Chromodomain helicase DNA-binding proteins and orofacial cleft

Abstract

Orofacial clefts (OFCs) are among the most common congenital anomalies, arising from a complex interplay of genetic and environmental factors that disrupt the precisely coordinated processes of craniofacial development. This review collects current evidence on the role of the chromodomain helicase DNA-binding (CHD) family of ATP-dependent chromatin remodelers in OFC etiology. We highlight the functions of the nine mammalian CHD proteins (CHD1–CHD9) during palatogenesis. There is strong evidence that mutations in CHD1, CHD3, CHD4, and CHD7 are directly causative for OFCs, frequently through alterations in critical developmental signaling pathways, including bone morphogenetic protein, wingless-related integration site, and retinoic acid, leading to disrupted cranial neural crest cell specification, migration, and differentiation. This review concludes that a specific subset of CHD proteins acts as essential epigenetic regulators of gene programs critical for palate formation. Understanding their distinct and overlapping functions provides novel insights into OFC pathogenesis.

INTRODUCTION

Orofacial clefts (OFCs), the most common congenital craniofacial anomalies with a global incidence of approximately 1 in 700 live births, represent a major public health challenge.[1] These anomalies are classified into three main types, including cleft lip only (CL), cleft palate only (CP), and CL with palate. The vast majority of cases (approximately 70%) are nonsyndromic, occurring without other associated anomalies.[2] The global burden of OFCs is unevenly distributed, with around 84% of cases and nearly 94% of the total disease burden concentrated in low- and middle-income countries.[3] This disparity results primarily from limited access to specialized medical care, reconstructive surgery, and essential multidisciplinary services. Socioeconomic factors and gender inequalities also contribute significantly to this unequal burden.[4]

From a developmental perspective, facial and palatal structures form within the first 10 weeks of pregnancy, requiring precise coordination between molecular signaling pathways and cellular processes.[5] The etiology of OFCs is complex and multifactorial, arising from interactions between genetic and environmental factors. Known environmental factors include smoking, micronutrient deficiencies, obesity, diabetes, and exposure to environmental pollutants.[5,6] Conversely, genetic factors play a primary role, as family and twin studies indicate that first-degree relatives of affected individuals have a 32- to 56-fold higher risk than the general population, and the concordance rate in monozygotic twins reaches 47%.[7,8]

Normal craniofacial development depends on complex molecular signaling networks involving wingless-related integration site (WNT), transforming growth factor-beta, bone morphogenetic protein (BMP), and fibroblast growth factor pathways. Disruption in any of these pathways can lead to OFCs. Genetic studies have associated variants in over 43 genes and loci, including interferon regulatory factor 6, transforming growth factor alpha, msh homeobox 1 (MSX1), and MAF bZIP transcription factor B, with the risk of nonsyndromic OFC.[2] However, a significant portion of phenotypic variability across populations remains unexplained, indicating the involvement of complex regulatory mechanisms, including epigenetic factors.[9]

Epigenetics refers to reversible changes in gene expression that do not alter the DNA sequence. One of the most important epigenetic mechanisms is chromatin remodeling, carried out by specialized protein complexes called ATP-dependent chromatin remodelers. These complexes regulate transcription factor access to DNA by altering nucleosome structure, thereby controlling the expression patterns of genes crucial during development.[10,11] Growing evidence from studies on human syndromes and animal models demonstrates that mutations in genes encoding subunits of chromatin remodeling complexes can disrupt delicate signaling networks and are associated with developmental anomalies, including craniofacial abnormalities.[12,13]

This review collects and interprets current evidence regarding the role of the chromodomain helicase DNA-binding (CHD) family in the development of OFCs to improve the understanding of their pathophysiology and regulatory mechanisms in these conditions. The CHD family is a principal group of ATP-dependent remodelers in mammals, playing a pivotal role in chromatin rearrangement and transcriptional regulation.[14] This family consists of nine members (CHD1 to CHD9), all sharing a common core architecture including two chromodomains in the N-terminal region and a central helicase/ATPase domain. Based on the presence of additional domains, the family is divided into three main subgroups.[15]

Subgroup I (chromodomain helicase DNA-binding 1 and chromodomain helicase DNA-binding 2)

Subgroup I (CHD1 and CHD2) possesses a DNA-binding domain (DBD) in the C-terminal region. These proteins act as multifaceted transcriptional regulators. Recruited to active genes by binding to the H3K4me3 mark, they facilitate RNA polymerase II movement, playing a role in transcriptional elongation.[16,17] They are also involved in transcription termination and splicing.[18,19]

