Atoh1 regulation in the cochlea: more than just transcription

Atoh1 regulation in the cochlea: more than just transcription Atoh1转录因子在耳蜗内的转译后调节 1 2 3 4 5 6 first-author †E-mail:yfcheng2@vghtpe.gov.tw †E-mail:yfcheng2@vghtpe.gov.tw yfcheng2@vghtpe.gov.tw Department of Otology and Laryngology, Harvard Medical School, Boston, MA 02115, USA Department of Otology and Laryngology, Harvard Medical School, Boston, MA 02115, USA Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA Department of Medical Research, Taipei Veterans General Hospital, Taipei 112, Taiwan, China Department of Medical Research, Taipei Veterans General Hospital, Taipei 112, Taiwan, China Department of Otolaryngology-Head and Neck Surgery, Taipei Veterans General Hospital, Taipei 112, Taiwan, China Department of Otolaryngology-Head and Neck Surgery, Taipei Veterans General Hospital, Taipei 112, Taiwan, China School of Medicine, Yang-Ming University, Taipei 112, Taiwan, China School of Medicine, Yang-Ming University, Taipei 112, Taiwan, China Department of Speech Language Pathology and Audiology, Taipei University of Nursing and Health Science, Taipei 112, Taiwan, China Department of Speech Language Pathology and Audiology, Taipei University of Nursing and Health Science, Taipei 112, Taiwan, China More than 80% of all cases of deafness are related to the death or degeneration of cochlear hair cells and the associated spiral ganglion neurons, and a lack of regeneration of these cells leads to permanent hearing loss. Therefore, the regeneration of lost hair cells is an important goal for the treatment of deafness. Atoh1 is a basic helix-loop-helix (bHLH) transcription factor that is critical in both the development and regeneration of cochlear hair cells. Atoh1 is transcriptionally regulated by several signaling pathways, including Notch and Wnt signalings. At the post-translational level, it is regulated through the ubiquitin-proteasome pathway. In vitro and in vivo studies have revealed that manipulation of these signaling pathways not only controls development, but also leads to the regeneration of cochlear hair cells after damage. Recent progress toward understanding the signaling networks involved in hair cell development and regeneration has led to the development of new strategies to replace lost hair cells. This review focuses on our current understanding of the signaling pathways that regulate Atoh1 in the cochlea. 概要 2 Atoh1属于bHLH转录因子家族成员,其对耳蜗毛细胞的胚胎发育及损伤后再生具有重要作用.许多讯号通道在转录水平上对Atoh1有调节作用,包括Notch和Wnt通道.在蛋白转译后水平,Atoh1是经由泛素-蛋白酶通道所调节.体外细胞实验及体内动物实验都显示:经由上述讯号通道的调节手段不仅影响耳蜗发育,也导致毛细胞的损伤后再生.本综述回顾了耳蜗内各个对Atoh1调节讯号通道研究的进展,并聚焦于泛素-蛋白酶通道对Atoh1进行转译后调节及其对毛细胞发育的影响. Atoh1 Huwe1 Cochlea Hair cells Regeneration Post-translational regulation Atoh1 Huwe1 耳蜗 毛细胞 再生 转译后调节 Introduction 1 Hair cell formation is dependent on the basic helix-loop-helix (bHLH) transcription factor Atoh1, which is both necessary and sufficient for hair cell differentiation during development (Bermingham et al., 1999 1999 ). Atoh1 overexpression in the inner ear via genetic or pharmacological manipulation has been shown to result in new hair cell formation in the cochlea and to restore hearing in experimental animal models (Zheng and Gao, 2000 2000 ; Izumikawa et al., 2005 2005 ; Gubbels et al., 2008 2008 ). The transcriptional and post-translational regulations of Atoh1 have been shown to involve several overlapping signaling pathways. Transcriptional regulation of Atoh1 by Wnt or Notch signaling or post-translational regulation by the Huwe1-ubiquitin-proteasome pathway has been shown to sucessfully convert inner ear progenitor cells into hair cells (Shi et al., 2010 2010 ; 2013 2013 ; 2014 2014 ; Mizutari et al., 2013 2013 ; Bramhall et al., 2014 2014 ; Cheng et al., 2016 2016 ). These studies have led to novel approaches for the regeneration of hair cells in the inner ear. This review focuses on recent advances in our understanding of the signaling pathways that regulate Atoh1 in the cochlea. Hearing impairment 1 Hearing impairment is one of the most prevalent disabilities in industrialized countries, and it is estimated that 360 million people have moderate to profound hearing deficiency globally (WHO, 2017 2017 ). Most cases of hearing impairment involve