Epigenetic Mechanisms of Inner Ear Development

Abstract

Epigenetic factors are critically important for embryonic and postnatal development. Over the past decade, substantial technological advancements have occurred that now permit the study of epigenetic mechanisms that govern all aspects of inner ear development, from otocyst patterning to maturation and maintenance of hair cell stereocilia. In this review, we highlight how three major classes of epigenetic regulation (DNA methylation, histone modification, and chromatin remodeling) are essential for the development of the inner ear. We highlight open avenues for research and discuss how new tools enable the employment of epigenetic factors in regenerative and therapeutic approaches for hearing and balance disorders.

1. Introduction

The mammalian inner ear is a highly specialized sensory organ that functions to perceive and transduce sound waves as well as detect changes in balance and orientation in space. The cochlea houses the organ of Corti which contains hair cells that respond to mechanical disruption from sound waves. Spiral ganglion neurons innervate the cochlear epithelium and send afferent auditory signals from the hair cells to the brain. In addition to the cochlea, the inner ear also houses the vestibular system, which contains hair cells that respond to vestibular cues and form synapses with neurons in the vestibular ganglion that project to the brain and enable the interpretation of changes in angular velocity and orientation. The formation of these auditory and vestibular structures is dependent on a series of finely tuned developmental events including elongation of the cochlear duct, differentiation of hair cells and surrounding supporting cells, and establishment of the proper neuron connections.

Epigenetic mechanisms are among the many ways in which this dynamic process of inner ear development is regulated. Epigenetic regulation is carried out via the processes of DNA methylation, post-translational histone modifications, nuclear non-coding RNAs, and ATP-dependent chromatin remodeling (Doetzlhofer and Avraham, 2017). The enzymes that carry out these processes are classically categorized as writers, erasers, and readers that create, edit, and translate the epigenetic code (Jenuwein and Allis, 2001; Ruthenburg et al., 2007; Strahl and Allis, 2000; Turner, 2000). While there are many transcription factors responsible for driving inner ear development, the epigenetic code imparted by writers, erasers and readers facilitates the temporal- and cell type-specific activities of key inner ear genes. The embryonic lethality of numerous epigenetic factor knockout mice illustrates the critical nature of epigenetic regulation of gene expression during development (Jaenisch and Bird, 2003).

In this review, we discuss known epigenetic mechanisms that govern gene expression in the development of the inner ear. We present the variety of ways that epigenetic modifiers and their associated complexes influence target gene expression in the inner ear, highlight studies where the functional roles of these genes have been explored, and propose how epigenetic modifiers may be employed therapeutically to restore hearing and balance.

2. Epigenetic Regulation of Chromatin

DNA methylation, histone modifications, and ATP-dependent chromatin remodeling are among the most prominent processes through which the assembly and structure of chromatin are regulated. These mechanisms regulate gene expression by modifying the accessibility of genetic material through coordinated interactions between transcription factors and chromatin.

2.1. DNA Methylation

DNA methylation involves the addition of methyl groups to cytosine at the 5’ position of CpG dinucleotides. This process is highly conserved and important for transcriptional silencing (Zemach and Zilberman, 2010). Patterns of DNA methylation fluctuate during aging and development in both genome-wide and gene-specific ways, and the DNA methyltransferase enzymes function uniquely to ensure proper regulation of these patterns. The placement and maintenance of methyl groups is managed by three DNA methyltransferase enzymes: DNMT1, DNMT3A, and DNMT3B which are vital for proper development. Deletion of these enzymes leads to embryonic lethality in mice stemming from significant decreases in genomic methylation (Li et al., 1992; Okano et al., 1999). Altered DNA methylation has been shown to induce a sensory hair cell-like phenotype in mouse utricular progenitor cells, highlighting the important role that DNA methylation plays in regulating stem cell fate in the inner ear (Zhou and Hu, 2016).

DNMT1 is a maintenance methyltransferase, working in concert with the UHRF1 protein to ensure accurate replication of methylation during mitosis (Bronner et al., 2019). Through regulation of gene expression and chromatin stability, DNMT1 influences neurogenesis in post-mitotic neurons (Fan et al., 2001) . While not directly implicated in the inner ear, human mutations in DNMT1 have been associated with deafness secondary to central and peripheral neuropathy (Klein et al., 2013; Klein et al., 2011). The ability of DNMT1 to ensure faithful replication of methylation marks during mitosis is essential for proper DNA replication and embryonic development; however, roles for DNMT1-mediated methylation activity in inner ear development have not been defined.

