Chromodomain Helicase DNA Binding Protein 4 in Cell Fate Decisions

Spiral ganglion neurons (SGNs) relay acoustic information from sensory hair cells to central auditory circuits. Ototoxic damage, loud noise exposure, viral infections, genetic mutations, and aging can result in SGN loss. The inability to regenerate SGNs leads to hearing loss. Hearing loss impacts the quality of life and imposes a significant socioeconomic burden on over 1.57 billion people worldwide (McDaid et al., 2021). Current treatments for individuals with hearing loss are limited. Hearing aids and cochlear implants require sufficient numbers of functional SGNs to augment existing auditory function but fall short of providing permanent cures achievable through restorative efforts. Strategies to regenerate, repair, and replace SGNs will inform on appropriate treatments for a broad spectrum of hearing loss.

To replace lost SGNs, two different strategies have been proposed: otic progenitor transplantation and lineage conversion of resident cells (Roehm and Hansen, 2005; Wang et al., 2021; Zhang et al., 2021). Otic progenitor transplantation uses directed differentiation of induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells by adding different growth factors in the medium to guide stem cells into otic progenitors. Engraftment and differentiation of otic progenitors into SGNs necessitate neuronal differentiation, axonogenesis, axon guidance, and synaptogenesis to replace lost SGNs and reform the auditory circuit. ES cell-derived neural progenitors that underwent neuronal differentiation can innervate hair cells in chemically denervated cochlear explants (Martinez-Monedero et al., 2006; Shi et al., 2007). Engraftment of otic neuronal progenitors into ouabain denervated gerbil cochleae in vivo showed neurite outgrowth and fasciculating nerve bundles that project through Rosenthal's canal into the osseous spiral lamina to synapse with cochlear hair cells (Corrales et al., 2006). Using an auditory neuropathy model, engrafted otic neural progenitors were shown to differentiate into neurons and innervate hair cells and the cochlear nucleus. Repopulating lost SGNs partially restored auditory-evoked responses in the animals (Chen et al., 2012). These studies demonstrate the feasibility of SGN replacement using stem cell-derived otic progenitors.

In contrast to directed differentiation, lineage conversion or transdifferentiation does not require cells to transition through an intermediate cell state. Instead, the process converts one cell type directly into another. Converting somatic cells into neurons can be achieved by overexpressing neurogenic transcription factors (Pang et al., 2011; Vierbuchen et al., 2010). Expression of neurogenic transcription factors, Ascl1 and Neurod1, converted non-neuronal cells from the early postnatal cochlea into induced neuronal cells. The nascent neurons harbored transcriptional profiles similar to SGNs and extended projections toward cochlear hair cells and cochlear nucleus neurons (Noda et al., 2018). Similarly, the inducible expression of neurogenic transcription factors, Neurog1 and Neurod1, converted a subset of neonatal cochlear glia to express SGN markers and harbor transcriptomes similar to type I SGNs (Li et al., 2020). Clarifying the molecular processes that promote or prevent SGN regeneration will improve regeneration efficiency, inform the strategies for functional recovery, and accelerate regenerative efforts.

Molecular processes that alter gene expression to facilitate cell fate changes are the core of directed differentiation and lineage conversion. Evidence from different systems suggests that epigenetic modifications can regulate gene expression to alter cell identity (Chen and Dent, 2014). Although the literature on epigenetic changes during auditory regeneration is sparse, changes in the epigenetic landscape during ES cell differentiation and reprogramming iPSCs are well-established (Brumbaugh et al., 2019; Ho and Crabtree, 2010). This molecular framework provides a basis for altering the epigenome to regenerate SGNs.

Nuclear DNA in eukaryotes wraps ∼147 base pairs of DNA around a histone octamer to form a nucleosome, the fundamental repeating unit of chromatin. Packing DNA into chromatin allows the entire genome to fit in the nucleus but restricts access to macromolecular complexes that bind DNA. Making chromatin accessible enables protein complexes to bind DNA and catalyze molecular processes (Kouzarides, 2007). Covalent modifications of histones correlate with different chromatin states. Histone readers, writers, and erasers are enzymes that recognize and alter histone modifications to change chromatin status. In parallel with histone modification, chromatin remodeling enzymes that mobilize, evict, or rearrange nucleosomes function can control chromatin accessibility. Changes in histone modifications or chromatin accessibility at cis-regulatory regions, such as promoters and enhancers, can be used to regulate gene expression.

