Atoh1 is repurposed from neuronal to hair cell determinant by Gfi1 acting as a coactivator without redistribution of its genomic binding sites
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE136909 | GEO | 2022/08/10
REPOSITORIES: GEO
Similar Datasets
Project description:Atoh1 is repurposed from neuronal to hair cell determinant by Gfi1 acting as a coactivator without redistribution of its genomic binding sites
| PRJNA564019 | ENA
Project description:GFI1 is a zinc finger transcription factor that is necessary for the differentiation and survival of hair cells in the cochlea. Deletion of Gfi1 in mice significantly reduces the expression of hundreds of hair cell genes: this is a surprising result, as GFI1 normally acts as a transcriptional repressor by recruiting histone demethylases and methyltransferases to its targets. To understand the mechanisms by which GFI1 promotes hair cell differentiation, we used CUT&RUN to identify the direct targets of GFI1 and ATOH1 in hair cells. We found that GFI1 regulates hair cell differentiation in two distinct ways-first, GFI1 and ATOH1 can bind to the same regulatory elements in hair cell genes, but while ATOH1 directly binds its target DNA motifs in many of these regions, GFI1 does not. Instead, it appears to enhance ATOH1's transcriptional activity by acting as part of a complex in which it does not directly bind DNA. Second, GFI1 can act in its more typical role as a direct, DNA-binding transcriptional repressor in hair cells; here it represses non-hair cell genes, including many neuronal genes. Together, our results illuminate the function of GFI1 in hair cell development and hair cell reprogramming strategies.
| S-EPMC9098437 | biostudies-literature
Project description:Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.
| S-EPMC7722738 | biostudies-literature
Project description:To gain new insights into the genetic networks regulating hair cell development, we developed a new transcriptional programming strategy to promote in vitro hair cell differentiation, starting from mouse embryonic stem cells (mESCs). In vivo, Atoh1 is the only transcription factor (TF) known to be necessary and sufficient for HC differentiation, but in vitro its overexpression induces neuronal rather than hair cell differentiation. We discovered that Atoh1 expression combined with two other TFs (Pou4f3, Gfi1) resulted in efficient hair cell generation. This work offers a new paradigm to understand the molecular mechanisms governing transcription factor specificity. For example, how do Pou4f3-Gfi1 modulate Atoh1 activity to orchestrate a HC differentiation program? To address this question, we have engineered several mESCs lines containing Atoh1, Gfi1 and/or Pou4f3 in a doxycycline-inducible locus to allow a controllable overexpression of every combination of these 3 transcriptional factors either using a polycistronic approach (mESCs lines: iG+A, iG+P, iP+A and iGPA) or by promoting their individual overexpression (mESCs lines: iAtoh1, iGfi1 and iPou4f3). These findings highlight the diversity of mechanisms by which one TF can redirect the activity of another to enable combinatorial control of cell identity.
2022-08-10 | GSE136836 | GEO
Project description:To gain new insights about the genetic networks controlling hair cell (HC) development, we previously developed a direct genetic programming strategy to generate an inexhaustible supply of HC-like cells (induced HCs, iHCs) in vitro, starting from mouse embryonic stem cells (ESC). We found that combined activity of three transcription factors, Gfi1, Pou4f3, and Atoh1, can program ESC-derived progenitors towards HC fate with efficiencies of 55%-80%. These iHCs express several HC markers and exhibit polarized structures that are highly reminiscent of the mechanosensitive hair bundles, with many microvilli-like stereocilia. Here, we describe the experimental design, methodology, and data validation for the microarray analysis used to characterize the transcriptome profile of iHCs at different stages of their differentiation. This approach based on FACS sorting and microarray analysis revealed a highly similar iHC transcriptome to that of endogenous HCs in vivo. The data obtained in this study is available in the Gene Expression Omnibus (GEO) database (accession number GSE60352).
| S-EPMC4664721 | biostudies-literature
Project description:Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1cre;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1, Atoh1 and Insm1). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation.
| S-EPMC7502595 | biostudies-literature
Project description:Mechanosensory hair cells of the mature mammalian organ of Corti do not regenerate; consequently, loss of hair cells leads to permanent hearing loss. Although nonmammalian vertebrates can regenerate hair cells from neighboring supporting cells, many humans with severe hearing loss lack both hair cells and supporting cells, with the organ of Corti being replaced by a flat epithelium of nonsensory cells. To determine whether the mature cochlea can produce hair cells in vivo, we reprogrammed nonsensory cells adjacent to the organ of Corti with three hair cell transcription factors: Gfi1, Atoh1, and Pou4f3. We generated numerous hair cell-like cells in nonsensory regions of the cochlea and new hair cells continued to be added over a period of 9 wk. Significantly, cells adjacent to reprogrammed hair cells expressed markers of supporting cells, suggesting that transcription factor reprogramming of nonsensory cochlear cells in adult animals can generate mosaics of sensory cells like those seen in the organ of Corti. Generating such sensory mosaics by reprogramming may represent a potential strategy for hearing restoration in humans.