Subgroup II (chromodomain helicase DNA-binding 3, chromodomain helicase DNA-binding 4, and chromodomain helicase DNA-binding 5)

Subgroup II (CHD3, CHD4, and CHD5) is characterized by two plant homeodomain (PHD) domains in the N-terminal region. These proteins are part of the large nucleosome remodeling and deacetylase (NuRD) complex, which integrates histone deacetylase activity with nucleosome remodeling activity.[15,20,21] These members are primarily associated with transcriptional repression. They compact chromatin and silence genes by integrating ATP-dependent remodeling activity with histone deacetylation.[22,23] This process is essential for cell fate decisions and differentiation during development.[24]

Subgroup III (chromodomain helicase DNA-binding 6, chromodomain helicase DNA-binding 7, chromodomain helicase DNA-binding 8, and chromodomain helicase DNA-binding 9)

Subgroup III (CHD6, CHD7, CHD8, and CHD9) contains Brahma and Kismet (BRK) domains with unclear function.[15] This group exhibits strong tissue-specific functions.

Previous studies have established that chromatin remodeling is important for craniofacial development and has implicated individual CHD genes (particularly CHD7) in OFCs; therefore, we aim to distinguish CHD family members that play a direct or causal role in palatogenesis from those with an indirect or no established role and to identify the main pathway influenced.

CHROMODOMAIN HELICASE DNA-BINDING FAMILY MEMBERS WITH DIRECT EVIDENCE

Chromodomain helicase DNA-binding 1

CHD1, comprising 1709 amino acids, plays a fundamental role in regulating the transcription of genes essential for zygotic genome activation (ZGA) and maintaining pluripotency in embryonic stem cells.[25] Based on UniProt and National Center for Biotechnology Information (NCBI) data, CHD1 contains, from N- to C-terminus, two chromodomains, a helicase ATP-binding domain (containing a DEAH box motif), a helicase C-terminal domain, and a CHD1 helical C-terminal (CHCT) domain [Figure 1]. CHD1 is recruited to genomic regions marked by H3K4me3, where its chromodomains play a regulatory role; it then utilizes energy from ATP hydrolysis to modify chromatin structure and provide transcription factors with access to DNA. This unique structure enables CHD1 to modify chromatin. It recognizes the H3K4me3 epigenetic mark (a sign of active transcriptional regions) and uses energy from ATP hydrolysis to remodel nucleosome structure.[17]

Figure 1.

Chromodomain helicase DNA-binding (CHD) proteins structure (based on UniProt and National Center for Biotechnology Information information). All CHDs share a common core architecture including two chromodomains in the N-terminal region and a central helicase/ATPase domain. Subgroup I (CHD1 and CHD2) possesses a DNA-binding domain (DBD) in the C-terminal region. Subgroup II (CHD3, CHD4, and CHD5) is characterized by two plant homeodomain (PHD) domains in the N-terminal region. Subgroup III (CHD6, CHD7, CHD8, and CHD9) contains Brahma and Kismet (BRK)

Functionally, CHD1 is involved in numerous cellular processes, including transcriptional regulation, splicing, and DNA repair.[19,26,27] This protein is essential for embryonic survival, as complete loss of its function in mice leads to developmental arrest after implantation. Studies show that CHD1 is necessary for maintaining the expression of key pluripotency genes such as Pou5f1 (Oct4), Nanog, and Cdx2 by regulating Hmgpi expression during ZGA.[25] In addition, CHD1 plays a role in incorporating the histone variant H3.3 into chromatin after fertilization, which is crucial for heterochromatin formation and proper chromosomal segregation.[28]

In the context of craniofacial development, CHD1 plays a critical role. Heterozygous mutations in the CHD1 gene are associated with Pilarowski–Bjornsson syndrome, characterized by intellectual disability, developmental delay, and facial dysmorphism [Table 1]. Wyatt et al. reported a patient affected by nonsyndromic cleft lip and palate with a heterozygous deletion overlapping the CHD1 gene locus. Functional studies in a Xenopus model show that reduced Chd1 expression leads to severe defects in cranial neural crest development and jaw cartilage morphology. In addition, decreased Chd1 levels resulted in increased apoptosis in the developing head, likely reducing the cell population necessary for forming facial structures.[29] Chd1 is regulated by retinoic acid (RA) signaling during craniofacial development. Expression levels of Chd1 mRNA in the head were increased by retinoic acid receptor (RAR) agonist exposure and decreased upon antagonist treatment. These results indicated that subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergize to cause orofacial defects.[29] This evidence indicates a direct role for CHD1 in OFC formation in both model animals and humans.