As embryonic development proceeds, cells progressively restrict their identities to acquire a specialized fate. Cell identity was initially thought to be unalterable in mature animals, but studies have shown that cell identity is malleable. Observations from reprogramming or lineage conversion suggest that somatic cells maintain their epigenetic landscape to preserve cell identity (Brumbaugh et al., 2019), while epigenetic modifications alter their cell identity (Vierbuchen and Wernig, 2011). Such changes were exemplified by reprogramming somatic cells into induced pluripotent stem cells that exhibit epigenetic states and gene expression profiles highly similar to embryonic stem cells (Mikkelsen et al., 2008). Similarly, comparing the epigenome of pluripotent stem cells to differentiated cells from different tissues implied dramatic changes in histone modifications during development (Hawkins et al., 2010; Mikkelsen et al., 2007). These studies suggest that reorganizing the epigenome facilitates cell fate changes.

Understanding epigenetic landscape changes during inner ear development or regeneration provides insight into cell-based hearing restoration. Epigenetic factors in the inner ear are challenging to identify due to the limited cell numbers and heterogeneity of cell types. Studies of congenital hearing loss have identified pathogenic variants in genes that code for histone modifying and chromatin remodeling proteins. Pathogenic variants in DNA methyltransferases (DNMT1), histone methyltransferases (EHMT1, KMT2D), acetyltransferase (KAT6B), and chromatin remodelers were some of the genetic alterations implicated in hearing loss (Layman and Zuo, 2014). Members of the Chromodomain Helicase DNA Binding Protein (CHD) family, CHD4 and CHD7, were also associated with syndromic hearing loss (Clapier et al., 2017; Micucci et al., 2015). CHD4 is noteworthy since it biochemically interacts with several other histone modifiers (Sharifi Tabar et al., 2022; Torrado et al., 2017). CHD4, with other epigenetic modifiers, can drastically alter the chromatin state, affect gene expression, and ultimately impact cell fate.

Murine cochlear supporting cells and hair cells expressed CHD4 during cochlear development from embryonic day (E) 18 to postnatal day (P) 21 (Layman et al., 2013). Gene expression analysis showed that CHD4 transcripts were present and dynamically changing in developing murine SGNs from E12 to P15 (Lu et al., 2011). In addition to CHD4 expression in SGNs, immunostaining showed the presence of CHD4 in non-neuronal cells that reside near SGNs. Immunofluorescent images displayed strong CHD4 expression in SGNs but not non-neuronal cells at P0. By P28, both non-neuronal cells and SGNs showed robust CHD4 expression (Fig. 1). The presence of CHD4 in SGNs and non-neuronal cells suggested that it could play a role in the development, maturation, or maintenance of these cell types. Although the function of CHD4 in the inner ear has not been directly tested, its physiological function was gleaned from missense mutations, truncating variants, and an in-frame deletion identified from de novo CHD4 heterozygous mutations. These pathogenic variants in CHD4 were identified in patients with Sifrim-Hitz-Weiss syndrome (SIHIWES) (Sifrim et al., 2016; Weiss et al., 2016). SIHIWES is an intellectual developmental disorder with variable congenital anomalies, including short stature, enlarged head circumference, non-specific dysmorphic facial features, and hearing loss (Sifrim et al., 2016; Weiss et al., 2016). The expression of CHD4 in cochlear cell types and identified genetic variants that cause hearing loss in SIHIWES patients suggest that CHD4 may have a critical cellular function in the inner ear.

Understanding how genetic variants in the human CHD4 gene could cause hearing loss provides insight into disease pathogenesis. CHD4 codes for a large polypeptide containing a high mobility group (HMG)-like domain in the N-terminus, a pair of plant homeodomain (PHD), two chromodomains, a central SNF2-like ATPase motif, two domains of unknown function (DUF 1086 and 1087) and a C-terminal CHDCT2 domain (Fig. 2A). A cryoelectron microscopy structure of human CHD4 protein bound to the nucleosome core allowed mapping of amino acid changes inferred from human disease mutations (Kovac et al., 2018; Sifrim et al., 2016; Weiss et al., 2016). Some missense mutations in the PHD2 and SNF2-like ATPase domains were identified (Farnung et al., 2020). A more comprehensive study showed that alterations in the SNF2-like ATPase domain, PHD2, chromodomain, or DUF1087 decreased chromatin remodeling activity. Many genetic alterations tested in the SNF2-ATPase domains correlated to SIHIWES patients with detectable conductive and sensorineural hearing loss (Weiss et al., 2020). These studies implicate that decreased CHD4 chromatin remodeling activity may contribute to hearing loss.

