Epigenetic modifications can heritably change gene expression without altering the underlying DNA sequence, which are mainly discovered in DNA, RNA, and histone, respectively. Previous literature has revealed that epigenetic modifications participate in regulating gene expression across multiple levels, including changes in chromatin structure and accessibility, as well as the regulation of mRNA transcription, post-transcription, and translation processes. Consequently, they impact various critical biological processes and diseases, such as transposon suppression, tissue development, and carcinogenesis (Skvortsova et al., 2018; Mohammad et al., 2019). For instance, 5mC exhibits a negative correlation with gene expression at the promoter and a positive correlation at the gene body (Siegfried and Simon, 2010), while 5hmC consistently shows a positive correlation with gene expression across most genomic regions (Li et al., 2016). Furthermore, different epigenetic modifications interact synergistically to regulate gene expression coherently. For example, the histone H3 Lys 4 (H3K4) methylation, especially H3K4me2/3, suppresses the activity of DNA methyltransferases 3A (DNMT3A) via its ADD domain, protecting active promoters and enhancers from DNMT3A-induced DNA methylation (Zhang et al., 2010b).

The cerebral cortex is the most complex structure and is responsible for the advanced functions of the brain (Sansom and Livesey, 2009). During the developmental process of the mammalian cerebral cortex, neurogenesis starts from embryonic day 9 (E9) and peaks at E17.5. The neonatal stage (postnatal days 1 to 10) is important for synapse formation and programmed cell death. Subsequently, the juvenile stage (3 to 8 weeks) becomes crucial for synaptic pruning and myelination. Finally, synaptogenesis and myelination are completed at the adult stage (after 15 weeks), symbolizing full brain development (Zeiss, 2021). Previous studies have revealed that multiple epigenetic mechanisms intricately govern the spatiotemporal expression of crucial factors influencing brain development and functions (Jobe et al., 2012). For example, DNA methylation at genes associated with neurological and cognitive functions dynamically changes during brain development (Guo et al., 2011a). Concurrently, DNA hydroxymethylation is abundant in synapse-related genes, aligning with neuronal maturation and synaptogenesis (Spiers et al., 2017). It is worth noting that abnormal changes in epigenetic modifications are associated with several neurodevelopmental and neurodegenerative disorders. Patients with Huntington's disease manifest significantly reduced levels of 5hmC in the cerebral cortex and striatum tissue, closely linked to neuron formation and function (Wang et al., 2013). The model of cerebral palsy, a neurodevelopmental disorder, exhibits a significant decrease in the overall 5hmC level in the cortex of rat pups (Zhang et al., 2019). Nevertheless, whether epigenetic dysregulation underlies abnormal cerebral cortex development and nervous system dysfunction remains unclear.

Primary microcephaly is a neurodevelopmental disorder presenting with microcephaly at birth, which primarily arises from mutations in microcephaly-related genes. Among these genes, the microcephalin 1 (MCPH1) gene was first discovered and is a classical model for abnormal cerebral cortex development (Ghafouri-Fard et al., 2015; Boonsawat et al., 2019). To understand the pathogenic mechanism of microcephaly, several Mcph1 knockout mouse models were established to mimic microcephaly (Kristofova et al., 2022). Notably, transgenic mice with knockout of exons 4–5 of the Mcph1 gene (Mcph1-del mice) exhibited reduced brain weight at birth and a significantly thinned cerebral cortex (Gruber et al., 2011). Disruption of Mcph1 was found to perturb the pattern of neural progenitor cell division (gradually changing from symmetric to asymmetric division), resulting in exhaustion of the precursor cell pool, subsequently impacting neuronal production in the cerebral cortex of Mcph1-del mice (Gruber et al., 2011). Mechanistically, MCPH1 deficiency prevents the CHK1 protein from localizing to the centrosome, triggering premature activation of CDK1 and early entry into mitosis, which leads to the mislocalization of the mitotic spindle and then alters the division plane of neural progenitors in Mcph1-del mice (Gruber et al., 2011). Additionally, MCPH1 could interact with the E3 ligase bTrCP2, promoting the degradation of CDC25A, which results in a reduced mitotic rate and NPC differentiation due to Mcph1 knockout (Liu et al., 2017). Hence, the Mcph1-del mouse faithfully recapitulates the primary clinical symptoms of microcephaly patients and is an excellent model to investigate whether abnormal development could reshape the epigenetic landscape of the cerebral cortex.

In this study, we employed Mcph1-del mice as a model to investigate whether and how the malformed cerebral cortex development could shift the DNA methylome/hydroxymethylome landscape and, consequently, impact brain function by using transcriptome sequencing (RNA-Seq), reduced-representation bisulfite sequencing (RRBS), and oxidative RRBS technologies, and by mining published histone modifications data to explore their association with major histone modifications. We uncovered a global decrease in 5hmC level in the cerebral cortex of Mcph1-del mice compared to wild-type ones at juvenile (6 weeks, 6w) and adult (18 weeks, 18w) stages. Notably, the differentially hydroxymethylated regions (DhMRs) are predominantly enriched in TET1-binding regions of the chromatin. Strikingly, genes implicated in establishing and maintaining the nervous system exhibited a delayed accumulation of 5hmC at the adult stage compared to the juvenile stage in Mcph1-del mice. Together, our study offers insight into the molecular mechanisms underlying cerebral cortex developmental diseases and provides a valuable resource for studying the epigenetic regulation of neurogenesis.