While DNMT1 maintains established methylation patterns, DNMT3A and DNMT3B work as de novo methyltransferases. These enzymes act on both hemi-methylated and unmethylated DNA to establish methylation patterns across the genome (Okano et al., 1998). DNMT3A/B are highly expressed in undifferentiated embryonic stem (ES) cells and then downregulated after differentiation (Charlton et al., 2020; Okano et al., 1999). DNMT3A is expressed in the presumptive otic region from the gastrula through neurula stages in the chick embryo, where it influences otic placode development by maintaining increased expression levels of the key otic placode specification genes Pax2 and Gbx2 as well as the later otic markers Sox10 and Soho1 (Roellig and Bronner, 2016). Interestingly, while DNMT3A knockdown significantly reduces expression of these markers and otic vesicle size, it does not affect early neural plate border or non-neural ectoderm marker expression. These findings suggest that DNMT3A works cell-autonomously to promote otic development, independent from its roles in neural tube patterning and closure (Fig 1).

Figure 1.

Epigenetic factors drive otic placode specification and otocyst formation. Beginning at approximately E8.5 in the mouse, the otic placode invaginates to form the otocyst that will develop into the entire inner ear. Histone modification, DNA methylation, and chromatin remodeling each play important roles in patterning the otic placode and in formation of the otocyst. Histone demethylase LSD1 (KDM1A) removes H3K9me3 marks to enable acetylation of these residues, ultimately activating the expression of key otic transcription factors Pax2, Sox8, and Etv (Ahmed and Streit, 2018). DNA methyltransferase DNMT3A activates Gbx2 expression by methylating the Gbx2 repressor binding site, thereby allowing GBX2 to activate expression of otic transcription factors Pax2, Sox10, and Soho1 (Roellig and Bronner, 2016). The chromatin remodeling activities of CHD7 and BRG1 coordinate neurogenic specification in the anteroventral region of the otocyst (Ahmed et al., 2012; Hurd et al., 2012; Hurd et al., 2010).

The removal of methyl marks from DNA is carried out by the DNA demethylating enzymes TET1, TET2, and TET3 (Ross and Bogdanovic, 2019). While the TET enzymes are known to be critical for early embryonic development and maintenance of pluripotency, little is known about how DNA demethylation is involved in inner ear development (Koh et al., 2011). Interestingly, a recent case report described compound heterozygous pathogenic variants in TET3 in a pair of siblings with bilateral sensorineural hearing loss (Seyama et al., 2021). This observation suggests the changes in DNA methylation may contribute to hearing loss, and further investigation in animal models is warranted.

Effective modulation of DNA methylation patterns by DNMT3A and DNMT3B is vital for ensuring proper development, with DNMT3A playing a particularly important role in inner ear morphogenesis. However, in addition to directly modifying DNA, transcription can also be regulated by adjusting the ease with which transcription factors are able to access the genetic material. Histone modifications and ATP-dependent chromatin remodeling both influence transcription in this way, with each method of epigenetic regulation modifying chromatin accessibility rather than DNA itself.

2.2. Histone Modifications

Nucleosomes form the fundamental unit of eukaryotic chromatin, with each nucleosome containing 8 histones, a globular region, at least one tail on each histone N-terminus, and roughly 200 bp of genomic DNA (Absolom and Van Regenmortel, 1977; Grover et al., 2018; Kornberg, 1974). Nucleosomes function to package large amounts of eukaryotic genetic material while still allowing for chromatin to be accessed (Kornberg, 1974; Lilley and Pardon, 1979). Histones are subject to a wide variety of post-translational modifications including methylation and acetylation, and histone tails are generally more accessible and susceptible than their globular regions. Altered histone modifications in the inner ear result in severe defects in inner ear development, likely due to erroneous repression of key progenitor genes (Fig 1) (Ahmed and Streit, 2018; Shin et al., 2018).

2.2.1. Histone Acetylation and Deacetylation

Post-translational modification of histone tails alters their interactions with DNA. One of these modifications, acetylation, involves the addition of an acetyl group to a lysine residue. Acetylation of histones results in a more relaxed chromatin configuration due to decreased electrostatic interactions. This relaxed configuration allows transcription factors and other cofactors to access chromatin and activate transcription of exposed genes. This dynamic process is regulated by two types of enzymes: histone acetyltransferases (HATs), which loosen the interactions between histones and DNA by transferring acetyl groups to lysine residues, and histone deacetylases (HDACs), which remove acetyl groups and cause histones to interact more tightly with DNA, thus reducing transcriptional activity (Bonnaud et al., 2016). Regulation of DNA-histone interactions is of critical importance to development of many structures, including the inner ear (Fig 1).

To date, there are no data directly implicating specific HAT or HDAC enzymes in inner ear development. However, several studies have reported altered histone acetylation near genes that regulate neurogenesis or hair cell formation. CCCTC binding factor (CTCF) encodes a transcription factor that can bind HAT or HDAC-containing complexes and function as a transcriptional activator or repressor, respectively. Loss of CTCF in the developing otocyst leads to a complete loss of spiral ganglion neurons and reduced expression of the proneural genes Neurog1 and Neurod1, along with a host of other structural and developmental defects in the inner ear (Shin et al., 2018). Ctcf deletion is also associated with decreased H3K27 acetylation at the promoter region of Neurog1. Together, these results suggest that neuronal differentiation in the inner ear is dependent upon transcriptional enhancement of Neurog1 via histone acetylation (Fig 2).