Like many chromatin remodelers, the conserved SNF2-like ATPase core in CHD4 facilitates ATP-dependent DNA translocation along the chromatin. Some variants identified in patients with SIHIWES disrupted CHD4 ATPase activity and chromatin remodeling activity (Weiss et al., 2020). When bound to chromatin, the SNF2-like ATPase domain of CHD4 catalyzed the ATP-dependent mobilization of nucleosomes along DNA (Morra et al., 2012; Ramirez et al., 2012; Watson et al., 2012). The translocation of CHD4 disrupted electrostatic interactions between the DNA and histone octamers (Zhong et al., 2020). Structural studies suggested that CHD4 can recognize different multivalent histone marks using the PHD domains (Musselman et al., 2012), facilitate nucleosome binding using the chromodomain, and reposition nucleosomes with the SNF2-like ATPase domain (Farnung et al., 2020). The enzymatic activities of CHD4 could alter the chromatin state to affect gene expression. The physiological consequences of CHD4 are likely context-dependent and determined by the protein complex composition, association with specific histone modifications, and recruitment to defined DNA sequences.

CHD4 is a well-established subunit in the Nucleosome Remodeling and Deacetylase (NuRD) complex (Tong et al., 1998; Xue et al., 1998; Zhang et al., 1998). The core NuRD complex harbors six protein subunits (Millard et al., 2016; Torchy et al., 2015). Paralogous proteins biochemically interacted to form distinct NuRD complexes. The ATP-dependent nucleosome remodeler CHD4 and histone deacetylases (HDAC) comprise the catalytic subunits of the NuRD complex. Other members included the Methyl CpG Binding Domain (MBD) proteins and structural proteins Metastasis Associated (MTA), GATA Zinc Finger Domain Containing 2 (GATAD2), and RB Binding Protein (RBBP) paralogs (Torrado et al., 2017) (Fig. 2B). NuRD complexes with individual CHD paralogs (CHD3, CHD4, and CHD5) have distinct functions. In brain development, NuRD complexes with CHD4 promoted progenitor proliferation, while CHD5 and CHD3 were involved in neuronal migration and cortical layer specification. Genetic ablation or shRNA knockdown of each CHD caused unique defects that could not be rescued by the remaining paralogous CHDs (Nitarska et al., 2016). CHD4-containing NuRD complexes in the inner ear may contain distinct catalytic, methyl-binding, and structural subunits.

The NuRD complex acts mainly, but not exclusively, as a transcriptional repressor. In ES cells, CHD4-containing NuRD complexes bound to promoters and enhancers with distinct histone marks to maintain the pluripotency network (Hu and Wade, 2012). The NuRD complex was localized to sites of active transcription by histone readers MBD2/3, while CHD4 repositioned nucleosomes. Other subunits of the NuRD complex, such as HDAC1/2, deacetylated histones to repress transcription (Morra et al., 2012; Watson et al., 2012). CHD4 function affected gene expression and had profound cellular effects. CHD4 maintained self-renewal and pluripotency of ES cells by repressing endoderm-associated genes such as Tbx3 (Zhao et al., 2017). CHD4 also played a role in neural differentiation in ES cells by suppressing p53 levels to regulate neural gene expression (Hirota et al., 2019). These findings suggest that CHD4 alters chromatin status at promoters and enhancers to influence transcriptional regulatory networks.

In addition to promoters and enhancers, genome-wide chromatin occupancy studies showed that CHD4 binds to thousands of other sites in the mammalian genome. The differences in subunit composition likely recruit protein complexes to distinct chromatin regions and confer different enzymatic activities. Indeed, a CHD4 protein complex, distinct from NuRD, was identified. Activity-Dependent Neuroprotector Homeobox (ADNP), a transcription factor that interacted with CHD4 and the architectural heterochromatin protein 1 (HP1), formed a stable complex known as ChAHP (CHD4, ADNP, HP1) (Ostapcuk et al., 2018). Genome-wide binding of ADNP, the DNA binding subunit, suggested that ChAHP perturbs CCCTC-binding factor (CTCF) binding. In ES cells, CTCF is a zinc finger protein that binds to insulators, cis-regulatory elements that serve as barriers between chromatin domains. The joining of two CTCF-bound sites formed a chromatin loop and established a topologically associating domain (TAD) (Merkenschlager and Nora, 2016). Deletion of ADNP altered long and short-range chromatin contacts in TADs (Kaaij et al., 2019). ES cells lacking ADNP showed precocious expression of lineage-specifying genes, spontaneously differentiated, and failed to differentiate toward a neuronal lineage (Ostapcuk et al., 2018). These results suggest that the ChAHP complex may regulate gene expression and cell fate decisions by maintaining and establishing TADs.