sensorineural hearing loss caused by the degeneration or loss of two specific inner ear cell types: cochlear hair cells, which act as the primary mechanoreceptors for sound transduction, and the connected auditory nerve neurons, which transmit electrical signals from the synapsed inner hair cells to the brain (Davis, 1983 1983 ). Loss of cochlear hair cells caused by genetic mutations, ototoxic medications, noise overexposure, autoimmune disease, or aging is irreversible in mammals because they have very limited ability to spontaneously regenerate hair cells after loss, and there are currently very few methods to regenerate hearing function after hair cell damage, especially in adult animals (Izumikawa et al., 2005 2005 ; Mizutari et al., 2013 2013 ). Vestibular hair cells have a limited regenerative capacity (Forge et al., 1993 1993 ; Warchol et al., 1993 1993 ; Wang et al., 2015 2015 ), but spontaneous hair cell regeneration after damage or hair cell loss has been reported only in the newborn murine cochlea (Bramhall et al., 2014 2014 ; Cox et al., 2014 2014 ). For deafness caused by loss of cochlear hair cells, cochlear implants are surgically implanted into the inner ear to stimulate the spiral ganglion. However, cochlear implants depend on the remaining functional spiral ganglion neurons, the loss of which can severely compromise their efficacy (Incesulu and Nadol, 1998 1998 ). The regeneration or replacement of damaged cochlear hair cells is thus the ultimate goal for the treatment of hearing loss. Several different approaches for hair cell regeneration have been attempted, including cell therapy, gene therapy, and pharmacological therapy. A few attempts at cell transplantation into the damaged cochlea have been reported, but most have shown extremely limited survival of transplanted cells and the inability of these cells to differentiate into mature hair cell types (Hu et al., 2005 2005 ; Hu and Ulfendahl, 2006 2006 ). Gene therapy involving the overexpression of Atoh1 has been shown to be a successful strategy for converting supporting cells into hair cells, thereby restoring hearing loss after damage (Izumikawa et al., 2005 2005 ; Richardson and Atkinson, 2015 2015 ). Gene augmentation (Akil et al., 2012 2012 ; Askew et al., 2015 2015 ; Pan et al., 2017 2017 ), as well as emerging CRISPR-Cas9 genome editing methods (Zou et al., 2015 2015 ; Zuris et al., 2015 2015 ; Mianné et al., 2016 2016 ), also provides promising experimental paradigms for treating genetic deafness. However, the complexity of the inner ear anatomy, as well as a lack of ideal vector systems and appropriate delivery methods, has made the delivery of foreign genetic material into the inner ear challenging (Husseman and Raphael, 2009 2009 ; Shu et al., 2016 2016 ). Pharmacotherapy using local delivery of small molecule compounds to convert inner ear progenitors into hair cells seems to be a more physiologically relevant approach to regenerating lost hair cells after damage (Mizutari et al., 2013 2013 ). However, a successful therapy will require a better understanding of the underlying mechanisms controlling hair cell formation and regeneration. Atoh1, a bHLH transcription factor, is required for hair cell differentiation and regeneration after damage 1 bHLH transcription factors orchestrate cell fate commitment and specification during development (Lo et al., 1991 1991 ; Ross et al., 2003 2003 ), and a homologous group of “proneural” bHLH transcription factors plays a central role in neurogenesis. Several proneural bHLH transcription factors, including Atoh1, Neurog1, Neurod1, and Ascl1, contribute to the development of inner ear neurons and hair cells (Bertrand et al., 2002 2002 ; Fritzsch, 2003 2003 ; Tiveron et al., 2003 2003 ; Jahan et al., 2013 2013 ). Atoh1 , the mammalian homolog of the Drosophila gene atonal (also known as Math1 for “mouse atoh1 ” and HATH1 for “human atoh1 ”) (Ben-Arie et al., 1996 1996 ), is a bHLH transcription factor that is both necessary and sufficient for the formation and differentiation of inner ear hair cells. Animal studies have shown that Atoh1 -knockout mice fail to form cochlear and vestibular hair cells (Bermingham et al., 1999 1999 ) as a result of progenitor cell apoptosis (Chen et al., 2002 2002 ). In gain-of-function studies, overexpression of Atoh1 in mice by viral delivery led to ectopic hair cell formation in organ of Corti explants (Zheng and Gao, 2000 2000 ). In vivo and in utero experiments in mice confirmed that forced upregulation of Atoh1 leads to the formation of extra hair cells (Woods et al., 2004 2004 ; Izumikawa et al., 2005 2005 ; Staecker et al., 2007 2007 ; Gubbels et al., 2008 2008 ; Liu et al., 2012 2012 ; 2014 2014 ; Atkinson et al., 2014 2014 ), but very little is known about how cells regulate the level of Atoh1 , especially in the cochlea. Atoh1 level is critical for hair cell development and regeneration in mice 1 Proper spatiotemporal expression of Atoh1 is key for the differentiation and viability of hair cells. Atoh1 is expressed in mice in the inner ear progenitors before they have committed to the hair cell fate. It is first detected in the prosensory cells of the basal cochlear turn of the embryonic cochlea at embryonic day (E) 13.5. The expression of Atoh1 surges at the base of the cochlea by E14.5 and starts to appear in the apical turn by E17.5. Atoh1 expression begins to decline immediately after birth in the basal turn and by postnatal day 4 in hair cells in the apical turn (Yang et al., 2010 2010 ; Pan et al., 2012 2012 ). Atoh1 affects not only hair cell differentiation, but also hair cell viability. Embryonic reduction of Atoh1 led to significant cochlear hair cell loss and abnormal hair cell bundle formation in a transgenic mouse model (Pan et al., 2012 2012 ). Temporally, Atoh1 is critical for the survival of hair cell progenitors at the base of the cochlea in a 72-h window from E13.5 to E16.5, and deletion of Atoh1 outside this developmental window does not affect survival (Cai et al., 2013 2013 ). Although Atoh1 overexpression in the supporting cells leads to ectopic hair cell formation, transgenic mouse models have shown that persistent Atoh1 overexpression in the cochlear hair cells leads to hair cell death and eventual hearing loss (Liu et al., 2012 2012 ). A similar phenotype was also seen when Atoh1 protein degradation mechanisms were disrupted (Cheng et al., 2016 2016 ). Atoh1 is widely expressed 1 In addition to hair cell formation, Atoh1 plays important roles in the differentiation and formation of several other cell types. Atoh1 knockout induces cell loss in several tissues, including cerebellar granule cells (Ben-Arie et al., 1997 1997 ; Flora et al., 2007 2007 ; Forget et al., 2014 2014 ), retrotrapezoid nucleus (RTN) neurons in the medulla (Wang et al., 2005 2005 ; Huang et al., 2012 2012 ; Ruffault et al., 2015 2015 ), dorsal commissural interneurons in the spine (Miesegaes et al., 2009 2009 ), intestinal goblet cells (Yang et al., 2001 2001 ; VanDussen and Samuelson, 2010 2010 ), and Merkel cells in the skin (Ben-Arie et al., 2000 2000 ; Fröhlich et al., 2009 2009 ; Morrison et al., 2009 2009 ; Maksimovic et al., 2014 2014 ; Wright et al., 2015 2015 ). The distribution of Atoh1 in cells other than inner ear hair cells and the associated phenotypes in loss-of-function assays are listed in Table 1 1 . Cell type Function of Atoh1 Phenotype in loss-of-function assays Reference Cerebellar granule cells Maturation and proliferation Missing external germinal layer (EGL) of the developing cerebellum Ben-Arie et al., 1997 1997 ; Wang et al., 2005 2005 ; Flora et al., 2007 2007 ; Forget et al., 2014 2014 Retrotrapezoid nucleus (RTN) neurons of the medulla Migration of neurons and establishment of essential connections with the pre-Bötzinger complex RTN neurons fail to develop and establish connections with the respiratory rhythm-generating center in mammals Rose et al., 2009 2009 ; Huang et al., 2012; Ruffault et al., 2015 2015 D1 commissural interneurons of the spinal cord Ventral migration and fate determination of D1 interneurons Progenitors adopting roof plate or D2 interneuron fates Miesegaes et al., 2009 2009 Intestinal goblet cells Secretory lineage fate determination Failure to form intestinal secretory cells Yang et al., 2001 2001 ; Shroyer et al., 2007 2007 ; VanDussen and Samuelson, 2010 2010 Merkel cells Specification of Merkel cells Absence of Merkel cells Ben-Arie et al., 2000 2000 ; Morrison