Figure 2.

Epigenetic factors involved in inner ear neurosensory cell patterning and maturation. Neurosensory progenitor cells are biased towards hair cell or neuronal cell fates by activation or repression of cell type-specific transcription factors (Atoh1 for hair cells, Ngn1, Neurod1, Eya1, and Six1 for neurons). In neurosensory progenitors, Atoh1 is in a bivalent or poised state based upon the presence of activating H3K4me3 marks and repressive H3K27me3 marks (Stojanova et al., 2015). As neurosensory progenitors differentiate into hair cells, H3K27me3 marks at the Atoh1 locus are replaced by activating H3K27ac marks, permitting substantial upregulation of Atoh1 (Stojanova et al., 2015). Atoh1 expression is dampened in mature hair cells by H3K27ac removal (Stojanova et al., 2015). Chromatin remodeler BRG1 regulates the accessibility of several genes in the planar cell polarity pathway (Jin et al., 2019; Jin et al., 2016). In neurosensory progenitors fated to the neuronal lineage, CHD7 augments expression of the key neurogenic transcription factors Ngn1 and Neurod1 (Hurd et al., 2012; Hurd et al., 2010). In ES cells, CHD7 is recruited to active enhancers by H3K4me1 marks (Schnetz et al., 2009; Schnetz et al., 2010), but whether this occurs in neurosensory progenitors in the inner ear is unknown. BRG1 additionally facilitates chromatin remodeling to stimulate the expression of neurogenic genes Eya1 and Six1 (Ahmed et al., 2012). Epigenetic regulation of neuronal maturation in the inner ear has not yet been explored.

Histone acetylation is also likely important for hair cell development. The prosensory gene Atoh1 is necessary and sufficient for hair cell formation in the developing ear(Bermingham et al., 1999; Izumikawa et al., 2005; Zheng and Gao, 2000). Expression of Atoh1 is regulated by histone acetylation. Atoh1 exhibits high levels of H3K9 acetylation in E14.5 progenitors as well E17. 5 hair cells (Fig 2), and inhibition of histone acetyltransferase activity correlates with decreased Atoh1 expression in the inner ear (Stojanova et al., 2015). Thus, histone acetylation appears essential for inner ear hair cell morphogenesis and development.

2.2.2. Histone Methylation and Demethylation

Methylation is another histone post-translational modification that is important for inner ear development. The most common targets of histone methylation are Arginine and Lysine residues on histones H3 and H4. Unlike the activating effects of histone acetylation, addition of methyl groups to histones can have either an activating or repressing effect on chromatin structure, depending on both the histone and residue being modified (Layman and Zuo, 2015). Studies have shown that many promoters in pluripotent ES cells express both active (H3K4me3) and repressive (H3K27me3) histone modifications, giving these domains a “bivalent” or “poised” classification (Voigt et al., 2013). Although the exact function of bivalency is not yet known, dual H3K4me3 and H3K27me3 expression in the promoter region of bivalent genes serves to regulate expression of important factors during development while protecting against inappropriately timed gene activation (Azuara et al., 2006). Below we describe several examples whereby changes in histone methylation result in or contribute to inner ear development.

Early in inner ear development, the histone demethylase LSD1 (also known as KDM1A) is required to preserve progenitor identity prior to cell-type specification. As otic epithelium progenitor cells begin to differentiate, LSD1 activity is crucial to maintain progenitor cell transcriptional profiles, preventing them from adopting improper fates or prematurely entering lineage specification. LSD1 maintains progenitor states by catalyzing H3K9 demethylation at several major otic progenitor genes (Sox8, Pax2, Etv, and Zbtb16, Fig 1) and allowing for maintenance of the active H3K9ac histone mark which confers a relaxed chromatin state and enables increased transcriptional activation of progenitor genes (Ahmed and Streit, 2018).

In addition to its H3K9 demethylating activity, LSD1 also acts as a H3K4 demethylase (Shi et al., 2004). Using the VOT-N33 mouse otic neuronal progenitor cell line, Patel et al. found that LSD1 directly interacts with the nuclear remodeling and deacetylase (NuRD) complex along with PAX2 to maintain otic progenitor cell fate. Inhibition of LSD1 enzymatic activity led to increased H3K4 methylation at several prosensory genes and increased gene expression, suggesting that H3K4 demethylation by LSD1 is critical for regulating the timing of otic proneurosensory differentiation (Patel et al., 2018).

