ABSTRACT
Introduction: Mutations in microRNA-96 (miR-96), a microRNA expressed within the hair cells (HCs) of the inner ear, result in progressive hearing loss in both mouse models and humans. In this study, we present the first HC-specific RNA-sequencing (RNA-seq) dataset from newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Methods: Bulk RNA-seq was performed on HCs of newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Differentially expressed gene analysis was conducted on Mir96Dmdo homozygous mutant HCs compared to wildtype littermate controls, followed by GO term and protein-protein interaction analysis on these differentially expressed genes. Results: We identify 215 upregulated and 428 downregulated genes in the HCs of the Mir96Dmdo homozygous mutant mice compared to their wildtype littermate controls. Many of the significantly downregulated genes in Mir96Dmdo homozygous mutant HCs have established roles in HC development and/or known roles in deafness including Myo15a, Myo7a, Ush1c, Gfi1, and Ptprq and have enrichment in gene ontology (GO) terms with biological functions such as sensory perception of sound. Interestingly, upregulated genes in Mir96Dmdo homozygous mutants, including possible miR-96 direct targets, show higher wildtype expression in supporting cells compared to HCs. Conclusion: Our data further support a role for miR-96 in HC development, possibly as a repressor of supporting cell transcriptional programs in HCs. The HC-specific Mir96Dmdo RNA-seq data set generated from this manuscript are now publicly available in a dedicated profile in the gene expression analysis resource (gEAR-https://umgear.org/p?l=miR96).
ABSTRACT
Age-related hearing loss (ARHL) is a major health concern among the elderly population. It is hoped that increasing our understanding of its underlying pathophysiological processes will lead to the development of novel therapies. Recent genome-wide association studies (GWASs) discovered a few dozen genetic variants in association with elevated risk for ARHL. Integrated analysis of GWAS results and transcriptomics data is a powerful approach for elucidating specific cell types that are involved in disease pathogenesis. Intriguingly, recent studies that applied such bioinformatics approaches to ARHL resulted in disagreeing findings as for the key cell types that are most strongly linked to the genetic pathogenesis of ARHL. These conflicting studies pointed either to cochlear sensory epithelial or to stria vascularis cells as the cell types most prominently involved in the genetic basis of ARHL. Seeking to resolve this discrepancy, we integrated the analysis of four ARHL GWAS datasets with four independent inner-ear single-cell RNA-sequencing datasets. Our analysis clearly points to the cochlear sensory epithelial cells as the key cells for the genetic predisposition to ARHL. We also explain the limitation of the bioinformatics analysis performed by previous studies that led to missing the enrichment for ARHL GWAS signal in sensory epithelial cells. Collectively, we show that cochlear epithelial cells, not stria vascularis cells, are the main inner-ear cells related to the genetic pathogenesis of ARHL.
Subject(s)
Presbycusis , Stria Vascularis , Aged , Humans , Stria Vascularis/pathology , Genome-Wide Association Study , Cochlea/pathology , Presbycusis/genetics , Presbycusis/pathology , Epithelium/pathologyABSTRACT
We explore the changes in chromatin accessibility and transcriptional programs for cochlear hair cell differentiation from postmitotic supporting cells using organoids from postnatal cochlea. The organoids contain cells with transcriptional signatures of differentiating vestibular and cochlear hair cells. Construction of trajectories identifies Lgr5+ cells as progenitors for hair cells, and the genomic data reveal gene regulatory networks leading to hair cells. We validate these networks, demonstrating dynamic changes both in expression and predicted binding sites of transcription factors (TFs) during organoid differentiation. We identify known regulators of hair cell development, Atoh1, Pou4f3, and Gfi1, and the analysis predicts the regulatory factors Tcf4, an E-protein and heterodimerization partner of Atoh1, and Ddit3, a CCAAT/enhancer-binding protein (C/EBP) that represses Hes1 and activates transcription of Wnt-signaling-related genes. Deciphering the signals for hair cell regeneration from mammalian cochlear supporting cells reveals candidates for hair cell (HC) regeneration, which is limited in the adult.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Cochlea , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Cell Differentiation/genetics , Organoids/metabolism , Mammals/metabolismABSTRACT
The cochlea consists of diverse cellular populations working in harmony to convert mechanical stimuli into electrical signals for the perception of sound. Otic mesenchyme cells (OMCs), often considered a homogeneous cell type, are essential for normal cochlear development and hearing. Despite being the most numerous cell type in the developing cochlea, OMCs are poorly understood. OMCs are known to differentiate into spatially and functionally distinct cell types, including fibrocytes of the lateral wall and spiral limbus, modiolar osteoblasts, and specialized tympanic border cells of the basilar membrane. Here, we show that OMCs are transcriptionally and functionally heterogeneous and can be divided into four distinct populations that spatially correspond to OMC-derived cochlear structures. We also show that this heterogeneity and complexity of OMCs commences during early phases of cochlear development. Finally, we describe the cell-cell communication network of the developing cochlea, inferring a major role for OMC in outgoing signaling.