Table 1.

Diseases related to the chromodomain helicase DNA-binding family indexed in OMIM

CHD protein	Role in human disease and link to orofacial clefts	Inheritance
CHD1	Pilarowski–Bjornsson syndrome	AD
CHD2	Developmental and epileptic encephalopathy 94	AD
CHD3	SNIBCPS	AD
CHD4	SIHIWES	AD
CHD5	Parenti-Mignot neurodevelopmental syndrome	AD
CHD7	CHARGE Syndrome and hypogonadotropic hypogonadism 5 with or without anosmia	AD
CHD8	Intellectual developmental disorder with autism and macrocephaly	AD

CHD=Chromodomain helicase DNA-binding; SNIBCPS=Snijders Blok–Campeau Syndrome; SIHIWES=Sifrim-Hitz-Weiss Syndrome

Chromodomain helicase DNA-binding 3

CHD3, also known as Mi-2α, is a core component and catalytic subunit of the NuRD chromatin remodeling complex. This protein plays a vital structural and functional role in regulating gene expression, including BMP signaling.[30,31] Based on UniProt and NCBI data, the CHD3 protein comprises 2000 amino acids and contains, from N- to C-terminus, two PHD-type zinc fingers, two chromodomains, a helicase ATP-binding domain (containing a DEAH box motif), and a C-terminal helicase domain. The CHD3 C-terminus contains a region required for interaction with pericentrin (PCNT) to regulate centrosome integrity.[31]

Heterozygous mutations in the CHD3 gene cause Snijders Blok–Campeau syndrome (SNIBCPS), a rare neurodevelopmental disorder with an autosomal dominant inheritance pattern.[32] This syndrome is characterized by intellectual disability (with a wide range of severity), global developmental delay, macrocephaly, impaired speech and language skills, and facial dysmorphisms.[33] Clinical facial features often include a prominent forehead, hypertelorism, midface hypoplasia, a prominent nose, and a high-arched palate.[32,34] This strong phenotypic overlap, particularly the presence of a high-arched palate and midface hypoplasia, indicates a fundamental role for CHD3 in palate and craniofacial structure development.

CHD3 acts through its vital role in the development of cranial neural crest cells (CNCCs), the mesenchymal origin of facial bones, cartilages, and connective tissues.[35] Studies on human induced pluripotent stem cell (iPSC) models lacking CHD3 have shown that its absence leads to severe impairment in CNCC specification. This defect results from an inability to respond appropriately to BMP signaling and a disruption in the precise Wnt/BMP balance. CHD3 regulates accessibility at enhancers bound by BMP-responsive transcription factors, including DLX and MSX families. In the absence of CHD3, the BMP response is ineffective, leading to a Wnt/BMP imbalance. Consequently, the CNCC specification fails and is replaced by mesodermal identity, which can be partially rescued by increasing Wnt levels.[36]

Given the shared pathways, the CHD3 function is indirectly linked to palate and craniofacial development. The BMP/Wnt signaling pathway regulated by CHD3 is fundamental in palate and cranial bone morphogenesis.[37] Disruption of this pathway due to CHD3 mutation leads to defects in CNCC migration and differentiation, resulting in shared features such as midface hypoplasia and a high-arched palate. These features may predispose to CP.

Chromodomain helicase DNA-binding 4

CHD4, also known as Mi-2β, is a catalytic subunit and core component of the NuRD chromatin remodeling complex. Based on UniProt and NCBI data, the CHD4 protein comprises 1912 amino acids and contains, from N-to C-terminus, a KIKL motif, two PHD-type zinc fingers, two chromodomains, a helicase ATP-binding domain (containing a DEAH box motif), a C-terminal helicase domain, and a region required for interaction with pericentrin. The KIKL motif positions the protein to bind other proteins containing an extra terminal (ET) domain, such as BRD3.[38] The primary function of CHD4, acting as the molecular motor of the NuRD complex, is to use energy from ATP hydrolysis to rearrange nucleosome structure and alter chromatin accessibility. This remodeling activity is critical for numerous cellular processes, including transcriptional regulation, genomic integrity, cell cycle progression, response to DNA damage, and especially cell differentiation and embryonic development.[39,40]