Another example illustrating the importance of histone methylation in ear development involves the Atoh1 gene. Prior to sensory epithelial cell differentiation, the Atoh1 gene body and enhancer regions in hair cell progenitors exhibit the bivalent H3K4me3/H3K27me3 “poised” state of histone methylation. Once hair cell differentiation is initiated, levels of activating H3K4me3 in Atoh1 increase whereas repressive H3K27me3 levels decrease. These changes are accompanied by increased H3K9ac and expression of Atoh1. As hair cell development continues, H3K9ac rapidly declines and is replaced by H3K9me3 upon completion of hair cell differentiation (Tao et al., 2021). These tightly regulated changes in histone acetylation and methylation highlight how histone modifications control gene expression in the inner ear in a developmental stage-specific manner (Fig 2).

2.3. ATP-Dependent Chromatin Remodeling

Unlike histone modifications in which singular histone-chromatin electrostatic interactions are manipulated, ATP-dependent chromatin remodeling uses energy derived from the hydrolysis of ATP to alter the larger chromatin structure, affecting gene transcription via the repositioning of nucleosomes (Hota and Bruneau, 2016; VandenBosch and Reh, 2020). ATP-dependent chromatin remodelers are commonly assigned to one of four groups: chromodomain helicase DNA-binding (CHD), switching/sucrose non-fermenting (SWI/SNF), imitation SWI (ISWI), and INO80 (Hota and Bruneau, 2016; Li et al., 2007; Mashtalir et al., 2018). The removal of nucleosomes from regulatory regions of the gene body, such as promoter or enhancer regions, is vital for the recruitment of transcription factors and for transcriptional elongation (Basson and van Ravenswaaij-Arts, 2015; Li et al., 2007). ATP-dependent chromatin remodelers are also capable of positioning nucleosomes such that chromatin accessibility is limited, allowing for stable gene repression (Raab et al., 2015). As a result, chromatin remodelers can either enhance or repress gene expression, allowing them to act as versatile regulators during development. Since chromatin remodelers are necessary to regulate the development of such a wide array of structures, loss of function of these protein complexes have wide-reaching effects. Mutations in chromatin remodeling proteins CHD7, SWI/SNF, and CERF have been shown to result in severe malformations in many structures, including the inner ear (Dawe et al., 2011; Hurd et al., 2011; Hurd et al., 2012; Jin et al., 2019).

2.3.1. CHD7 in the inner ear

The CHD7 gene encodes a chromodomain helicase DNA-binding ATP-dependent chromatin remodeler. CHD7 contains a Snf2 helicase-like ATPase for catalyzing nucleosome translocation and is comprised of DEXDc and HELICc domains that are conserved throughout the CHD family of proteins (Basson and van Ravenswaaij-Arts, 2015). The CHD7 gene also consists of a SANT-SLIDE unit that recognizes and binds extranucleosomal DNA longer than 40 base pairs. This SANT-SLIDE module, particularly the C-terminus, is thought to allow chromatin remodelers to influence the direction of nucleosome sliding to generate evenly spaced nucleosomes (Bouazoune and Kingston, 2012; Ryan et al., 2011). Deletion of SANT-SLIDE from CHD proteins results in a significant decrease in nucleosome sliding, suggesting that it plays a vital role in facilitating CHD7 activity (Bouazoune and Kingston, 2012; Ryan et al., 2011).

The tandem chromodomains located at the N terminus of CHD7 are also critical for mediating chromatin remodeling activity. These tandem chromodomains are thought to mediate the recruitment of CHD7 to specific sites along the chromatin in a pattern that is highly correlated to histone methylation, particularly H3K4 methylation and epigenetic signatures common to enhancer regions (Schnetz et al., 2009). In addition, most CHD7 binding sites are located distal to transcription start sites and within open chromatin. These binding sites also commonly exhibit co-localization of CHD7 with OCT4, SOX2, and NANOG, all of which play important roles in regulating ES cells (Engelen et al., 2011; Schnetz et al., 2010). The CHD7 protein additionally contains a pair of BRK domains, the function of which are poorly understood (Bouazoune and Kingston, 2012). Although CHD7 is capable of positively and negatively modulating gene expression, the primary effect of CHD7 binding appears to be gene repression (Schnetz et al., 2010).

Heterozygous mutations in CHD7 cause CHARGE syndrome, a multiple anomaly congenital condition characterized by Coloboma of the eye, Heart defects, choanal Atresia, Retardation of growth, Genital abnormalities, and Ear abnormalities including deafness and vestibular dysfunction (Bosman et al., 2005; Vissers et al., 2004). Heterozygous Chd7 loss in the Chd7^Gt/+ mouse model of CHARGE syndrome recapitulates many of these hallmark clinical features, including ear abnormalities (Hurd et al., 2011). Chd7^Gt/+ adult animals exhibit malformations of the middle and inner ear, vestibular dysfunction evidenced by circling behaviors, as well as deficits in hearing as measured by both auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) (Hurd et al., 2011). Numerous studies have revealed a critical role for CHD7 in regulating the expression of genes required for proper vestibular and cochlear development.