To directly test the effects of CHD4 on 3D chromatin structure, genetic ablation of CHD4 in cerebellar granule cells was done. Genome architecture profiling demonstrated that CHD4 conditional knockout cells allowed precocious interactions between contact domains and chromatin loops to alter TADs and perturb gene expression (Goodman et al., 2020). Depleting CHD4 in ES cells using an inducible degron system provided mechanistic insight into CHD4 function. Lack of CHD4 disrupted TAD organization due to aberrant CTCF binding and increased chromatin loop contacts. The proposed mechanism suggested that CHD4 maintained TADs by increasing chromatin accessibility at aberrant CTCF sites to allow deposition of the repressive histone mark H3K9me3. H3K9me3 concealed these sites to prevent aberrant CTCF binding (Han et al., 2021). These results suggest that CHD4 organizes 3D chromatin architecture to regulate gene expression. Perturbed TADs may affect chromatin loop formation to alter gene expression (Fig. 2C). The studies implicate CHD4 in regulating gene expression by employing different molecular mechanisms.

CHD4 is involved in diverse cellular processes, including establishing cell identity, DNA damage repair, cell cycle progression, and cancer (Bornelov et al., 2018; Hosokawa et al., 2013; Hung et al., 2012; Pan et al., 2012; Polo et al., 2010; Xia et al., 2017; Yang et al., 2016). Investigating CHD4 function during cell fate transitions has provided mechanistic insight. Knockout of murine CHD4 implicated its role in early fate decisions during embryogenesis. Zygotes undergoing symmetrical cell division maintained the same cell fate in pre-implantation embryos. At the 32-cell stage, the developing embryo formed a blastocyst composed of trophectoderm, a single epithelial layer surrounding a fluid-filled cavity, and the inner cell mass. The commitment to become trophoectoderm was the first cell fate decision made by the developing embryo. Cells in the blastocysts lacking CHD4 could not make the initial cell fate decision to form functional trophoectoderm. CHD4 influenced the frequency of unspecified blastocyst cells to express lineage markers before making the first cell fate decision. The absence of CHD4 increased aberrant lineage marker expression in 16-cell stage embryos. By the blastocyst stage, these cells failed to adopt an appropriate trophoectoderm gene expression pattern (O'Shaughnessy-Kirwan et al., 2015). CHD4 guided cell fate decisions in vivo by preventing inappropriate expression of alternative lineage-specifying genes while allowing trophoectoderm gene expression.

Lineage commitment during B-cell development also implicated CHD4. Sequential induction of lineage-specifying transcription factors and signaling pathways advanced B-cell differentiation. CHD4 suppressed transcriptional programs that promoted alternative cell fates, while other transcriptional regulatory events encouraged B-cell precursor survival, expansion, and differentiation. Deletion of CHD4 arrested differentiation and derepressed transcription of genes not associated with B-cell development. CHD4 activity repressed alternative lineage-specific transcriptional regulatory networks and prevented the adoption of alternative cell lineages (Yoshida et al., 2019). The studies showed that CHD4 facilitates differentiation by preventing alternative cell fates through transcriptional repression.

Muscle regeneration, a process that requires cell fate changes, also implicated CHD4. Resident progenitors called satellite cells can repopulate muscle cells after injury. Muscle cells failed to regenerate after CHD4 conditional ablation in satellite cells because of increased satellite cell plasticity and lineage infidelity. The cellular changes in the absence of CHD4 derepressed transcription of non-muscle cell lineage genes (Sreenivasan et al., 2021). Together the studies implicate CHD4 in guiding cell fate decisions during development and regeneration by suppressing the expression of alternative lineage-specifying genes.

CHD4 binding at distinct chromatin regions has not been investigated in cochlear cell types. CHD4 expression was shown in different cell types in the cochlear sensory epithelium (Layman et al., 2013) and the spiral ganglion (Fig. 1). The small cell numbers obtained from individual cochlea make identifying CHD4 binding sites challenging, while distinct CHD4 binding in different cochlear cell types may confound interpretation. Cell fate changes during inner ear development or regeneration provided insights into the underlying epigenetic changes. Some epigenetic changes observed in inner ear cell types align well with CHD4 function and deserve discussion.