ABSTRACT
HYPOTHESIS: Metformin treatment will protect mice from noise-induced hearing loss (NIHL). BACKGROUND: We recently identified metformin as the top-ranking, Food and Drug Administration-approved drug to counter inner ear molecular changes induced by permanent threshold shift-inducing noise. This study is designed to functionally test metformin as a potential otoprotective drug against NIHL. METHODS: Male and female B6CBAF1/J mice were obtained at 7 to 8 weeks of age. A cohort of the females underwent ovariectomy to simulate menopause and eliminate the effect of ovarian-derived estrogens. At 10 weeks of age, mice underwent a permanent threshold shift-inducing noise exposure (102.5 or 105 dB SPL, 8-16 kHz, 2 h). Auditory brainstem response (ABR) thresholds were obtained at baseline, 24 h after noise exposure, and 1 week after noise exposure. Mice were administered metformin (200 mg/kg/d) or a saline control in their drinking water after the baseline ABR and for the remainder of the study. After the 1-week ABR, mice were euthanized and cochlear tissue was analyzed. RESULTS: Metformin treatment reduced the 1-week ABR threshold shift at 16 kHz ( p < 0.01; d = 1.20) and 24 kHz ( p < 0.01; d = 1.15) as well as outer hair cell loss in the 32-45.5 kHz range ( p < 0.0001; d = 2.37) in male mice. In contrast, metformin treatment did not prevent hearing loss or outer hair cell loss in the intact or ovariectomized female mice. CONCLUSIONS: Metformin exhibits sex-dependent efficacy as a therapeutic for NIHL. These data compel continued investigation into metformin's protective effects and demonstrate the importance of evaluating the therapeutic efficacy of drugs in subjects of both sexes.
Subject(s)
Deafness , Ear, Inner , Hearing Loss, Noise-Induced , Metformin , Female , Male , United States , Animals , Mice , Hearing Loss, Noise-Induced/prevention & control , Cochlea , Metformin/pharmacology , Metformin/therapeutic useABSTRACT
Recent studies have identified sex-differences in auditory physiology and in the susceptibility to noise-induced hearing loss (NIHL). We hypothesize that 17ß-estradiol (E2), a known modulator of auditory physiology, may underpin sex-differences in the response to noise trauma. Here, we gonadectomized B6CBAF1/J mice and used a combination of electrophysiological and histological techniques to study the effects of estrogen replacement on peripheral auditory physiology in the absence of noise exposure and on protection from NIHL. Functional analysis of auditory physiology in gonadectomized female mice revealed that E2-treatment modulated the peripheral response to sound in the absence of changes to the endocochlear potential compared to vehicle-treatment. E2-replacement in gonadectomized female mice protected against hearing loss following permanent threshold shift (PTS)- and temporary threshold shift (TTS)-inducing noise exposures. Histological analysis of the cochlear tissue revealed that E2-replacement mitigated outer hair cell loss and cochlear synaptopathy following noise exposure compared to vehicle-treatment. Lastly, using fluorescent in situ hybridization, we demonstrate co-localization of estrogen receptor-2 with type-1C, high threshold spiral ganglion neurons, suggesting that the observed protection from cochlear synaptopathy may occur through E2-mediated preservation of these neurons. Taken together, these data indicate the estrogen signaling pathways may be harnessed for the prevention and treatment of NIHL.