Chd7 is highly expressed throughout the ventral and dorsal mouse otocyst at embryonic day 9.5 (E9.5) before becoming restricted to the neurogenic domain at E10.5 (Table 1) (Hurd et al., 2010). In mature mice, Chd7 expression is high throughout cochlear hair cells, spiral ganglion neurons, vestibular sensory epithelia, and middle ear ossicles (Hurd et al., 2011). Heterozygous loss of Chd7 disrupts expression of semicircular canal-associated genes (Bmp2, Bmp4, Msx1, and Fgf10), semicircular canal development, and vestibular nerve innervation (Adams et al., 2007; Hurd et al., 2012; Hurd et al., 2010).

Table 1.

Expression of Epigenetic Factors During Inner Ear Development

Function	Gene	E10.5	E15.5–16.5	Postnatal-Adult	References
DNA methyltransferase	Dnmt3a	Robust throughout otocyst	Not reported	Enriched in the organ of Corti	(Layman et al., 2013; Roellig and Bronner, 2016)
Zinc finger transcriptional regulator	Ctcf	Widely expressed throughout the otocyst and delaminating neuroblasts	Not reported	Not reported	(Shin et al., 2018)
Histone demethylase	Lsd1	Robustly expressed throughout otocyst	Not reported	Enriched in organ of Corti E18.5-P0 and subsequently down-regulated, up-regulated again by P7	(Layman et al., 2013; Patel et al., 2018)
Chromatin remodeler	Chd7	Restricted to the otocyst neurogenic domain, robustly expressed in delaminating neuroblasts	Expression persists in the cristae, CVG, organ of Corti	Highly expressed in vestibular and spiral ganglia neurons and hair cells	(Bosman et al., 2005; Hurd et al., 2011; Hurd et al., 2007; Hurd et al., 2010)
	Brg1	Widely expressed throughout the otocyst and delaminating neuroblasts	Hair cells, supporting cells, spiral ganglion neurons	Highly expressed in differentiating hair cells and supporting cells in the organ of Corti	(Jin et al., 2019; Xu et al., 2021)
	Cecr2	Not reported	Localized to longitudinal axis of cochlear duct by E18.5	Not reported	(Dawe et al., 2011; Kooistra et al., 2012)

Heterozygous or complete deletion of Chd7 in the developing otocyst disrupts formation of both the semicircular canals and vestibular sensory organs (Adams et al., 2007; Hurd et al., 2011; Hurd et al., 2012). These morphological disruptions are accompanied by reduced or absent expression of Otx1, Jagged1, Bmp2, Bmp4, Lmo4, Msx1, and Sox2, all of which play critical roles in regulating inner ear development (Hurd et al., 2012). Additionally, these disruptions in gene expression appear to occur in a regionally restricted manner, with genes expressed in the presumptive crista (Bmp4, Fgf10, Gata3, Jag1, Lmo4, Msx1, and Sox2) being downregulated in Chd7 conditional knockout tissue while genes in the dorsomedial otocyst are largely unaffected. The disruption of crista-expressed genes leads to malformation of the crista and ultimately results in improper development of the semicircular canals (Hurd et al., 2012). Artificial decreases in retinoic acid synthesis via citral can rescue the lateral semicircular canal phenotype evident in Chd7^Gt/+ mice, suggesting that the interaction between retinoic acid and CHD7 is critical for inner ear development (Micucci et al., 2014; Yao et al., 2018). Thus, CHD7 functions upstream of inner ear regulatory genes and in coordination with retinoic acid signaling in a dose-dependent and tissue-specific manner to modulate inner ear morphogenesis.

Loss of Chd7 throughout the developing otocyst has also been shown to result in severe defects in cochlear morphology. Foxg1Cre;Chd7^Gt/flox ears exhibit shortened and hypoplastic cochleae, suggesting that Chd7 is important for regulating cochlear duct extension (Hurd et al., 2010). CHD7 is also necessary for the proliferation of inner ear neuroblasts and regulation of neuronal cell fate specification. In addition to the changes in expression mentioned above, complete loss of Chd7 leads to decreased expression of patterning and pro-neural genes (Otx2, Fgf10, Neurog1, Neurod1 and Islet1) in the otic epithelium and cochleovestibular ganglion (Hurd et al., 2010). Chd7 is expressed throughout the subventricular zone in adult mice and loss of function mutations have been shown to produce defects in proliferation, self-renewal, and neurogenesis in both adult and neonatal mice (Micucci et al., 2014). Chd7 is essential for ensuring proper gross cochlear morphology and neuronal organization in the embryonic and early postnatal inner ear. As in the vestibular system, interactions between Chd7 and retinoic acid likely mediate proper development of the cochlea, since decreasing retinoic acid via citral treatment rescues the cochlear morphological defects observed with Chd7 loss (Yao et al., 2018).

