Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression

The Prosensory Function of Sox2 in the Chicken Inner Ear Relies on the Direct Regulation of Atoh1

The proneural gene Atoh1 is crucial for the development of inner ear hair cells and it requires the function of the transcription factor Sox2 through yet unknown mechanisms. In the present work, we used the chicken embryo and HEK293T cells to explore the regulation of Atoh1 by Sox2. The results show that hair cells derive from Sox2-positive otic progenitors and that Sox2 directly activates Atoh1 through a transcriptional activator function that requires the integrity of Sox2 DNA binding domain. Atoh1 activation depends on Sox transcription factor binding sites (SoxTFBS) present in the Atoh1 3′ enhancer where Sox2 directly binds, as shown by site directed mutagenesis and chromatin immunoprecipitation (ChIP). In the inner ear, Atoh1 enhancer activity is detected in the neurosensory domain and it depends on Sox2. Dominant negative competition (Sox2HMG-Engrailed) and mutation of the SoxTFBS abolish the reporter activity in vivo. Moreover, ChIP assay in isolated otic vesicles shows that Sox2 is bound to the Atoh1 enhancer in vivo. However, besides activating Atoh1, Sox2 also promotes the expression of Atoh1 negative regulators and the temporal profile of Atoh1 activation by Sox2 is transient suggesting that Sox2 triggers an incoherent feed-forward loop. These results provide a mechanism for the prosensory function of Sox2 in the inner ear. We suggest that sensory competence is established early in otic development through the activation of Atoh1 by Sox2, however, hair cell differentiation is prevented until later stages by the parallel activation of negative regulators of Atoh1 function

Effects of DAPT and Atoh1 Overexpression on Hair Cell Production and Hair Bundle Orientation in Cultured Organ of Corti from Neonatal Rats

BACKGROUND: In mammals, hair cells do not undergo spontaneous regeneration when they are damaged and result in permanent hearing loss. Previous studies in cultured Organ of Corti dissected from neonatal animals have shown that both DAPT (r-secretase inhibitor in the Notch signal pathway) treatment and Atoh1 overexpression can induce supernumerary hair cells. The effects of simultaneous DAPT treatment and Atoh1 over expression in the cells of cultured Organ of Corti from neonatal rats are still obscure. PRINCIPAL FINDINGS: In this study, we set out to investigate the interaction of DAPT treatment and Atoh1 overexpression as well as culture time and the location of basilar fragment isolated form neonatal rat inner ear. Our results showed that DAPT treatment induced more hair cells in the apical turn, while Atoh1 overexpression induced more extra hair cells in the middle turn of the cultured Organ of Corti. When used together, their effects are additive but not synergistic. In addition, the induction of supernumerary hair cells by both DAPT and Atoh1 overexpression is dependent on the treatment time and the location of the cochlear tissue. Moreover, DAPT treatment causes dramatic changes in the orientation of the stereociliary bundles of hair cells, whereas Atoh1 overexpression didn't induce drastic change of the polarity of stereociliary bundles. CONCLUSIONS/SIGNIFICANCE: Taken together, these results suggest that DAPT treatment are much more potent in inducing supernumerary hair cells than Atoh1 overexpression and that the new hair cells mainly come from the trans-differentiation of supporting cells around hair cells. The orientation change of stereociliary bundle of hair cells may be attributed to the insertion of the newly formed hair cells. The immature hair bundles on the newly formed hair cells may also contribute to the overall chaos of the stereociliary bundle of the sensory epithelia

Sp1 Is Essential for p16(INK4a) Expression in Human Diploid Fibroblasts during Senescence

BACKGROUND: p16 (INK4a) tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16 (INK4a) gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from −466 to −451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts. METHODOLOGY/PRINCIPAL FINDINGS: Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16 (INK4a) gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16 (INK4a). Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16 (INK4a) gene expression. In addition, the enhanced binding of Sp1 to p16 (INK4a) promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level. CONCLUSIONS/SIGNIFICANCE: All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16 (INK4a) expression during cell aging

Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea

Abstract\ud \ud Background\ud Culturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy.\ud \ud \ud Methods\ud Organs of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFα). Immunofluorescence assays were conducted for phenotype characterization.\ud \ud \ud Results\ud The TGFα group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells.\ud \ud \ud Conclusions\ud Dissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage

Stemness of the Organ of Corti Relates to the Epigenetic Status of Sox2 Enhancers