et al., 2009 2009 ; van Keymeulen et al., 2009 2009 ; Maksimovic et al., 2014 2014 Atoh1 expression has also been reported to be associated with tumorigenesis. Atoh1 acts as a tumor suppressor gene in the colon and skin, as it antagonizes tumor formation and growth, and many colorectal cancer and Merkel cell carcinoma patients show genetic mutations of Atoh1 (Bossuyt et al., 2009 2009 ). However, Atoh1 acts as an oncogene in medulloblastoma (Zhao H. et al., 2008 2008 ; Flora et al., 2009 2009 ; Ayrault et al., 2010 2010 ), indicating its multiple roles in differentiation and proliferation in different cell types. Signaling pathways that regulate Atoh1 1 Notch signaling pathway 2 Notch signaling plays a key role in Atoh1 regulation and inner ear development (Jarriault et al., 1995 1995 ; Lanford et al., 1999 1999 ; Brooker et al., 2006 2006 ). The major role of Notch signaling is to regulate cell fate decisions through lateral inhibition during development. It acts as a mediator between prosensory cells during development, leading these cells to differentiate into hair cells and supporting cells. Thus, Notch signaling is utilized to establish the structural pattern of hair cells and supporting cells in the cochlea (Kelley, 2003 2003 ). The inner ear is derived from the otic placode, which is recognized initially as a thickening near the hindbrain at E8 in mice. The otic placode invaginates by E10.5 to form an otocyst that contains prosensory cells and ganglion neuroblasts. The determination of hair cell or supporting cell fate is influenced by Notch signaling, and developing hair cells express Atoh1 along with the Notch ligands Jag2 and Delta1 . These ligands bind to Notch1 in adjacent cells and induce the release of Notch intracellular domain (NICD) from their membranes. Upregulation of inhibitory bHLH transcription factors such as Hes1 and Hes5 follows after the increase of NICD, and expression of Atoh1 is blocked, leading to inhibition of hair cell fate and development of supporting cells (Kelley, 2003 2003 ; 2006 2006 ). Pharmacological inhibition of Notch signaling by γ-secretase inhibitors increases the number of cochlear hair cells in neonatal cochlear explants (Yamamoto et al., 2006 2006 ; Takebayashi et al., 2007 2007 ; Bramhall et al., 2014 2014 ; Li et al., 2015 2015 ; Maass et al., 2015 2015 ) and increases the differentiation of inner ear stem cells into hair cells (Jeon et al., 2011 2011 ). In an in vitro lineage-tracing study involving genetically marking supporting cells with a fluorescent reporter, treatment with γ-secretase inhibitors after aminoglycoside-induced hair cell damage has been shown to promote hair cell regeneration through the direct transdifferentiation of supporting cells in the cochlea (Bramhall et al., 2014 2014 ). An in vivo study also showed that direct round-window application of γ-secretase inhibitors resulted in recovery of cochlear function after noise-induced hair cell damage. This was evidenced by increased Atoh1 expression and direct conversion of supporting cells into hair cells, as measured by a similar genetic tagging model (Mizutari et al., 2013 2013 ). The responsiveness of the cochlea to γ-secretase inhibitors has been shown to be position-dependent and age-dependent. The apical region of the cochlea exhibits a stronger response in terms of transdifferentiation of supporting cells into hair cells compared to the basal region, and the response declines rapidly with age (Mizutari et al., 2013 2013 ; Bramhall et al., 2014 2014 ; Maass et al., 2015 2015 ). Wnt/β-catenin signaling pathway 2 The Wnt pathway plays a critical role in patterning and cell fate specification in the early development of the inner ear (Stevens et al., 2003 2003 ; Gregorieff and Clevers, 2005 2005 ; Riccomagno et al., 2005 2005 ; Ohyama et al., 2006 2006 ). In canonical Wnt signaling, Wnt binds to receptors on the cell membrane surface to disrupt the destruction complex consisting of axin, adenomatosis polyposis coli (APC), glycogen synthase kinase 3β (GSK-3β), and Disheveled (Dvl). This prevents β-catenin from being degraded in the cytoplasm. β-Catenin then translocates into the nucleus and subsequently forms nuclear β-catenin/Tcf complexes that drive the expression