Chd7 is essential for development of the mammalian inner ear (Fig 1, 2). CHD7 regulates gene expression by carrying out nucleosome repositioning activity at the enhancer and promoter regions of proneural genes, allowing for the accumulation of H3K4 methyl marks, which are associated with transcriptional regulation. CHD7-mediated transcriptional regulation plays early roles in patterning the neurogenic domain of the developing otocyst beginning at E10.5 (Hurd et al., 2010). Chd7 loss of function is associated with morphological changes within the neurogenic domain of the inner ear, specifically a significant decrease in proliferating and migrating neuroblasts and results in a smaller cochleovestibular ganglion (Hurd et al., 2010). As the ear develops, CHD7 regulates the expression of genes that are critical for proper development of the crista ampullaris in the vestibular system as well as extension of the cochlear duct as evidenced by the severely stunted and hypoplastic inner ears found in Chd7 conditional knockout mice (Hurd et al., 2010).

2.3.2. SWI/SNF Complexes in the Inner Ear

SWI/SNF chromatin remodeling complexes (also known as BRG1/BRM associated factor (BAF) complexes in mammals) are large, multi-subunit assemblies (~1–1.5 mDa) generated from the products of 29 genes working in combination (Mashtalir et al., 2018). Whereas CHD7 requires at least 40 bp of extranucleosomal DNA to efficiently bind and remodel nucleosomes, SWI/SNF enzymes are capable of remodeling nucleosome core substrates alone. SWI/SNF regulation of transcriptional activation has been implicated in several processes including DNA damage repair, tumor suppression, cell differentiation, and lineage specification. SWI/SNF complexes are highly enriched at enhancer and promoter regions along the genome and are essential for modulation of chromatin accessibility, modulating the ability of transcription factors to bind target genes (Mittal and Roberts, 2020). The subunit most essential for SWI/SNF complex chromatin remodeling activity is the ATPase, encoded by BRG1 (SMARCA4). BRG1 has been implicated in otocyst formation, inner ear neurogenesis, and maturation of vestibular and cochlear hair cells.

Brg1 is expressed as early as E8.5 throughout the otic placode and otocyst as well as in delaminating neurons and persists until roughly E10.5 (Table 1) (Xu et al., 2021). At this early development time point, the primary function of BRG1 appears to be regulating neurogenesis by activating expression of the key otocyst patterning genes Eya1 and Six1. Indeed, knockdown of BRG1 activity in the otocyst precluded both endogenous and Eya1/Six1 overexpression-induced ectopic neurogenesis, suggesting that SWI/SNF enzymatic complexes are necessary for proper EYA1/SIX1-mediated transcriptional activation of Neurog1 and Neurod1 (Ahmed et al., 2012). Based on these results, BRG1 is required to ensure transcriptional upregulation of the Eya1 and Six1 genes, both of which are necessary for neurogenesis and morphological development in the inner ear (Fig 2).

As the inner ear continues to develop, Brg1 is highly expressed in cochlear hair cells and supporting cells, a pattern that continues into adult stages. BRG1 plays important roles in maintaining hair cell polarity, anchoring outer hair cells to Deiters’ cells, and proper scar formation in the auditory epithelium (Jin et al., 2016). Conditional deletion of Brg1 at E14.5 in developing mouse hair cells causes significant hearing loss at P20 and profound deafness by P40 (Jin et al., 2016). In the same study, histological analysis of Atoh1Cre;-Brg1^−/− cochleae revealed progressive, basal-to-apical gradient of outer and inner hair cell loss over the postnatal period. Spiral ganglion neuron density considerably decreased by 4 months of age in Atoh1Cre;Brg1^−/− cochleae due to a lack of innervation targets following widespread hair cell death. Importantly, the cause of hair cell loss varies depending on developmental timepoint. Initial hair cell loss (from P8 to P14), is due to apoptosis as confirmed by the presence of Cleaved-Caspase3-positive outer hair cells. However, from P14 onward the method of hair cell death changes from apoptosis to necrosis due to hair cell cuticular plate loss and subsequent leakage of endolymph into the organ of Corti (Jin et al., 2016). BRG1 also functions in the maintenance of hair cell stereociliar orientation and organization, as Atoh1Cre;Brg1^−/− ears present with disrupted hair cell-intrinsic polarity accompanied by abnormal expression of stereocilia-related genes, including Gαi, mINSC, LGN, and aPKC (Jin et al., 2016). Widespread expression of Brg1 in the developing cochlea is critical for proper maintenance of hair cell survival (and, secondarily, spiral ganglion neuron survival) as well as stereocilia organization and orientation (Fig 2).

