MYT1L is required for suppressing earlier neuronal development programs in the adult mouse brain

Abstract

In vitro studies indicate the neurodevelopmental disorder gene myelin transcription factor 1-like (MYT1L) suppresses non-neuronal lineage genes during fibroblast-to-neuron direct differentiation. However, MYT1L's molecular and cellular functions in the adult mammalian brain have not been fully characterized. Here, we found that MYT1L loss leads to up-regulated deep layer (DL) gene expression, corresponding to an increased ratio of DL/UL neurons in the adult mouse cortex. To define potential mechanisms, we conducted Cleavage Under Targets & Release Using Nuclease (CUT&RUN) to map MYT1L binding targets and epigenetic changes following MYT1L loss in mouse developing cortex and adult prefrontal cortex (PFC). We found MYT1L mainly binds to open chromatin, but with different transcription factor co-occupancies between promoters and enhancers. Likewise, multiomic data set integration revealed that, at promoters, MYT1L loss does not change chromatin accessibility but increases H3K4me3 and H3K27ac, activating both a subset of earlier neuronal development genes as well as Bcl11b, a key regulator for DL neuron development. Meanwhile, we discovered that MYT1L normally represses the activity of neurogenic enhancers associated with neuronal migration and neuronal projection development by closing chromatin structures and promoting removal of active histone marks. Further, we showed that MYT1L interacts with HDAC2 and transcriptional repressor SIN3B in vivo, providing potential mechanisms underlying repressive effects on histone acetylation and gene expression. Overall, our findings provide a comprehensive map of MYT1L binding in vivo and mechanistic insights into how MYT1L loss leads to aberrant activation of earlier neuronal development programs in the adult mouse brain.

Figure 1.

MYT1L controls cortical neuron layer specification. (A) GSEA showed an up-regulation of DL genes in Myt1l mutant E14 CTX. (B) UL genes showed no significant change in Myt1l mutant E14 CTX. (C) GSEA showed an up-regulation of DL genes in Myt1l Het P60 PFC. (D) UL genes showed subtle but not significant down-regulation in Myt1l Het P60 PFC. (E) Representative images of DL neuronal marker BCL11B staining on the P60 mouse cortex. (F) Representative images of UL neuronal marker POU3F2 staining on the P60 mouse cortex. (G) Myt1l Het mice had reduced brain weights compared to WTs. (H) Myt1l Het mice had increased BCL11B⁺ neuron density in cortex. (I) POU3F2⁺ neuron density remains the same between Hets and WTs. (*) P < 0.05, (**) P < 0.01. Data were represented as Mean ± SEM. Scale bar, 100 µM.

Figure 2.

CUT&RUN identifies MYT1L-specific binding targets in E14 mouse cortex and adult mouse PFC. (A) Workflow of CUT&RUN experiments on E14 CTX and adult prefrontal cortex (PFC) created with BioRender (https://www.biorender.com/). (B) Representative Integrative Genomics Viewer (IGV) tracks showing a reproducible MYT1L peak at the Hes1 promoter region in all 3 WT E14 biological replicates but not in IgG and KO samples. (C) Heatmaps of CUT&RUN signals of MYT1L, IgG, and histones at MYT1L-bound regions in PFC. (D) Annotations of PFC ATAC-seq peaks showed the genome-wide distribution of promoters, active enhancers, and poised enhancers in open chromatin regions. (E) Annotations of MYT1L targets in PFC showed MYT1L mainly binds to active enhancers. (F) Representative genome tracks of MYT1L-bound promoter (left), active enhancer (middle), and poised enhancer (right).

Figure 3.

MYT1L co-occupies with different sets of TFs at promoter and enhancer regions. (A) monaLisa motif analysis revealed that MYT1L co-occupies with transcriptional activators such as ELK1 at promoter regions, whereas it co-occupies with neurogenic TFs such as MEF2A at enhancer regions. (B) Both MYT1L-bound promoters and enhancers were significantly enriched for MYT1L core binding motif, AAGTT. (C) Overlapping between MYT1L CUT&RUN targets and TFs ChIP-seq peaks showed that more MYT1L promoter targets were also bound by transcriptional activators like SP1 and (D) ELK1 than enhancer targets, whereas more enhancer targets were bound by (E) the neurogenic TF MEF2A and (F) activity-dependent protein JUNB. (G) NEUROD1 and (H) NEUROD2 had stronger presence at MYT1L promoter targets than enhancer targets.