of Wnt target genes. In non-canonical Wnt signaling, Wnt proteins can work in a β-catenin-independent manner via the planar cell polarity (PCP) and Wnt/calcium pathways (Jansson et al., 2015 2015 ). Wnt/β-catenin signaling induces patches of hair cells and supporting cells in the auditory sensory epithelium and is involved in establishing or maintaining the distinction between sensory domains that contain hair cells and non-sensory domains in the inner ear (Stevens et al., 2003 2003 ). Overexpression of β-catenin increases Atoh1 expression in neuroblastoma cells and neural progenitor cells. This upregulation is due to the interaction of β-catenin with the 3'-enhancer of the Atoh1 gene (Shi et al., 2010 2010 ). Conditional knockout of β-catenin inhibits hair cell formation from sensory progenitors, while constitutive upregulation of β-catenin expands sensory progenitors and results in the formation of extra hair cells (Shi et al., 2012 2012 ; 2014 2014 ; McLean et al., 2017 2017 ). The Notch and Wnt/β-catenin signaling pathways interact with each other. As mentioned earlier, β-catenin increases Atoh1 expression through interaction with the 3' enhancer of Atoh1 , and this accounts for the effect of Notch inhibition on Atoh1 . Shi et al. ( 2010 2010 ) found that inhibition of Notch signaling increases β-catenin expression, while simultaneous inhibition of β-catenin abolishes the increase in Atoh1 expression caused by Notch inhibition. Thus, β-catenin expression is required not only for increased expression of Atoh1 , but also for the mitotic generation of hair cells in the cochlea after Notch inhibition (Li et al., 2015 2015 ). Interestingly, a combination of Notch inhibition and Wnt signaling activation seems to have synergistic effects on the proliferation and transdifferentiation of progenitors or supporting cells in the neonatal cochlea, especially in the basal turn that usually shows only limited ability to form new hair cells after birth (Ni et al., 2016 2016 ; McLean et al., 2017 2017 ). Post-translational regulation of Atoh1: the CK1-Huwe1-ubiquitin-proteasome pathway 2 Increasing evidence has shown that Atoh1 gene expression is tightly regulated by overlapping pathways, some of which have been described above. Post-translational regulation of Atoh1, however, is only now beginning to be understood. The ubiquitin-proteasome pathway (UPP) is the major post-translational regulatory system in eukaryotes, and it plays an important role in the development and physiology of eukaryotic cells (Tai and Schuman, 2008 2008 ). The UPP has been implicated in the regulation of stem cell differentiation and lineage commitment via proteolytic degradation of key regulatory proteins. Specific protein substrates are conjugated by polyubiquitylation in an adenosine triphosphate (ATP)-dependent process and then targeted by the proteasome, the machinery by which proteins are degraded. The system is highly selective and tightly regulated. It not only degrades misfolded or damaged proteins, but is also essential for regulating cell-signaling pathways (Naujokat and Šarić, 2007 2007 ). The UPP consists of three key enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). E3 catalyzes polyubiquitin (and sometimes monoubiquitin) chain formation by transferring ubiquitin that has been activated by E1 and E2 onto the lysine residues of specific substrates. Huwe1 (HECT, UBA, and WWE domain containing 1) is a large 4371-amino acid HECT-domain E3 ubiquitin ligase that is involved in proteasomal degradation of several protein substrates, including Atoh1 in the cerebellum and in the cochlea (Forget et al., 2014 2014 ; Cheng et al., 2016 2016 ). Huwe1 binds to Atoh1 and forms a lysine 48-cojugated polyubiquitin chain on Atoh1 as a signal for proteasomal degradation (Cheng et al., 2016 2016 ). As a result, overexpression of Huwe1 decreases Atoh1 levels, while inhibiting Huwe1 increases the half-life of the Atoh1 protein. A “degron” is a sequence element or modification that is sufficient to be recognized or targeted by an E3 ligase to promote ubiquitylation (Varshavsky, 1991 1991 ). Atoh1 is found to be enriched in serine residues in the C-terminus, and many of the serines are conserved among different