Germline mutations in the CHD4 gene cause Sifrim-Hitz-Weiss syndrome (SIHIWES), a rare neurodevelopmental disorder with an autosomal dominant inheritance pattern. This syndrome is characterized by intellectual disability, macrocephaly, congenital heart defects, hypogonadism, and facial dysmorphisms. Clinical facial features often include ophthalmic abnormalities, midface hypoplasia, a prominent nose, and palatal abnormalities, which can manifest as a high-arched, dome-shaped palate or a submucosal CP.[41,42] This strong phenotypic overlap, particularly the presence of palatal abnormalities and midface hypoplasia, indicates a fundamental role for CHD4 in palate and craniofacial structure development.

A large-scale genomic study of syndromic OFCs further investigated the pivotal role of CHD4 in palatogenesis. This study identified CHD4 as one of the most highly connected hubs in a protein–protein interaction (PPI) network of syndromic cleft genes. The study proposed that genes involved in chromatin remodeling, such as CHD4, represent a key molecular pathway distinguishing syndromic OFCs from nonsyndromic forms. This positions CHD4, through its role in the NuRD complex, as a master regulator of critical gene expression programs during embryonic development; its disruption leads concurrently to CP and the additional neurodevelopmental and physical anomalies seen in patients.[43]

Chromodomain helicase DNA-binding 7

CHD7, comprising 2997 amino acids, is a member of Subgroup III of ATP-dependent chromatin remodeling proteins. Based on UniProt and NCBI data, the CHD7 protein contains, from N- to C-terminus, two chromodomains, a helicase ATP-binding domain (containing a DEAH box motif), a C-terminal helicase domain, and two BRK domains in the C-terminus.[15] This protein acts as a key regulator of the transcription of genes involved in the migration and differentiation of CNCCs.[44,45,46] Mutations in the CHD7 gene are the primary cause of CHARGE syndrome, a congenital disorder with various features including coloboma, heart defects, choanal atresia, ear abnormalities, and CP or lip. CP is observed in approximately 33%–48% of patients with CHARGE syndrome.[47,48] Disruption of CHD7 function leads to defects in the specification, migration, or differentiation of CNCCs, which are essential for normal facial structure formation and palatal shelf fusion.[13] CHD7 hypomorphic mouse models also faithfully recapitulate orofacial abnormalities such as CP.[49] CHD7 binds to enhancer regions of its target genes and modulates chromatin accessibility to control their expression.[50] Disruption of this epigenetic regulation can lead to aberrant expression of these factors and, consequently, defects in palate formation.

Some studies indicate that impaired palate formation is a common feature in animal models with defects in the Chd7 gene.[13,49] These experiments include generating conditional knockout mice for Chd7, where deletion occurs specifically in CNCC populations or the palatal epithelium. These mice show severe defects in secondary palate closure, including complete CP and thickening and vaulting of the palatal shelves.[13] The CHD7 protein is essential for forming the multipotent migratory neural crest, a transient cell population of ectodermal origin that undergoes transcriptional reprogramming to acquire broad differentiation potential, giving rise to craniofacial bones and cartilages.[51] CHD7 is critical for activating the core neural crest transcriptional circuitry, including key genes such as Sox9, Twist, and Slug. In human neural crest cells, CHD7 associates with the PBAF chromatin-remodeling complex, and together, they occupy specific distal enhancers, such as one for the SOX9 gene. This collaboration between CHD7 and PBAF promotes neural crest gene expression and cell migration during embryogenesis.[51] Consequently, disruption of CHD7 function impairs the genetic programs necessary for proper neural crest cell migration and differentiation, ultimately leading to craniofacial anomalies, including palate defects, as seen in CHARGE syndrome.[46] These findings comprehensively demonstrate the causal role of CHD7 mutations in craniofacial abnormalities, including CP, through disruption of the epigenetic program regulating CNCC development.