Subject(s)
Cochlea , Estradiol/pharmacology , Evoked Potentials, Auditory/drug effects , Hearing Loss, Noise-Induced , Animals , Cochlea/metabolism , Cochlea/pathology , Cochlea/physiopathology , Female , Hearing Loss, Noise-Induced/metabolism , Hearing Loss, Noise-Induced/pathology , Hearing Loss, Noise-Induced/physiopathology , Hearing Loss, Noise-Induced/prevention & control , Mice , OvariectomyABSTRACT
Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.
Subject(s)
Cell Lineage/genetics , Cochlea/cytology , Genetic Association Studies , Mitosis , Protein Biosynthesis , Regeneration/genetics , Stem Cells/cytology , Stem Cells/metabolism , Animals , Cell Differentiation , Cell Survival , Epithelial Cells/cytology , Gene Expression Regulation , Integrases/metabolism , Mice , Multigene Family , Receptors, G-Protein-Coupled/metabolismABSTRACT
Noise-induced hearing loss (NIHL) results from a complex interplay of damage to the sensory cells of the inner ear, dysfunction of its lateral wall, axonal retraction of type 1C spiral ganglion neurons, and activation of the immune response. We use RiboTag and single-cell RNA sequencing to survey the cell-type-specific molecular landscape of the mouse inner ear before and after noise trauma. We identify induction of the transcription factors STAT3 and IRF7 and immune-related genes across all cell-types. Yet, cell-type-specific transcriptomic changes dominate the response. The ATF3/ATF4 stress-response pathway is robustly induced in the type 1A noise-resilient neurons, potassium transport genes are downregulated in the lateral wall, mRNA metabolism genes are downregulated in outer hair cells, and deafness-associated genes are downregulated in most cell types. This transcriptomic resource is available via the Gene Expression Analysis Resource (gEAR; https://umgear.org/NIHL) and provides a blueprint for the rational development of drugs to prevent and treat NIHL.
Subject(s)
Ear, Inner/metabolism , Hair Cells, Auditory/metabolism , Hearing Loss, Noise-Induced/metabolism , Hearing Loss, Noise-Induced/physiopathology , Spiral Ganglion/metabolism , Animals , Cochlea/metabolism , Cochlea/physiopathology , Ear, Inner/physiopathology , Evoked Potentials, Auditory, Brain Stem/physiology , Hearing Loss, Noise-Induced/genetics , Mice , Neurons/metabolism , Noise , Spiral Ganglion/cytology , Spiral Ganglion/physiopathologyABSTRACT
OBJECTIVE: The cellular diversity of the inner ear has presented a technical challenge in obtaining molecular insight into its development and function. The application of technological advancements in cell type-specific expression enable clinicians and researchers to leap forward from classic genetics to obtaining mechanistic understanding of congenital and acquired hearing loss. This understanding is essential for development of therapeutics to prevent and reverse diseases of the inner ear, including hearing loss. The objective of this study is to describe and compare the available tools for cell type-specific analysis of the ear, as a means to support decision making in study design. STUDY DESIGN: Three major approaches for cell type-specific analysis of the ear including fluorescence-activated cell sorting (FACS), ribosomal and RNA pulldown techniques, and single cell RNA-seq (scRNA-seq) are compared and contrasted using both published and original data. RESULTS: We demonstrate the strength and weaknesses of these approaches leading to the inevitable conclusion that to maximize the utility of these approaches, it is important to match the experimental approach with the tissue of origin, cell type of interest, and the biological question. Often, a combined approach (eg, cell sorting and scRNA-seq or expression analysis using 2 separate approaches) is required. Finally, new tools for visualization and analysis of complex expression data, such as the gEAR platform (umgear.org), collate cell type-specific gene expression from the ear field and provide unprecedented access to both clinicians and researchers. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:S1-S16, 2021.