Similar to the cochlea, Brg1 deletion in the vestibular epithelium precedes vestibular hair cell death (Jin et al., 2019). Unlike the cochlea however, vestibular hair cell loss is observed in a regionally specific manner and over a much longer timespan. Hair cell death occurs throughout the striolar region of the utricle and saccule of 12-month-old Brg1 conditional knockout ears but there is no difference in hair cell number in the extrastriolar region relative to controls. Hair cell loss is also observed in the central, but not peripheral, region of the cristae. Much like SWI/SNF-mediated regulation of cochlear hair cells, BRG1 does not simply manage vestibular hair cell survival but also controls stereocilia organization and kinocilium orientation. Atoh1Cre;Brg1^−/− cristae hair cells exhibit stereocilia bundle fusion, with all of the stereocilia of a single hair cell fusing to form one or several “giant” stereocilia (Jin et al., 2019).

SWI/SNF complexes containing the BRG1 ATPase are initially widely expressed in the developing otocyst before becoming restricted to specific cochlear and vestibular structures. In adult mice, BRG1 is expressed in both hair cells and supporting cells throughout the inner ear and is critical for vestibular and cochlear hair cell survival. Additionally, BRG1 appears to be responsible for upregulating expression of genes that play important roles in initiating stereocilia formation and regulating kinocilium orientation in both vestibular and cochlear hair cells. The regulation of Eya1 and Six1 by BRG1 further implicates SWI/SNF complexes in neurogenesis pathways, as without transcriptional upregulation from the SWI/SNF ATPase, EYA1 and SIX1 are unable to adequately stimulate transcription of the basic helix-loop-helix factors Neurog1 and Neurod1.

2.3.3. CERF complex in the Inner ear

ISWI (Imitation Switch) protein complexes SNF2L and SNF2H in mammals are chromatin remodeling complexes encoded by Smarca1 and Smarca5 genes, respectively. The main functional subunits of ISWI chromatin remodelers are an N-terminal ATPase domain and a C-terminal HAND-SANT-SLIDE domain, which regulates extranucleosomal DNA binding, similar to the SANT-SLIDE unit in CHD remodelers (Goodwin and Picketts, 2018). In mammals, the SNF2H proteins are far more widely and abundantly expressed than SNF2L (Lazzaro and Picketts, 2001). There have been 5 novel ISWI complexes identified in mammals, but for the purpose of this review, we will focus on CECR2-containing remodeling factor (CERF) as CECR2 has been directly implicated in the development of the inner ear.

CECR2 is a chromatin remodeling protein that forms a CERF complex with the SNF2L ATPase (Banting et al., 2005). The CECR2 protein sequence contains many domains common to ISWI-associated factors. Among those domains are the DDT domain, AT-hook, and two bromodomains. The DDT domain is a putative DNA binding domain present in the vast majority of ISWI binding partners as well as other chromatin-associating factors (Doerks et al., 2001). Similar to the ARID1A/B DNA binding domains in SWI/SNF complexes, the DDT binding domain appears to deal with DNA damage; however, rather than initiating DNA repair, research has shown that DDT domains are critical for DNA damage tolerance (Noguchi et al., 2012). AT-Hook domains are also DNA binding sequences thought to produce a rigid surface that serves to tether CERF complexes to AT-rich regions of DNA (Liu and McKeehan, 2002).

At E18.5, Cecr2 expression is localized along the longitudinal axis of the cochlear duct, specifically concentrated within the medial cochlear floor (Dawe et al., 2011; Kooistra et al., 2012). Mutations in Cecr2 have been shown to result in inner defects and inner ears of homozygous Cecr2^Gt45Bic mice dissected at E18.5 revealed shortened and widened cochlear ducts along with significant decreases in overall size of the inner ear (Dawe et al., 2011). This phenotype is similar to, though markedly less severe than, those noted in Chd7 conditional knockout mice at similar ages (Hurd et al., 2010). Both hypomorphic (Cecr2^Gt45Bic) and loss of function (Cecr2^{tm1. 1Hemc}) homozygous mutations are associated with an increased number of improperly aligned cochlear outer hair cells. Heterozygous mutations in both genotypes produce an intermediate hair cell disorganization phenotype (Dawe et al., 2011). Both homozygous mutation genotypes exhibit a severity gradient along the length of the cochlea, with the more mature basal hair cells presenting with less misalignment than the relatively immature apical sensory cells. The inner hair cells of all mutant ears show no significant phenotype. In addition to defects in hair cell organization along the cochlear epithelium, both hypomorphic and deletion mutations of Cecr2 also result in defects in stereocilia bundle orientation along with previously reported exencephaly phenotypes. Although these inner ear defects are reminiscent of those seen in mice with planar cell polarity (PCP) mutations, microarray analysis did not reveal any significant interaction between Cecr2 and the PCP pathway (Dawe et al., 2011).