CHROMODOMAIN HELICASE DNA-BINDING FAMILY MEMBERS WITH INDIRECT EVIDENCE

Chromodomain helicase DNA-binding 2

CHD2, comprising 1828 amino acids, has a structure similar to CHD1. Based on UniProt and NCBI data, its domain architecture from N- to C-terminus includes two chromodomains, a helicase ATP-binding domain (containing a DEAH box motif), a helicase C-terminal domain, and a CHCT domain. Although research on CHD2 has primarily focused on its role in neurodevelopmental disorders and epileptic encephalopathies, growing evidence suggests its potential role in craniofacial development.[52]

Patients with heterozygous mutations in CHD2 show developmental and epileptic encephalopathy 94.[53] Murine Chd2 mutant models exhibit neurodevelopmental and skeletal abnormalities.[54,55] For example, Chd2+/− deficient mice exhibit spinal abnormalities, including abnormal lordosis, kyphosis, and scoliosis, consistent with clinical findings in human patients with CHD2 deficiency. These mice also show reduced body fat, growth delay, and decreased viability.[54] In humans, CHD2 gene disruption during translocation is associated with developmental delay, intellectual disability, epilepsy, and mild facial dysmorphism. Although CP has not been consistently reported, abnormal facial features such as a high-arched palate have been observed in some patients with t(X;15)(p22.2;q26.1). As only the 15q26.1 breakpoint disrupts the CHD2 gene, while the Xp22.2 breakpoint affects no known or predicted genes, CHD2 may contribute to palatal development.[54]

Regarding its mechanism of action, CHD2 acts as an epigenetic regulator by recognizing epigenetic marks such as H3K4me (albeit with lower affinity than CHD1) and depositing the histone variant H3.3. This protein also plays a role in DNA damage repair through nonhomologous end joining and is recruited to double-strand DNA break sites by PARP1. Furthermore, CHD2 regulates the expression of developmental genes through interaction with key transcription factors like repressor element 1-silencing transcription factor.[52]

In summary, although direct evidence is less extensive than for CHD1, available data support a role for CHD2 in craniofacial development and its potential contribution to CP pathogenesis as part of a more complex syndrome. Further studies on animal models and more detailed phenotypic examinations of human patients are essential to fully understand the extent and mechanism of this involvement.

Chromodomain helicase DNA-binding 5

CHD5, a member of Subgroup II of ATP-dependent chromatin remodeling proteins from the CHD family, has a structure similar to CHD3 and CHD4 and comprises 1954 amino acids. Based on UniProt and NCBI data, the CHD5 protein contains, from N- to C-terminus, two PHD-type zinc fingers, two chromodomains, a helicase ATP-binding domain (containing a DEAH box motif), and a C-terminal helicase domain. The PHD domains mediate specific binding to histone H3 unmethylated at Lys-4 and may preferentially recruit the protein to transcriptionally inactive genes.[56] The chromodomains mediate specific binding to histone H3 trimethylated at Lys-27 (H3K27me3) and may be required for proper gene regulation during neuron differentiation.[57] Unlike the widely expressed CHD3 and CHD4, CHD5 expression is primarily restricted to the nervous system and testes, indicating its specialized role in neurogenesis and cell differentiation.[58] The primary function of CHD5, the catalytic subunit of a specialized NuRD-like complex, is to modulate chromatin structure and regulate gene transcription through its ATP hydrolysis activity. This protein interacts with repressive epigenetic marks such as H3K27me3 through its PHD and chromodomains.[57] Heterozygous mutations in the CHD5 gene cause the rare neurodevelopmental disorder Parenti-Mignot syndrome, characterized by developmental delay, intellectual disability, cleft lip and palate, hydrocephalus, and renal pathology.[59]

Although direct and definitive evidence linking CHD5 to CP in humans is very rare and reported in only one case, functional studies in a zebrafish model have shown an important role for Chd5 in overall craniofacial development.[58,59] Knockdown of Chd5 expression using antisense morpholinos (chd5MO1 and chd5MO2) that inhibit chd5 mRNA translation results in clear phenotypes, including a small head, small eyes, and severely reduced head cartilage formation. Interestingly, coinjection of chd5 mRNA (lacking the morpholino target sequence) partially rescues these phenotypes, proving the specificity of these defects.[58] Although these studies did not directly examine CP, the general defect in craniofacial cartilage formation and reduced head size create an unfavorable environment for successful palatal fusion. These studies suggest that CHD5 is a general modifier of craniofacial development rather than a direct regulator of palatal morphogenesis.