Subject(s)
Ear, Inner/cytology , Flow Cytometry/methods , Gene Expression Profiling , RNA/isolation & purification , Sequence Analysis, RNA/methods , Animals , Decision Making , Fluorescent Dyes , Gene Expression , Hearing Loss/congenital , Hearing Loss/genetics , Humans , Mice , Mice, Transgenic , Organ of Corti/cytology , Pyridinium Compounds , Quaternary Ammonium Compounds , Ribosomes/metabolism , Single-Cell Analysis/methods , Tight JunctionsABSTRACT
Age-related hearing impairment (ARHI), one of the most common medical conditions, is strongly heritable, yet its genetic causes remain largely unknown. We conducted a meta-analysis of GWAS summary statistics from multiple hearing-related traits in the UK Biobank (n = up to 330,759) and identified 31 genome-wide significant risk loci for self-reported hearing difficulty (p < 5x10-8), of which eight have not been reported previously in the peer-reviewed literature. We investigated the regulatory and cell specific expression for these loci by generating mRNA-seq, ATAC-seq, and single-cell RNA-seq from cells in the mouse cochlea. Risk-associated genes were most strongly enriched for expression in cochlear epithelial cells, as well as for genes related to sensory perception and known Mendelian deafness genes, supporting their relevance to auditory function. Regions of the human genome homologous to open chromatin in epithelial cells from the mouse were strongly enriched for heritable risk for hearing difficulty, even after adjusting for baseline effects of evolutionary conservation and cell-type non-specific regulatory regions. Epigenomic and statistical fine-mapping most strongly supported 50 putative risk genes. Of these, 39 were expressed robustly in mouse cochlea and 16 were enriched specifically in sensory hair cells. These results reveal new risk loci and risk genes for hearing difficulty and suggest an important role for altered gene regulation in the cochlear sensory epithelium.
Subject(s)
Cochlea/cytology , Genetic Loci , Genetic Predisposition to Disease , Hearing Loss/genetics , Adult , Animals , Biological Specimen Banks , Chromatin/genetics , Cohort Studies , Epigenome , Epithelial Cells/physiology , Female , Genome-Wide Association Study , Hair Cells, Auditory/cytology , Hair Cells, Auditory/physiology , Humans , Mice, Inbred ICR , Mice, Inbred Strains , Polymorphism, Single Nucleotide , Single-Cell Analysis , United KingdomABSTRACT
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.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Hair Cells, Auditory, Inner/metabolism , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA-Binding Proteins/genetics , Hair Cells, Auditory, Inner/cytology , Mice , Mice, Transgenic , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factor Brn-3A/genetics , Transcription Factor Brn-3A/metabolism , Transcription Factors/geneticsABSTRACT
The sensory cells that are responsible for hearing include the cochlear inner hair cells (IHCs) and outer hair cells (OHCs), with the OHCs being necessary for sound sensitivity and tuning1. Both cell types are thought to arise from common progenitors; however, our understanding of the factors that control the fate of IHCs and OHCs remains limited. Here we identify Ikzf2 (which encodes Helios) as an essential transcription factor in mice that is required for OHC functional maturation and hearing. Helios is expressed in postnatal mouse OHCs, and in the cello mouse model a point mutation in Ikzf2 causes early-onset sensorineural hearing loss. Ikzf2cello/cello OHCs have greatly reduced prestin-dependent electromotile activity, a hallmark of OHC functional maturation, and show reduced levels of crucial OHC-expressed genes such as Slc26a5 (which encodes prestin) and Ocm. Moreover, we show that ectopic expression of Ikzf2 in IHCs: induces the expression of OHC-specific genes; reduces the expression of canonical IHC genes; and confers electromotility to IHCs, demonstrating that Ikzf2 can partially shift the IHC transcriptome towards an OHC-like identity.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation, Developmental/genetics , Hair Cells, Auditory, Outer/cytology , Hair Cells, Auditory, Outer/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Transcriptome/genetics , Animals , Base Sequence , Biomarkers/metabolism , Female , Male , Mice , Mice, Inbred C57BLABSTRACT
BACKGROUND: Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used. RESULTS: We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a 'gold standard' control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit. CONCLUSION: RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts.
