The ChAHP chromatin remodelling complex regulates a network of neurodevelopmental disorder risk genes to scale the production of neocortical layers (cut&run-seq)

Although chromatin remodellers are among the most important risk genes associated with neurodevelopmental disorders (NDDs), the roles of these complexes during brain development are in many cases unclear. Here, we focused on the recently discovered ChAHP chromatin remodelling complex. The zinc finger and homeodomain transcription factor ADNP is a core subunit of this complex, and de novo ADNP mutations lead to intellectual disability and autism spectrum disorder. However, germline Adnp knockout mice were previously shown to exhibit early embryonic lethality, obscuring subsequent roles for the ChAHP complex in neurogenesis. Here, we employed single cell transcriptomics, cut&run-seq, and histological approaches to characterize mice conditionally ablated for the ChAHP subunits Adnp and Chd4. We show that during neocortical development, Adnp and Chd4 orchestrate the production of late-born, upper-layer neurons through a two-step process. First, Adnp is required to sustain progenitor proliferation specifically during the developmental window for upper-layer cortical neurogenesis. Accordingly, we found that Adnp recruits Chd4 to genes associated with progenitor proliferation. Second, in postmitotic differentiated neurons, we define a network of risk genes linked to NDDs that are regulated by Adnp and Chd4. Taken together, these data demonstrate that ChAHP is critical for driving the expansion upper-layer cortical neurons, and for regulating neuronal gene expression programs, suggesting that these processes may potentially contribute to NDD etiology. Cut&run-seq was performed using the Cutana cut&run kit (14-0500; Epicypher), following the manufacturer’s instructions. Briefly, E13.5 embryos were harvested and the dorsal telencephalon was microdissected. Cells were dissociated with mechanical trituration in order to retain the glycoproteins necessary for adhering to the concavanalin beads. 500 000 cells were used in each reaction. Cut&run seq was performed using antibodies against Chd4 (Abcam ab72418) or Ctcf (Cell Signaling Technologies 3418). Non-specific IgG was used as a negative control. Libraries were prepared using the NEBNext® UltraTM II Library Prep Kit (New England Biolabs). Paired-end 150 sequencing was performed using the NextSeq 500 platform (Illumina) to a read-depth of ~20-35 million reads per sample. Bioinformatic analysis was performed using Galaxy. FASTQ files were processed via Fastq Groomer and Trimmomatic, and then mapped to the mm9 genome using Bowtie2. To normalize samples to the E. coli spike-in, the trimmed reads were mapped to the E. coli K12 genome using Bowtie2. Coverage was calculated using Samtools Flagstat. Mapped BAM files were then scaled using Deeptools to generate scaled Bedgraph files. Peak calling was performed with Macs2.