief, on DIV17, cortical cultures derived by CD1 mice were pretreated with either DMSO or 25 µM MM401. After 30min of preincubation with the reagents, cells were treated with bicuculline-methiodide (Abcam, ab120108, 40 µM) or vehicle (sterile water) for 1 h or 4 h. All cells were treated with 2 mM bromouridine (Bru, Sigma, 18670, dissolved in PBS) for the last 30 min of incubation. Cultures were lysed in Tri-reagent BD (Sigma, T3809) and frozen for future procedures. RNA was purified by phenol-chloroform extraction and isopropaonol precipitation, treated with DNase-I (NEB, M0303) then fragmented by high-magnesium, high-temperature incubation. One 1µg of Bru-containing  RNA was used for library preparation. The library was prepared in collaboration with the BrU-seq lab of the University of Michigan (Paulsen et al., 2013, 2014). Finally, samples were sequenced by 150 bp paired-end on Illumina Novaseq. Experiments were conducted in three biological replicates with mixed sex