Subject(s)
Gene Library , High-Throughput Nucleotide Sequencing/methods , Protein Biosynthesis , RNA, Messenger/metabolism , Ribosomes/metabolism , Sequence Analysis, RNA/veterinary , Transcriptome , Animals , Immunoprecipitation , Mice , Quality Control , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Ribosomes/geneticsABSTRACT
BACKGROUND: Noise-induced hearing loss (NIHL) is the most prevalent form of acquired hearing loss and affects about 40 million US adults. Among the suggested therapeutics tested in rodents, suberoylanilide hydroxamic acid (SAHA) has been shown to be otoprotective from NIHL; however, these results were limited to male mice. METHODS: Here we tested the effect of SAHA on the hearing of 10-week-old B6CBAF1/J mice of both sexes, which were exposed to 2 h of octave-band noise (101 dB SPL centered at 11.3 kHz). Hearing was assessed by measuring auditory brainstem responses (ABR) at 8, 16, 24, and 32 kHz, 1 week before, as well as at 24 h and 15-21 days following exposure (baseline, compound threshold shift (CTS) and permanent threshold shift (PTS), respectively), followed by histologic analyses. RESULTS: We found significant differences in the CTS and PTS of the control (vehicle injected) mice to noise, where females had a significantly smaller CTS at 16 and 24 kHz (p < 0.0001) and PTS at 16, 24, and 32 kHz (16 and 24 kHz p < 0.001, 32 kHz p < 0.01). This sexual dimorphic effect could not be explained by a differential loss of sensory cells or synapses but was reflected in the amplitude and amplitude progression of wave I of the ABR, which correlates with outer hair cell (OHC) function. Finally, the frequency of the protective effect of SAHA differed significantly between males (PTS, 24 kHz, p = 0.002) and females (PTS, 16 kHz, p = 0.003), and the magnitude of the protection was smaller in females than in males. Importantly, the magnitude of the protection by SAHA was smaller than the effect of sex as a biological factor in the vehicle-injected mice. CONCLUSIONS: These results indicate that female mice are significantly protected from NIHL in comparison to males and that therapeutics for NIHL may have a different effect in males and females. The data highlight the importance of analyzing NIHL experiments from males and females, separately. Finally, these data also raise the possibility of effectors in the estrogen signaling pathway as novel therapeutics for NIHL.
Subject(s)
Hearing Loss, Noise-Induced/prevention & control , Noise/adverse effects , Protective Agents/pharmacology , Sex Characteristics , Vorinostat/pharmacology , Animals , Evoked Potentials, Auditory, Brain Stem , Female , Male , MiceABSTRACT
Studies of developmental and functional biology largely rely on conditional expression of genes in a cell type-specific manner. Therefore, the importance of specificity and lack of inherent phenotypes for Cre-driver animals cannot be overemphasized. The Gfi1Cre mouse is commonly used for conditional hair cell-specific gene deletion/reporter gene activation in the inner ear. Here, using immunofluorescence and flow cytometry, we show that the Gfi1Cre mice produce a pattern of recombination that is not strictly limited to hair cells within the inner ear. We observe a broad expression of Cre recombinase in the Gfi1Cre mouse neonatal inner ear, primarily in inner ear resident macrophages, which outnumber the hair cells. We further show that heterozygous Gfi1Cre mice exhibit an early onset progressive hearing loss as compared with their wild-type littermates. Importantly, vestibular function remains intact in heterozygotes up to 10 months, the latest time point tested. Finally, we detect minor, but statistically significant, changes in expression of hair cell-enriched transcripts in the Gfi1Cre heterozygous mice cochleae compared with their wild-type littermate controls. Given the broad use of the Gfi1Cre mice, both for gene deletion and reporter gene activation, these data are significant and necessary for proper planning and interpretation of experiments.