Figure 4.

MYT1L directly binds to and controls promoters that are associated with early neuronal development genes. (A) Mean MYT1L CUT&RUN signals showed decreased MYT1L binding at promoters in Het PFC. (B) No chromatin accessibility change was observed at MYT1L-bound promoters. (C) Mean H3K4me3 CUT&RUN signals showed increased H3K4me3 at MYT1L-bound (MYT1L⁺) promoters in Het PFC. (D) Mean H3K27ac CUT&RUN signals showed increased H3K27ac at MYT1L-bound promoters in Het PFC. (E) MYT1L coimmunoprecipitated with SIN3B and HDAC2 but not with HDAC1/3/4 and MEF2A in WT mouse cortex. (F) MYT1L loss increased its promoter targets’ expression in PFC. (****) P < 0.0001. (G) Venn diagram showing the overlaps among dDEGs, MYT1L promoter targets, and uDEGs. No biased overlap was observed (P = 0.36). (H) GO analysis on 85 uDEGs whose promoters were bound by MYT1L. (I) Representative genome browser track showed MYT1L-bound Bcl11b promoter had higher H3K4me3 and H3K27ac levels in Het PFC than WT.

Figure 5.

MYT1L suppresses enhancers that regulate neuronal migration and neuron projection development. (A) Majority of MYT1L-bound active enhancers were PFC-specific. (B) Heatmaps of MYT1L-bound active enhancers in PFC. (C) MYT1L loss increased its bound active enhancers but not poised enhancers’ chromatin accessibility. (D) MYT1L loss increased H3K4me1 levels at MYT1L-bound active enhancers but did not significantly increase H3K4me1 levels at MYT1L-bound poised enhancers. (E) MYT1L loss increased both its bound active and poised enhancers’ H3K27ac levels. (F) MYT1L active enhancer target genes showed increased expression in Het PFC. (G) Venn diagram showing the overlaps among dDEGs, MYT1L active enhancer targets, and uDEGs. There were more overlaps between MYT1L active enhancer targets and uDEGs than dDEGs (P = 0.01). (H) Functions of MYT1L active enhancer targets showing down-regulation in RNA-seq. (I) Functions of MYT1L active enhancer targets showing up-regulation in RNA-seq. (J) GO analysis on genes associated with MYT1L-bound (MYT1L⁺) E14 CTX/PFC overlapped active enhancers. (K) GO analysis on genes associated with MYT1L⁺ PFC-specific active enhancers. (L) Representative genome browser track showed MYT1L-bound Dcx active enhancer has higher ATAC-seq signals, H3K4me1 and H3K27ac levels in Het PFC than WT.

Figure 6.

MYT1L loss alters the H3K27ac landscape across the genome. (A) Volcano plot showing differential enrichment analysis identified 3487 diff-H3K27ac peaks in Het PFC. (B) Distribution of down- and up-regulated diff-H3K27ac peaks within the non-MYT1L (MYT1L⁻) and MYT1L-bound (MYT1L⁺) categories. (****) P < 0.0001. (C) GO analysis on down-regulated diff-H3K27ac peaks bound by MYT1L and (D) up-regulated diff-H3K27ac peaks bound by MYT1L. (E) Mean ATAC-seq signals of MYT1L⁺ diff-H3K27ac promoter peaks (left: up-regulated peaks, right: down-regulated peaks). (F) Mean ATAC-seq signals of MYT1L⁺ diff-H3K27ac non-promoter peaks (left: up-regulated peaks, right: down-regulated peaks). (G) Functions of MYT1L targets showing up-regulation in both RNA-seq and H3K27ac.

Figure 7.

The model for MYT1L repressing earlier neuronal development programs to facilitate neuronal maturation.