3. Crosstalk Between Epigenetic Mechanisms

While DNA methylation, histone modifications, and chromatin remodeling are each important for proper development, coordination between these mechanisms is also essential for developmental gene regulation, but is not well understood (Bird, 2002; Cedar and Bergman, 2009; Jobe et al., 2012). Below we describe two examples of interactions between DNA methylation and histone modification that appear to contribute to normal ear development.

Several proteins function to mediate the link between DNA methylation and histone modifications, including methyl CpG binding protein 2 (MeCP2) (Bird, 2002). MeCP2 contains a methylated DNA binding domain that targets methylated promoters and recruits histone deacetylases to mediate transcriptional repression (Jones et al., 1998; Nan et al., 1998). Pathogenic variants in MECP2 are the main cause of Rett syndrome, a dominant X-linked neurodevelopmental disorder that can be associated with sensorineural hearing loss (Ellaway and Christdoulou, 1999; Pillion et al., 2003). Despite the robust expression of MeCP2 in the developing spiral ganglion (Diez-Roux et al., 2011), its role in regulating inner ear gene expression has not been determined.

DNA methyltransferase DNMT3L also serves as a link between DNA methylation and histone modification. DNMT3A complexes with the C-terminus of DNMT3L, a closely related family member that lacks DNA methyltransferase activity (Bourc’his et al., 2001). The N-terminus of DNMT3L directly binds H3K4, but only in its unmethylated state, thus serving as an inhibitor of DNA methylation at CpG sites where H3K4me is located (Jia et al., 2007; Ooi et al., 2007). Despite numerous studies that demonstrate an inverse correlation between DNA methylation and H3K4me in neural development, it is unknown whether DNMT3L serves as an epigenetic crosstalk mediator in inner ear development (Meissner et al., 2008; Mohn et al., 2008).

4. New Areas of Investigation

We have highlighted the critical importance of epigenetic regulation for proper inner ear development. However, the temporal and spatial regulation of expression and function of these epigenetic factors have not been fully defined. Otocyst patterning (the neurogenic region in particular) relies on activation of key otic genes via the epigenetic modifiers DNMT3A, LSD1, CHD7, BRG1, and CECR2. While these modifiers perform diverse biochemical functions, they are nearly ubiquitously expressed in very early embryonic development and gradually become restricted to specific domains and cell types. Understanding this regulation will have substantial impact on development of new therapies to restore hearing and balance in congenital disorders affecting the inner ear.

As single cell technologies improve in accessibility, cost efficiency, and optimization for low cell numbers, researchers can profile the epigenetic landscape of nearly every cell of the developing inner ear. This technology is helping to uncover complex transcriptional programs that govern inner ear development and define how they are epigenetically orchestrated. For example, recent studies revealed the transcriptional diversity of Type I spiral ganglion neurons in the postnatal mouse cochlea and showed that the diversification of neuronal subtypes is at least in part due to physiological activity (Shrestha et al., 2018; Sun et al., 2018). Given the critical roles epigenetic factors play in inner ear neural development, particularly CHD7 and BRG1, it seems likely that subtype delineation of spiral ganglion neurons and many other cell types is driven by epigenetic regulation.

Restoration of hearing and balance in cases of trauma, aging, or abnormal is a motivating factor for exploring genetic and epigenetic mechanisms of inner ear development. Transdifferentiation of supporting cells into hair cells is one promising therapeutic approach for regeneration of damaged or missing cochlear hair cells. A recent study characterized the epigenetic landscape of hair cells and supporting cells through early postnatal mouse development and demonstrated an enhancer decommissioning phenomenon in cochlear supporting cells (Tao et al., 2021). In this study, enhancers of key hair cell genes were primed by H3K4 mono-methylation while their respective promoters were poised by H3K4 tri-methylation. These same hair cell-specific genes were silenced by H3K27 tri-methylation in supporting cells. Replacement of H3K27ac with H3K27me3 resulted in supporting cell transdifferentiation into cochlear hair cells. This study illustrates how epigenetic transitions that drive neurosensory cell type specification and differentiation can be subverted to replace cell populations in the postnatal inner ear.

5. Conclusions

Over the past several years, we have witnessed major advances in research exploring epigenetic regulators and their functions in development of the inner ear. Orchestrators of DNA methylation, histone modification, and chromatin remodeling are all required to ensure appropriate patterning of the early otocyst then later specification, differentiation, and maturation of neurosensory cells in the vestibular and auditory components of the inner ear. As additional new and emerging technologies are applied to the inner ear, we will gain even greater understanding of the epigenetic and transcriptional transitions that occur during the development of this marvelously complex structure.