CHROMODOMAIN HELICASE DNA-BINDING FAMILY MEMBERS WITH NO ESTABLISHED CAUSAL ROLE

Chromodomain helicase DNA-binding 6

CHD6, a member of Subgroup III of ATP-dependent chromatin remodeling proteins from the CHD family, comprises 2715 amino acids. Based on UniProt and NCBI data, the CHD6 protein contains, from N-to C-terminus, two chromodomains, a helicase ATP-binding domain (containing a DEAH box motif), a C-terminal helicase domain, and Myb-like domains. One function of CHD6 is to respond to oxidative stress and DNA repair. This protein is recruited to DNA damage sites caused by oxidative stress and acts as a key factor in the response to oxidative damage. Loss of CHD6 function leads to accumulation of DNA damage, increased reactive oxygen species levels, impaired repair, and severe sensitivity to PARP inhibitors.[60] Currently, no direct functional association has been established between the CHD6 gene and human diseases, and no related diseases are reported in online mendelian inheritance in man (OMIM). However, cases with chromosomal translocations involving the CHD6 genetic locus have been identified in specific patients, including one individual with Pitt–Hopkins syndrome,[61] a case of intellectual disability,[62] and sporadic instances of acute myeloid leukemia.[63] Kargapolova et al. reported a missense mutation in the CHD6 gene in a patient exhibiting symptoms related to Hallermann–Streiff syndrome.[64] In Hallermann–Streiff syndrome, craniofacial and dental anomalies are typical symptoms. Based on a systematic review, a high-arched palate is present in 44% of patients with Hallermann–Streiff syndrome.[65] Therefore, based on available evidence, CHD6 is not considered a direct causative factor for palatal formation; a high-arched palate in these patients may result from other complications such as micrognathia and skull anomalies.

Regarding the role of CHD6 in craniofacial development, no specific experiments have examined its role in CP. Mouse models with mutations in CHD6 (such as deletion of exon 12) primarily show cerebellar defects and ataxia, but no craniofacial abnormalities or CP have been reported.[66] However, further experiments are required to investigate the role of CHD6 in embryogenesis and development.

Chromodomain helicase DNA-binding 8

CHD8, like CHD7, is a member of Subgroup III of the CHD family and shares a similar structure, which includes two chromodomains, an ATP-binding domain (containing a DEAH box motif), a C-terminal helicase domain, a region for interaction with FAM124B,[67] and two BRK domains in the C-terminus.[15] Although mutations in the CHD8 gene are recognized as a strong risk factor for autism spectrum disorders (ASD) and intellectual disability, CP is not a common or defining feature of CHD8-related syndromes.[68]

The function of CHD8 focuses on regulating critical signaling pathways, such as Wnt/β-catenin, which are essential for neurogenesis.[68] Disruption of CHD8 function leads to defects in the proliferation and differentiation of neural progenitor cells, as well as gastrointestinal disturbances.[69,70] Although Chd8 hypomorphic mouse models show macrocephaly and subtle craniofacial abnormalities (such as a long, broad forehead and prominent glabella), CP has not been reported in these models.[71]

Given shared pathways, although CHD8 function is not directly related to palate development, it may indirectly influence palatal morphogenesis through physical interaction with CHD7 and participation in shared chromatin remodeling complexes.[72] By binding to promoters enriched with H3K4me2 and H3K4me3, this protein modulates chromatin accessibility and controls the expression of vast gene networks.[73] Disruption of this epigenetic regulation can lead to misexpression of target genes and, consequently, defects in overall craniofacial growth, such as macrocephaly.[74,75]

Experiments investigating the role of CHD8 in facial development have primarily used mouse models to understand its overall role in neurogenesis and body growth. These experiments include generating heterozygous knockout mice for Chd8, which exhibit macrocephaly, behavioral abnormalities, and subtle changes in facial features, but do not display CP.[74,76] Histological and immunohistochemical analysis of these models shows reduced cell proliferation in the developing brain and changes in the expression of cell cycle-related markers.[68] Furthermore, experiments on human pluripotent stem cells with CHD8 deletion show severe defects in neural ectoderm differentiation and the activation of neural specification genes.[69] CHD8 plays a critical role as a master regulator of p53-mediated apoptosis during early embryonic development. CHD8 physically interacts with both p53 and histone H1, using these interactions to recruit histone H1 to the promoters of p53 target genes. This recruitment leads to the suppression of gene expression and, consequently, the inhibition of apoptosis that would otherwise be triggered by genotoxic stress. This model is strongly supported by the finding that the developmental arrest caused by CHD8 loss in mice is ameliorated by the simultaneous deletion of p53. Therefore, the primary physiological role of CHD8 appears to be to act as a crucial brake on p53 activity, preventing unwanted cell death and ensuring proper embryonic progression.[77] These findings comprehensively demonstrate the key role of CHD8 in neural development and overall body growth but have not identified a direct and causal role in CP pathogenesis, unlike CHD7.