species. Casein kinase 1 (CK1), a serine/threonine selective protein kinase, phosphorylates Atoh1 at its serine-enriched C-terminal region, specifically serine 334, to form a phosphodegron that enhances the Atoh1-Huwe1 interaction and the subsequent ubiquitylation of Atoh1 (Cheng et al., 2016 2016 ). The overexpression of CK1 increases the degradation of Atoh1, while inhibition of CK1 stabilizes Atoh1. Interestingly, other sites, including other conserved serines at the C-terminus, have been reported to contribute to Atoh1 stability. Forget et al. ( 2014 2014 ) found that sonic hedgehog (SHH) controls the phosphorylation of serine 328 and serine 339 of Math1 through protein phosphatase 2A (PP2A) activity to protect Atoh1 from degradation. Tsuchiya et al. ( 2007 2007 ) reported that Atoh1 protein is expressed in normal colon tissue, but it is degraded by GSK3β in cancer tissue through phosphorylation of serine 54 and serine 58 of Hath1 (the equivalent of serine 52 and serine 56 of Math1). The biological roles of Huwe1 in development and neurogenesis have recently been described (Zhao X. et al., 2008 2008 ; 2009 2009 ; D'Arca et al., 2010 2010 ; Dominguez-Brauer et al., 2016 2016 ; Urbán et al., 2016 2016 ). Atoh1 degradation by Huwe1 is required for proper neuronal migration and differentiation in the cerebellum (Forget et al., 2014 2014 ) because uncontrolled Atoh1 expression in the cerebellum leads to early postnatal lethality and interferes with cerebellar development (Helms et al., 2001 2001 ). Spatiotemporal control of the Atoh1 protein level by Huwe1 is essential for cochlear hair cell fate determination and survival (Cheng et al., 2016 2016 ), and conditional knockout of Huwe1 in the cochlear supporting cells at the embryonic or early postnatal stage leads to extra hair cells in the cochlea. However, hair cell death is observed when Huwe1 is conditionally knocked out in the cochlear hair cells, and this phenotype appears to be correlated with an increase in Atoh1 protein expression. This is in line with a previous study showing that Atoh1 overexpression in the hair cell causes hair cell death (Liu et al., 2012 2012 ). Thus, the termination of Atoh1 activity after hair cell differentiation and maturation seems to be critical for normal cell patterning in the cochlea. Although several substrates of Huwe1 have been found to be associated with cell apoptosis, including Mcl-1, protein phosphatase 5 (PP5), p53, Myc, and cdc6 (Chen et al., 2005 2005 ; Hall et al., 2007 2007 ; D'Arca et al., 2010 2010 ; Kurokawa et al., 2013 2013 ), further studies are required to unravel the mechanisms of hair cell death underlying Huwe1 deletion and Atoh1 overexpression. Concluding remarks 1 Atoh1 is a key transcription factor in the development and regeneration processes of the inner ear. Several signaling pathways have been shown to be involved in the regulation of its expression in the cochlea, including the Notch, Wnt, and ubiquitin-proteasome pathways. The studies reviewed in this article have shown that proper spatiotemporal control of Atoh1 expression is essential for inner ear development, not only at the transcriptional level, but also at the post-translational level (concluded in Fig. 1 1 ). Forcing Atoh1 overexpression or manipulating Atoh1-enhancing signaling pathways is one of most efficient strategies known for regenerating damaged hair cells. Incorporating knowledge of how Atoh1 is regulated will lead to a better understanding of cochlear development and to more insights into therapeutic strategies for hair cell regeneration. Wnt signaling activates Atoh1 expression through interactions between β-catenin and the 3' enhancer of Atoh1 . Overexpression of Notch signaling decreases β-catenin and decreases Atoh1 expression, while inhibition of Notch leads to upregulation of β-catenin and increased Atoh1 expression. At the post-translational level, CK1 binds to and phosphorylates Atoh1 to facilitate interaction with the E3 ubiquitin ligase Huwe1, which orchestrates ubiquitin transfer events from E1 (ubiquitin-activating enzyme) and E2 (ubiquitin-conjugating enzyme). This in turn leads to the polyubiquitylation and proteasomal degradation of Atoh1. 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