In the mature cochlea, regeneration is severely limited. After an acoustic injury in adult animals, acute loss of synapses with inner hair cells and gradual loss of SGNs were observed (Kujawa and Liberman, 2006, 2009; Lin et al., 2011). The inability to regain a full complement of SGNs suggested limited regeneration after acoustic damage. Similarly, killing hair cells in the mature cochlea did not reveal observable spontaneous regeneration (Oesterle et al., 2008). In neonatal mice, genetic ablation of newborn hair cells allowed the spontaneous conversion of supporting cells into hair cells through mitotic proliferation and direct transdifferentiation. By one week of age, the animals lost the ability to spontaneously regenerate hair cells (Cox et al., 2014). Probing the molecular changes in supporting cells during this period suggested that epigenetic changes at enhancers prevented regeneration (Tao et al., 2021). In the cochlea, satellite and Schwann cells are glia that serve as potential cell pools for neuronal regeneration (Jessen and Mirsky, 2005; McLean et al., 2016; Meas et al., 2018; Wan and Corfas, 2017). The conversion efficiency of the glial population declined with age. Lineage conversion of glia into SGN-like cells occurred in perinatal but not in 2-week-old cochleae (Li et al., 2020). Epigenetic changes as glia matured likely prevented conversion.

Taken together, supporting cells in the cochlea epithelium or glia in the spiral ganglion may serve as a cell source for regeneration. The inability to convert cells in the mature cochlea is a significant roadblock to regeneration. Active mechanisms that maintain cell fate likely prevent the conversion of supporting cells or glia. Inhibiting the molecules that safeguard cell fate could render cells amenable to conversion. In addition to overcoming mechanisms that maintain cell fate, introducing factors that specify cell fate, and prevent alternative lineages may facilitate lineage conversion. After generating nascent cells, reinstating the cellular process that maintains cell fate may help avoid additional changes (Fig. 2D). Controlling cis-regulatory elements to modulate these events could serve as a strategy for regeneration.

Among the cis-regulatory elements, enhancers drive cell-specific gene expression programs to specify cell fates (Ong and Corces, 2012). The number of active enhancers for each cell type has been estimated to be in the thousands (Heintzman et al., 2009). Large clusters of enhancers, known as super-enhancers, define cell identity by promoting cell type-specific gene expression (Hnisz et al., 2013; Whyte et al., 2013). Transcription factor binding at enhancers governs cell-specific gene expression patterns and generates different cell types (Bulger and Groudine, 2011; Maston et al., 2006). Increasing chromatin accessibility through pioneer transcription factor binding initiates enhancer activation (Adams and Workman, 1995; Boyes and Felsenfeld, 1996). The increased chromatin accessibility allows the recruitment of other transcription factors and protein complexes to activate enhancers. Enhancer activation ultimately enables a cell to change gene expression and acquire the transcriptome of the desired cell type. Conversely, silencing a different set of enhancers may prevent alternative cell fates.

The age-dependent ability of cochlear supporting cells to transdifferentiate provided a paradigm for probing the epigenetic changes at enhancers in supporting cells (Cox et al., 2014). Hair cell-specific enhancers were identified as chromatin-accessible regions bound by ATOH1 in FACS-purified P1 hair cells. Hair-cell enhancers harbored different histone marks in hair cells and supporting cells. H3K27ac and H3K4me1 marked the active enhancers in P1 hair cells. In contrast, the same enhancers in P1 supporting cells only harbored H3K4me1 marks (Tao et al., 2021). The presence of only H3K4me1 corresponded to a primed enhancer state (Calo and Wysocka, 2013; Heinz et al., 2015), which allowed low-level expression of hair cell genes and permitted supporting cells to transdifferentiate into hair cells. As supporting cells matured, the hair cell-specific enhancers were decommissioned by losing H3K4me1 marks (Tao et al., 2021). The study highlighted that decommissioning enhancers is an epigenetic roadblock that prevents supporting cell transdifferentiation.

The Lysine-Specfic Demethylase (LSD1) may be a candidate that decommissions enhancers in supporting cells. LSD1 was associated with the NuRD complex and was essential for decommissioning enhancers in differentiating ES cells (Whyte et al., 2012). In supporting cells, the NuRD complex may recruit LSD1 to remove the methyl marks on histones (H3K4me1) associated with primed enhancers. Inhibiting H3K4me1 demethylation using an LSD1-specific inhibitor extended the transdifferentiation potential of perinatal supporting cells (Tao et al., 2021). The NuRD complex with LSD1 may function to decommission hair-cell enhancers in supporting cells.

