Histone Crotonylation in Neurobiology: To Be or Not to Be?
Epigenetic regulation is a fundamental mechanism that governs gene transcription and cell fate. Over the past decades, it has become evident that modifications to histones, DNA, and RNA play critical roles in determining the fate of neural stem cells (NSCs). These modifications include well-studied processes such as histone acetylation and methylation, as well as DNA and RNA methylation. Non-coding RNAs also contribute significantly to neural differentiation. Beyond acetylation, other types of histone lysine acylations, including crotonylation, propionylation, succinylation, and malonylation, have been identified. However, the roles of these histone acylations in neuroscience remain largely unexplored.
Histone crotonylation, a type of short-chain lysine acylation, was first characterized by Zhao’s lab a decade ago as a hallmark of active transcription. This modification is reversibly regulated by acetyltransferases and deacetylases. Specifically, P300 and GCN5 act as writers of histone crotonylation, while class I histone deacetylases and Sirtuins 1–3 serve as erasers. The intracellular concentration of crotonyl-CoA, the substrate for crotonylation, is controlled by enzymes such as short-chain enoyl-CoA hydratase (ECHS1) and chromodomain-Y-like (CDYL) protein. These enzymes modulate the extent of histone crotonylation, thereby influencing transcriptional activity.
Several key histone lysine crotonylation (Kcr) sites have been identified, including H3K18cr, H2BK12cr, H3K9cr, and H3K27cr. These sites are involved in transcriptional regulation, and their distribution and dynamics differ from those of histone acetylation, suggesting distinct functional roles despite shared regulatory machinery. The temporal and spatial differences between crotonylation and acetylation in chromatin further underscore the unique contributions of each modification to gene expression.
Recent studies have highlighted the critical roles of histone crotonylation in various biological processes, including cardiac dysfunction, spermatogenesis, tumor biology, infection, and embryonic development. For example, histone crotonylation at H3K18cr and H2BK12cr has been implicated in cardiac hypertrophy in both humans and rodents. Additionally, crotonylation has been shown to promote endodermal commitment in pluripotent stem cells in humans and mice. These findings suggest that histone crotonylation may play significant roles in development and neurobiology. However, the genome-wide distribution, dynamic changes, and gene expression associations of histone crotonylation during developmental processes, particularly in the central nervous system, remain poorly understood.
To address these gaps, researchers have employed multi-omics approaches, including bulk RNA sequencing (RNA-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), to investigate the role of histone crotonylation in neural stem cell biology. For instance, Liu’s lab at the Institute of Zoology, Chinese Academy of Sciences, conducted genome-wide multi-omics analyses and identified the critical role of histone crotonylation in regulating NSC biology. Their studies focused on H3K9cr in the embryonic forebrain and revealed that H3K9cr-targeted genes are associated with stemness maintenance and neural differentiation. The researchers demonstrated that histone crotonylation regulates the expression of genes involved in the metabolism and proliferation of neural progenitor/stem cells (NPSCs). Specifically, enrichment of histone crotonylation activates bivalent promoters, facilitating chromatin openness and recruiting RNA polymerase II. This reprogramming of the transcriptome promotes neuronal differentiation.
In a follow-up study, the same group described the dynamic profiling and functional interpretation of histone lysine crotonylation and lactylation during neural development. These studies provided epigenetic maps for histone acetylation, methylation, crotonylation, lactylation, and DNA methylation, shedding light on the complex regulatory mechanisms underlying NPSC differentiation. The findings expanded our understanding of the epigenetic mechanisms governing NPSC fate decisions and their clinical implications.
Despite these advances, several open questions remain. For example, while pan-crotonylation of histones has been shown to regulate genes involved in neural stemness maintenance, the specific histone lysine crotonylation sites that play pivotal roles in determining NPSC fate in vitro and in vivo are yet to be fully elucidated. Additionally, the mechanisms underlying histone crotonylation-mediated functions in neurobiology are not fully understood. Although the involvement of the histone Kcr-miR-203-Bmi1 regulatory axis has been suggested, further studies are needed to identify which genes are regulated by specific Kcr sites and how these modifications reshape the transcriptome to determine NPSC fate.
The integration of multi-omics data with single-cell RNA sequencing data presents an opportunity to uncover the principles governing the interactions of various epigenetic modifications in modulating NPSC stemness. Researchers have already identified the participation of histone acetylation (H3K9ac and H3K27ac), methylation (H3K4me1/2/3, H3K9me3, H3K27me3, and H3K36me3), lactylation (H3K18la), and DNA methylation in NPSC maintenance and differentiation. Integrating these datasets could provide a more comprehensive understanding of the epigenetic landscape governing neural development.
The clinical implications of histone crotonylation in brain development and diseases are also yet to be fully addressed. Some experimental and genetic findings provide clues about its potential role in neurobiology. For instance, elevated Kcr levels have been observed in the brains of BTBR T+Itpr3tf/J mice, which exhibit developmental disorders in the central nervous system and aberrant neuroanatomical structures. Additionally, ECHS1, a regulator of histone crotonylation, is critical for human brain development. Mutations in ECHS1 cause developmental defects, such as Leigh syndrome, a severe neurodegenerative disease in children. Germline knockout of ECHS1 in mice leads to embryonic death, which may be related to developmental defects in the neuronal system. These findings suggest that dysregulation of histone crotonylation may contribute to developmental defects in the neuronal system and result in neuropathy.
CDYL, another enzyme that controls histone crotonylation and methylation, has also been implicated in neurobiology. In mice, CDYL suppresses epileptogenesis by repressing axonal Nav1.6 sodium channel expression, while CDYL deficiency disrupts neuronal migration and increases susceptibility to epilepsy. A genome-wide association study identified an association locus at 6p25.1, 61 kb upstream of CDYL, in individuals with drug-resistant epilepsy. However, further exploration with larger replication cohorts is needed to clarify whether CDYL and dysregulated histone crotonylation are causal factors underlying human epilepsy and associated drug resistance. Additionally, CDYL-mediated histone crotonylation has been shown to regulate stress-induced depression, although the underlying mechanisms and clinical implications remain unclear.
Interestingly, crotonate, a metabolite of gut microbes, contributes to histone crotonylation. This raises the possibility of a link between crotonate, histone crotonylation, and the gut-brain axis, suggesting that gut-mediated brain health and diseases may be influenced by these epigenetic modifications.
In conclusion, recent studies on the role of histone crotonylation in neurobiology have opened new avenues for research and therapeutic advancement. Investigating the complex roles of histone crotonylation in neural development and diseases, as well as exploring crotonylation-targeted strategies for treating human neural diseases, will be critical for advancing our understanding of neurobiology. The integration of multi-omics approaches and single-cell sequencing technologies will provide deeper insights into the epigenetic mechanisms governing neural stem cell fate and their implications for brain health and disease.
doi.org/10.1097/CM9.0000000000001945
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