| S-EPMC10835112 | biostudies-literature
Project description:Reprogramming of the cochlea with hair-cell-specific transcription factors such as ATOH1 has been proposed as a potential therapeutic strategy for hearing loss. ATOH1 expression in the developing cochlea can efficiently induce hair cell regeneration but the efficiency of hair cell reprogramming declines rapidly as the cochlea matures. We developed Cre-inducible mice to compare hair cell reprogramming with ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3. In newborn mice, all transcription factor combinations tested produced large numbers of cells with the morphology of hair cells and rudimentary mechanotransduction properties. However, 1 week later, only a combination of ATOH1, GFI1 and POU4F3 could reprogram non-sensory cells of the cochlea to a hair cell fate, and these new cells were less mature than cells generated by reprogramming 1 week earlier. We used scRNA-seq and combined scRNA-seq and ATAC-seq to suggest at least two impediments to hair cell reprogramming in older animals. First, hair cell gene loci become less epigenetically accessible in non-sensory cells of the cochlea with increasing age. Second, signaling from hair cells to supporting cells, including Notch signaling, can prevent reprogramming of many supporting cells to hair cells, even with three hair cell transcription factors. Our results shed light on the molecular barriers that must be overcome to promote hair cell regeneration in the adult cochlea.
| S-EPMC9708077 | biostudies-literature
Project description:Overexpression of the transcription factor (TF) ATOH1 is known to induce the transformation of nonsensory cells in the organ of Corti into hair cells (HCs). Evaluating DNA 5?Πto the coding sequence of the pou4f3 gene, a target of ATOH1 in HCs, we identified in three regions containing clustered binding sites for ATOH1 and several other TFs that are expressed in developing inner ear sensory epithelia at the time of HC specification. These regions and sites are highly conserved across evolutionarily distant mammalian species. To test the hypothesis that the identified TFs act in combination to regulate the pou4f3 gene, we transfected by electroporation neonatal cochlear sensory epithelium from mice expressing green fluorescent protein (GFP) under the control of an 8.5-kb 5' pou4f3 genomic fragment. Plasmids encoding 21 TFs c-transfected with human ATOH1 (hATOH1). Cotransfection with hETV4, hNMYC, or hETS2 produced significantly more pou4f3/GFP and myosin 7A-positive nonsensory cells than hATOH1 alone. Co-transfection of hATOH1 with hHES1, hHES5, or hNEUROD1 reduced the effects of hATOH1. Chromatin immunoprecipitation (ChIP)of DNA from an inner ear cell line transfected with hNMYC,hETV4, or hETS2 revealed binding to a conserved region immediately proximal to the coding sequence. ChIP similarly revealed binding of hGATA3, hNMYC, and hTFE2 to a region several kilobases distal to the coding sequence, which we have previously shown to bind ATOH1. The results suggest that ATOH1 acts in concert with a subset of other TFs to directly regulate the pou4f3 gene and more broadly to regulate the HC phenotype.
| S-EPMC4291311 | biostudies-literature
Project description:Stem cell pluripotency gene Sox2 stimulates expression of proneural basic-helix-loop-helix transcription factor Atoh1. Sox2 is necessary for the development of cochlear hair cells and binds to the Atoh1 3' enhancer to stimulate Atoh1 expression. We show here that Sox2 deletion in late embryogenesis results in the formation of extra hair cells, in contrast to the absence of hair cell development obtained after Sox2 knockout early in gestation. Sox2 overexpression decreased the level of Atoh1 protein despite an increase in Atoh1 mRNA. Sox2 upregulated E3 ubiquitin ligase, Huwe1, by direct binding to the Huwe1 gene. By upregulating its cognate E3 ligase, Sox2 disrupts the positive feedback loop through which Atoh1 protein increases the expression of Atoh1. We conclude that Sox2 initiates expression, while also limiting continued activity of bHLH transcription factor, Atoh1, and this inhibition represents a new mechanism for regulating the activity of this powerful initiator of hair cell development.
| S-EPMC11813075 | biostudies-literature