Epigenetic changes during hair cell development provided insight into how active enhancers can guide cell fate transition (Yu et al., 2021). In the cochlea, sensory progenitor cells initially express ATOH1, but most become supporting cells, while only some differentiate into hair cells (Driver et al., 2013; Yang et al., 2010). ATOH1 is an essential transcription factor for hair cell differentiation and survival (Bermingham et al., 1999; Woods et al., 2004; Zheng and Gao, 2000). In sensory progenitors, ATOH1 was unable to bind to its enhancers because of the inaccessible chromatin state. The inability of ATOH1 to bind to its cognate enhancers prevented hair cell differentiation. POU4F3, a transcription factor required for auditory and vestibular hair cell development, increased chromatin accessibility as a pioneer factor to allow ATOH1 binding (Xiang et al., 1997). POU4F3 and ATOH1 were pioneer and cell lineage-determining transcription factors that activated the hair cell-specific enhancer network, directed cell-specific gene expression, and conferred hair cell identity (Yu et al., 2021). The study highlights that changing chromatin accessibility can activate specific enhancers to facilitate differentiation.

In parallel, there are likely molecular processes that suppress enhancers during sensory progenitor development. Repressing supporting-cell enhancer networks may help reinforce hair cell identity. In contrast, supporting cells may suppress the hair-cell enhancer network to maintain cell identity. CHD4 expression was observed in hair cells and supporting cells (Layman et al., 2013) and likely works with other chromatin-remodeling proteins during inner ear development (Chohra et al., 2023). CHD4 may help maintain cell identity by repressing enhancer networks for alternative cell types.

Similar to hair cell development and supporting cell transdifferentiation, transcription factor-mediated conversion of somatic cells into hair cells likely employs the same principles. Pioneer and lineage-determining factors facilitate cell fate conversion, while other proteins may reinforce cell identity and suppress alternative cell fates. Hair cell conversion from different cell types by expressing different combinations of ATOH1, POU4F3, and GFI1 is nicely summarized (Iyer and Groves, 2021). Here we discuss the epigenetic changes at enhancers to facilitate cell fate transitions for inner ear cell types.

Initial reports suggested the combination of ATOH, POU4F3, and GFI1 formed a core group of transcription factors necessary for hair cell conversion (Costa and Henrique, 2015; Costa et al., 2015). Additional transcription factors such as SIX1 increased the efficiency of converting mouse embryonic fibroblasts, adult tail-tip fibroblasts, and postnatal supporting cells into induced hair cells. The induced hair cells displayed morphology, transcriptomic and epigenetic profiles, electrophysiological properties, and hair-cell transduction channel expression (Menendez et al., 2020).  ATOH1 and POU4F3 may activate hair cell-specific enhancers in embryonic fibroblasts during lineage conversion, similar to hair cell development or supporting cell transdifferentiation. Activation of hair cell enhancers allows fibroblasts to acquire gene expression profiles and hair cell properties.

In addition to activating enhancers, an active mechanism that suppresses the originating cell identity or inhibits alternative fates may be necessary. GFI1 was first identified as a transcription factor for hair cell differentiation and survival (Wallis et al., 2003). GFI1 functioned as a transcriptional repressor, and ablation of GFI1 resulted in ectopic neuronal gene expression (Jen et al., 2022; Matern et al., 2020). During lineage conversion of inner ear cell types into hair cells, GFI1 may repress gene expression programs corresponding to alternative cell identities. Adenoviral delivery and overexpression of ATOH1 and GFI1 in neonatal cochlear explants showed significantly more hair cells than ATOH1 alone. Ectopic hair cell-like cells emerged from regions medial to inner hair cells and in the stria vascularis. In vivo, expression of both ATOH1 and GFI1 in adult mice cochleae increased the number of hair cell-like cells over 6-fold compared to ATOH1 alone (Lee et al., 2020). The study suggests that transcriptional repression by GFI1 in specific inner ear cell types facilitates ATOH1-mediated hair cell conversion.

Whether GFI1 interacts with the NuRD complex during hair cell conversion has not been established, but there may be a potential biochemical interaction. In myeloid progenitors, GFI1 interacted with CHD4 and other subunits of the NuRD complex. GFI1 recruited CHD4 complexes to open chromatin sites to repress transcription (Helness et al., 2021). GFI1 and the NuRD complex may reinforce hair cell identity during lineage conversion by silencing alternative gene expression programs.