Subject(s)
DNA-Binding Proteins/genetics , Ear, Inner/pathology , Hearing Loss/genetics , Hearing Loss/pathology , Integrases/genetics , Recombination, Genetic , Transcription Factors/genetics , Animals , DNA-Binding Proteins/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Integrases/metabolism , Mice , Transcription Factors/metabolismABSTRACT
Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.
Subject(s)
DNA-Binding Proteins/metabolism , Hair Cells, Auditory/metabolism , Hearing/physiology , Transcription Factors/metabolism , Animals , Animals, Newborn , Biological Evolution , Chromatin Immunoprecipitation , Female , Gene Expression Regulation , Hair Cells, Auditory/ultrastructure , Male , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Knockout , Multigene Family , Regulatory Factor X Transcription Factors , Regulatory Factor X1 , Sequence Analysis, DNA , Transcriptome , ZebrafishABSTRACT
BACKGROUND: Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification. RESULTS: We developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin "states", individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, we detected diverse "exceptions" from the conventional rules. We found a profound paucity of chromatin modifications in a major fraction of closed chromatin, and identified a number of loci where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, we observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin. CONCLUSIONS: These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromatin/genetics , Chromosome Mapping , Drosophila/genetics , Genome, Insect , Animals , Binding Sites , Chromatin/metabolism , Computational Biology/methods , Deoxyribonuclease I/metabolism , Protein BindingABSTRACT
Theoretical models suggest that gene silencing at the nuclear periphery may involve "closing" of chromatin by transcriptional repressors, such as histone deacetylases (HDACs). Here we provide experimental evidence confirming these predictions. Histone acetylation, chromatin compactness, and gene repression in lamina-interacting multigenic chromatin domains were analyzed in Drosophila S2 cells in which B-type lamin, diverse HDACs, and lamina-associated proteins were downregulated by dsRNA. Lamin depletion resulted in decreased compactness of the repressed multigenic domain associated with its detachment from the lamina and enhanced histone acetylation. Our data reveal the major role for HDAC1 in mediating deacetylation, chromatin compaction, and gene silencing in the multigenic domain, and an auxiliary role for HDAC3 that is required for retention of the domain at the lamina. These findings demonstrate the manifold and central involvement of class I HDACs in regulation of lamina-associated genes, illuminating a mechanism by which these enzymes can orchestrate normal and pathological development.
Subject(s)
Chromatin/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation , Histone Deacetylase 1/genetics , Histone Deacetylases/genetics , Histones/genetics , Nuclear Lamina/genetics , Acetylation , Animals , Blotting, Western , Cell Line , Chromatin/enzymology , Chromatin Immunoprecipitation , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/enzymology , Gene Silencing , Histone Deacetylase 1/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Lamin Type B/antagonists & inhibitors , Lamin Type B/genetics , Lamin Type B/metabolism , Multigene Family , Nuclear Lamina/enzymology , RNA, Double-Stranded/genetics , Transcription, GeneticABSTRACT
BACKGROUND: A marked decrease in the level of zinc is a consistent characteristic of prostate cancer; which results from down-regulation of ZIP1 zinc transporter. The aim of this study was to determine if RREB-1 transcription is involved in the down-regulation of ZIP1 gene expression; and to determine the expression of RREB-1 in benign and cancerous prostate in situ. METHODS: Overexpression and siRNA knock down of RREB-1 were used to determine the effect of RREB-1 on hZIP1 abundance in PC-3 cells. Immunohistochemistry with tissue microarrays (TMAs) and tissue sections was used to determine the levels of RREB-1 expression in prostate in situ. RESULTS: Overexpression of RREB-1 resulted in a decrease in the abundance of hZIP1 in the plasma membrane of PC-3 cells; whereas siRNA knock down significantly increased hZIP1 expression. Prostate TMAs and tissue sections showed an inverse relationship between RREB-1 and hZIP1 staining. CONCLUSIONS: RREB-1 overexpression results in down-regulation of hZIP1 and contributes to the loss of hZIP1 expression and zinc in prostate cancer. This is an early event in prostate carcinogenesis.