In the adult mammalian auditory epithelium, the organ of Corti, loss of sensory hair cells results in permanent hearing loss. The underlying cause for the lack of regenerative response is the depletion of otic progenitors in the cell pool of the sensory epithelium. Here, we show that an increase in the sequence-specific methylation of the otic Sox2 enhancers NOP1 and NOP2 is correlated with a reduced self-renewal potential in vivo and in vitro; additionally, the degree of methylation of NOP1 and NOP2 is correlated with the dedifferentiation potential of postmitotic supporting cells into otic stem cells. Thus, the stemness the organ of Corti is related to the epigenetic status of the otic Sox2 enhancers. These observations validate the continued exploration of treatment strategies for dedifferentiating or reprogramming of differentiated supporting cells into progenitors to regenerate the damaged organ of Corti

NFATc1 Regulation of TRAIL Expression in Human Intestinal Cells

TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter

Hes5 Expression in the Postnatal and Adult Mouse Inner Ear and the Drug-Damaged Cochlea

The Notch signaling pathway is known to have multiple roles during development of the inner ear. Notch signaling activates transcription of Hes5, a homologue of Drosophila hairy and enhancer of split, which encodes a basic helix-loop-helix transcriptional repressor. Previous studies have shown that Hes5 is expressed in the cochlea during embryonic development, and loss of Hes5 leads to overproduction of auditory and vestibular hair cells. However, due to technical limitations and inconsistency between previous reports, the precise spatial and temporal pattern of Hes5 expression in the postnatal and adult inner ear has remained unclear. In this study, we use Hes5-GFP transgenic mice and in situ hybridization to report the expression pattern of Hes5 in the inner ear. We find that Hes5 is expressed in the developing auditory epithelium of the cochlea beginning at embryonic day 14.5 (E14.5), becomes restricted to a particular subset of cochlear supporting cells, is downregulated in the postnatal cochlea, and is not present in adults. In the vestibular system, we detect Hes5 in developing supporting cells as early as E12.5 and find that Hes5 expression is maintained in some adult vestibular supporting cells. In order to determine the effect of hair cell damage on Notch signaling in the cochlea, we damaged cochlear hair cells of adult Hes5-GFP mice in vivo using injection of kanamycin and furosemide. Although outer hair cells were killed in treated animals and supporting cells were still present after damage, supporting cells did not upregulate Hes5-GFP in the damaged cochlea. Therefore, absence of Notch-Hes5 signaling in the normal and damaged adult cochlea is correlated with lack of regeneration potential, while its presence in the neonatal cochlea and adult vestibular epithelia is associated with greater capacity for plasticity or regeneration in these tissues; which suggests that this pathway may be involved in regulating regenerative potential

Conditional Ablation of Ezh2 in Murine Hearts Reveals Its Essential Roles in Endocardial Cushion Formation, Cardiomyocyte Proliferation and Survival

Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development

Expression of Neurog1 Instead of Atoh1 Can Partially Rescue Organ of Corti Cell Survival

In the mammalian inner ear neurosensory cell fate depends on three closely related transcription factors, Atoh1 for hair cells and Neurog1 and Neurod1 for neurons. We have previously shown that neuronal cell fate can be altered towards hair cell fate by eliminating Neurod1 mediated repression of Atoh1 expression in neurons. To test whether a similar plasticity is present in hair cell fate commitment, we have generated a knockin (KI) mouse line (Atoh1KINeurog1) in which Atoh1 is replaced by Neurog1. Expression of Neurog1 under Atoh1 promoter control alters the cellular gene expression pattern, differentiation and survival of hair cell precursors in both heterozygous (Atoh1+/KINeurog1) and homozygous (Atoh1KINeurog1/KINeurog1) KI mice. Homozygous KI mice develop patches of organ of Corti precursor cells that express Neurog1, Neurod1, several prosensory genes and neurotrophins. In addition, these patches of cells receive afferent and efferent processes. Some cells among these patches form multiple microvilli but no stereocilia. Importantly, Neurog1 expressing mutants differ from Atoh1 null mutants, as they have intermittent formation of organ of Corti-like patches, opposed to a complete ‘flat epithelium’ in the absence of Atoh1. In heterozygous KI mice co-expression of Atoh1 and Neurog1 results in change in fate and patterning of some hair cells and supporting cells in addition to the abnormal hair cell polarity in the later stages of development. This differs from haploinsufficiency of Atoh1 (Pax2cre; Atoh1f/+), indicating the effect of Neurog1 expression in developing hair cells. Our data suggest that Atoh1KINeurog1 can provide some degree of functional support for survival of organ of Corti cells. In contrast to the previously demonstrated fate plasticity of neurons to differentiate as hair cells, hair cell precursors can be maintained for a limited time by Neurog1 but do not transdifferentiate as neurons