From these studies, two key molecular processes were crucial for cell type conversion, enhancer network activation, and alternative cell fate suppression. POU4F3 activity and ATOH1 binding activate hair cell enhancers for hair cell conversion. In some instances, the enhancers may already be accessible in the originating cell type, thus making the pioneer factor dispensable. ATOH1 can then bind to these enhancers and promote the expression of hair cell genes. GFI1 may prevent newly-converted hair cells from assuming their original or alternative cell identities. Together these molecular processes may contribute to efficient lineage conversion.

Using the guiding principles gleaned from hair cell studies, activating SGN enhancers along with repressing alternative cell type enhancers may contribute to generating new SGNs. For in vivo SGN conversion, glia that reside next to SGNs are good cellular targets for regeneration (Chen et al., 2021; Li et al., 2020). Glia such as Schwann and satellite cells are neural crest-derived, while SGNs arise from the otic epithelium (Fritzsch et al., 2015; Groves et al., 2013; Kelley, 2006; Ritter and Martin, 2019). The epigenetic landscape of the glia and SGNs may be very different because they originate from separate cell populations. The active enhancers are likely unique for each cell type and help define the transcriptome for glia or SGNs. The glial enhancers may be active, while SGN enhancers are inactive in glia. Lineage conversion of glia into SGNs may require pioneer and lineage-specifying transcription factors to activate SGN enhancers and other factors to decommission glial enhancers.

Conversion of non-neuronal cells in the spiral ganglion into SGN-like cells used pioneer factors such as ASCL1 and NEUROD1. The converted non-neuronal cells displayed transcriptomes and electrophysiological properties similar to SGNs (Nishimura et al., 2014; Noda et al., 2018). ASCL1 was initially identified as a potent neurogenic transcription factor that converted fibroblasts into neurons (Pang et al., 2011; Vierbuchen et al., 2010). ASCL1 functioned as a pioneer factor that occupied sequence-specific sites with a constellation of histone marks and recruited other transcription factors to enable lineage conversion (Wapinski et al., 2013). NEUROD1 also served as a pioneer factor when converting ES cells into neurons. In ES cells, expression of NEUROD1 displaced MBD3, a component of the NuRD complex, and conferred stable epigenetic changes that activated enhancers. NEUROD1 bound enhancers displayed increased chromatin accessibility, loss repressive histone marks (H3K27me3), and gained primed (H3K4me1) and active (H3K27ac) marks (Pataskar et al., 2016). The results suggested that the NuRD complex in ES cells repressed neuronal enhancers. The expression of NEUROD1 displaced the NuRD complex and activated the neuronal enhancers during conversion. Expression of ASCL1 or NEUROD1 may activate neuronal enhancers in non-neuronal cells, encourage neuron-specific gene expression programs, and help specify neuronal identity.

Neuronal lineage conversion also used other transcription factor combinations. In the developing cochlea, NEUROG1 and NEUROD1 were required for SGN development (Liu et al., 2000; Ma et al., 2000). Inducible NEUROG1 and NEUROD1 expression in neonatal cochlear glia employed a glial-specific Cre driver (Plp CreER) and a transgene containing a floxed STOP cassette between a strong promoter and the NEUROG1 and NEUROD1 coding sequences. Glia-derived nascent SGN-like cells expressed SGN markers, displayed transcriptomes similar to type I SGNs, and showed decreased expression of glial cell markers (Li et al., 2020). For lineage conversion, NEUROG1 likely served as a lineage-specifying transcription factor, while NEUROD1 functioned as a pioneer factor to activate gene expression networks that recapitulated SGN-like cell identity.

Two processes were notable in transcription factor-mediated conversion of cochlear glia: repression of glial marker expression in SGN-like cells and the inability to convert glia in one-month-old animals (Li et al., 2020). Although these two processes may not be mutually exclusive, the observations suggested one mechanism to repress glial gene expression in newly converted SGN-like cells in perinates and a second mechanism to maintain glial fate by suppressing alternative cell identities in older animals.

Since perinatal glia were competent for conversion, neuronal enhancers may reside in a primed state permissible for activation by NEUROG1 and NEUROD1. The downregulation of glial gene expression may be due to an intrinsic transcriptional repressive activity. In mature glia, the same neuronal enhancers may be decommissioned to maintain glial cell identity and thus prevent lineage conversion. Suppressing mechanisms safeguarding glial fate may make a cell permissible to lineage conversion. Upon completion of neuronal differentiation, processes that maintain nascent SGN identity may be required to prevent further cell fate changes. Re-establishing mechanisms that safeguard cell identity may help the long-term survival of newly converted cells. Understanding the two transcriptional repressive processes and whether they employ the same molecules to function may provide insight into converting adult cochlear glia into SGNs.