Chromodomain helicase DNA-binding 9

CHD9 is a member of Subgroup III of ATP-dependent chromatin remodeling proteins. Based on data from UniProt and NCBI, the CHD9 protein contains, from N-to C-terminus, two chromodomains, a helicase ATP-binding domain (containing a DEAH box motif), a C-terminal helicase domain, a region that binds A/T-rich DNA, and two BRK domains in the C-terminus.[15] Unlike other members of this subgroup (e.g., CHD7 and CHD8), CHD9 is not essential for development and survival in mice. Mice lacking CHD9 (Chd9−/−) are viable and fertile and show no obvious developmental defects. Conversely, acute depletion of CHD9 in cancer cells indicates that CHD9 is a context-dependent chromatin remodeler.[78] Detailed histopathological examinations of large cohorts of these mice at different ages (3 and 12 months) revealed only mild inflammation in some organs, related to the mouse strain’s genetic background or aging; none of the reported pathologies were related to craniofacial abnormalities. Furthermore, gene expression analysis (RNA-seq) in embryonic tissues from these mice showed very limited gene expression changes, primarily restricted to genes involved in the complement system and peroxisomes, with no significant enrichment observed in known signaling pathways associated with CP.[78]

The primary function of CHD9 has been described in the regulation of tissue-specific gene transcription, particularly in osteogenic differentiation (e.g., upregulation of the RUNX2 transcription factor) and in the regulation of ribosomal gene transcription by RNA polymerase I in the nucleolus.[79,80] RUNX2 is essential for intramembranous ossification, which forms the facial skeleton.[81,82] Since palate and facial development depend on the correct differentiation and organization of mesenchymal cells, any factor disrupting these pathways could theoretically impact craniofacial development.

The absence of a severe phenotype in knockout mice suggests that CHD9 is not essential or irreplaceable for palate development. This finding may imply that either other members of the CHD protein family compensate for its function, or CHD9 acts only within specific cellular contexts or under particular conditions.

CONCLUSION

The CHD family of proteins acts as epigenetic guardians of embryonic development. A comprehensive functional analysis of each member suggests that their involvement in CP etiology lies on a spectrum. CHD1, CHD3, CHD4, and CHD7 have the strongest evidence. Mutations in these genes lead directly to CP by disrupting neural crest cell (CNCC) fate and key signaling pathways (BMP, Wnt, and RA). CHD2 and CHD5 are involved in overall facial development, and their disruption can lead to palatal dysmorphism indirectly or in combination with other factors, but CP is not a fixed and defined feature of their phenotype. CHD6, CHD8, and CHD9 are focused on completely different pathways (stress response, brain development, and tissue-specific regulation), and studies do not indicate any direct causative role for them in CP. A key distinction is functional dependence on the NuRD complex. Subgroup II members (CHD3, CHD4, and CHD5), which are part of the NuRD complex, play a more prominent role in the initial specification of CNCCs through regulation of the BMP pathway. In contrast, other members (such as CHD7) may more likely affect the subsequent migration or differentiation of these cells.

From a diagnostic perspective, as mutations in CHD1, CHD3, CHD4, and CHD7 are directly causative for OFCs, these four genes should be prioritized in clinical testing for patients presenting with CP accompanied by additional features such as intellectual disability, facial dysmorphism, midface hypoplasia, or congenital anomalies. Involvement of these genes in specific targeted gene sequencing panels for patients with syndromic OFCs can improve diagnostic yield, inform accurate genetic counseling regarding recurrence risk, and guide prognosis based on the specific CHD-related syndrome.

Genetic testing and early detection are important in determining the therapeutic approach for affected children with syndromic OFCs, particularly regarding neurodevelopmental disorders. Early detection of mutation in CHD1, CHD3, CHD4, and CHD7 may help provide suitable medical care or early intervention in speech apraxia, autism, and seizure disorders.

Future research should focus on a more detailed understanding of the regulatory networks these proteins control in craniofacial development, as well as exploring possible interactions between them. In addition, future studies on animal models can help clarify the specific role of other members of this family.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was supported by the research fund of Isfahan University of Medical Sciences.