Cochlea glia are neural crest-derived cells. By E10.5 in mouse development, neural-crest-derived glial precursors have migrated into the otic vesicle. The glial precursors developed into satellite glia that resided next to the SGN soma and Schwann cells that ensheathed the SGN processes (Sandell et al., 2014). Neural crest-derived glial precursors intermingled with SGN cell bodies during cochlear vestibular ganglion development (Druckenbrod et al., 2020). CHD4 was expressed in TUBB3-marked SGNs, but not in non-neuronal cells at P0. At P28, non-neuronal cells expressing CHD4 were seen next to TUBB3 and CHD4 labeled SGN somas (Fig. 1). Although the identity of CHD4 expressing non-neuronal cells still needs to be established, CHD4 may play a role in preventing lineage conversion of these non-neuronal cells in older animals. Even though the epigenetic function of CHD4 in cochlear glia has yet to be determined, CHD4 is known to associate with enhancers (Wang et al., 2009; Whyte et al., 2012). CHD4 recruitment to enhancers remains unexplored in inner ear cells.

CHD4 has protein domains that bind nucleosomes but not a sequence-specific DNA binding domain. CHD4 interacts with DNA binding proteins, such as transcription factors, to associate with cis-regulatory elements. The interaction of CHD4 to specific transcription factors is likely cell-type dependent. PAX2 is a transcription factor essential for inner ear development (Burton et al., 2004). Using a mouse ventral otocyst cell line (VOT-N33), immunoprecipitation of LSD1 showed biochemical interaction with CHD4, components of NuRD, and PAX2 to form a variant of the NuRD complex. The NuRD protein complex maintained an undifferentiated otic progenitor state by repressing transcription (Patel et al., 2018). CHD4 and the NuRD complex may be associated with a different transcription factor in other cell types. GFI1 tethered CHD4 and components of the NuRD complex to promoters and enhancers in myeloid progenitors (Helness et al., 2021). Distinct transcription factors may recruit CHD4 and the NuRD complex to specific enhancers in different inner ear cell types based on the protein complex composition.

CHD4 recruitment by transcription factors to specific DNA sequences may result in enhancer decommissioning or inactivation. HDACs and lysine-specific demethylase (LSD1) associated with the NuRD complex (Tong et al., 1998; Wang et al., 2009; Xue et al., 1998; Zhang et al., 1998) can deacetylate and demethylate histones to decommission enhancers. In mature cochlear glia, CHD4 may help the NuRD complex decommission neuronal enhancers to maintain glial identity. Inhibiting CHD4 activity may prevent enhancer decommissioning, render neuronal enhancers amenable to activation, and facilitate lineage conversion. Overexpressing combinations of transcription factors such as ASCL1, NEUROG1, and NEUROD1 can turn on neuronal-specifying enhancer networks. Decommissioning enhancers that correspond to alternative and originating cell types may require CHD4. Finally, after completing lineage conversion, maintaining the epigenomic landscape of the new cell may require CHD4. For regenerative efforts, genome editing tools may provide temporal control to sequentially repress and activate CHD4 expression to facilitate distinct molecular steps in lineage conversion.

The fundamental cellular and molecular strategies for replacing lost SGNs are particularly interesting for treating hearing loss. Although CHD4 function is not well delineated in inner ear development, stem cell differentiation, and lineage conversion, CHD4 may contribute to epigenetic changes that prevent or promote regenerative efforts. In addition to changes in histone marks and chromatin accessibility ascribed to CHD4 protein complexes, additional post-translational histone modifications and epigenetic changes such as DNA methylation are likely involved. Despite significant progress toward SGN restoration, generating SGN subtypes (Grandi et al., 2020; Petitpre et al., 2018; Shrestha et al., 2018; Sun et al., 2018) may be imminent for functional recovery. In the cochlea, type I SGNs exhibit varied ion channel content and electrophysiologic properties (Adamson et al., 2002). Single-fiber recordings of the cat auditory nerve highlighted the diversity of sound sensitivities and spontaneous firing rates (Kiang et al., 1965; Liberman, 1978). SGNs with different spontaneous firing rates also form synapses at varying positions along the basolateral surface of inner hair cells (Liberman, 1982). The neuronal diversity and innervation patterns may be required for encoding a wide dynamic range of sound intensities necessary for hearing in complex environments and serve as additional considerations for future regenerative efforts.

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