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The molecular mechanisms allowing hair follicles to periodically activate their stem cells (HFSCs) are incompletely characterized. Here, we identify the transcription factor IRX5 as a promoter of HFSC activation. Irx5-/- mice have delayed anagen onset, with increased DNA damage and diminished HFSC proliferation. Open chromatin regions form near cell cycle progression and DNA damage repair genes in Irx5-/- HFSCs. DNA damage repair factor BRCA1 is an IRX5 downstream target. Inhibition of FGF kinase signaling partially rescues the anagen delay in Irx5-/- mice, suggesting that the Irx5-/- HFSC quiescent phenotype is partly due to failure to suppress Fgf18 expression. Interfollicular epidermal stem cells also show decreased proliferation and increased DNA damage in Irx5-/-mice. Consistent with a role for IRX5 as a promoter of DNA damage repair, we find that IRX genes are upregulated in many cancer types and that there is a correlation between IRX5 and BRCA1 expression in breast cancer.

Previous studies have established that transmural gradients of the fast transient outward K+ current (Ito,f) correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high Ito,f expression in the epimyocardium (EPI) being associated with short APs and low contractility and vice versa. Herein, we investigated the effects of altering the Ito,f gradients on transmural contractile properties using mice lacking Irx5 (Irx5-KO) or lacking Kcnd2 (KV4.2-KO) or both. Irx5- KO mice exhibited decreased global LV contractility in association with elevated Ito,f, as well as reduced cell shortening and Ca2+ transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of Irx5 ablation on ECC-related genes was to increase Ito,f gene expression (i.e., Kcnd2 and Kcnip2) in the ENDO, but not the EPI. By contrast, KV4.2-KO mice showed selective increases in cell shortening and Ca2+ transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both Irx5 and Kcnd2 displayed elevated ventricular contractility, comparable to KV4.2-KO mice, demonstrating a dominant role of Irx5- dependent modulation of Ito,f in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the Irx5-dependent Ito,f gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart. [ABSTRACT FROM AUTHOR]

Aims Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. Methods and results Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. Conclusion Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases. [ABSTRACT FROM AUTHOR]

Background/Aims: The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene was first found to be activated in colorectal neoplasia. Now, it also has been found to be upregulated in many other solid tumors. Whether CRNDE affects tumorigenesis remains unknown. Methods: We conducted bioinformatics, real-time polymerase chain reaction (PCR), Western blot analysis, cell proliferation assay, colony formation assay, wound healing assay, cell migration and invasion assays, RNA immunoprecipitation, and reporter vector construction and luciferase assays. Results: CRNDE was upregulated in hepatocellular carcinoma (HCC). The overexpression of CRNDE promoted HCC cellular proliferation, migration, and invasion in intro and in vivo, and acted as an oncogene in HCC progression. Furthermore, CRNDE impaired miR-136-5P expression in a RISC manner, and a reciprocal repression feedback loop was possible between CRNDE and miR-136-5P. We found that the neighboring mRNA of CRNDE was IRX5, and IRX5 increased the tumorigenicity of HCC cells. IRX5 was a potential downstream target gene of miR-136-5P. MiR-136 regulated IRX5 by interacting with its 3’UTR. In addition, miR-136-5P was involved in the CRNDE-regulated expression of IRX5. Conclusion: CRNDE acted as a tumor oncogene by exhibiting oncogenic properties of human HCC and revealed a novel CRNDE-miR-136-5P-IRX5 regulatory network in HCC. CRNDE may be considered to be a potential target for HCC therapies based on its ability to upregulate IRX5, and it deserves further investigation.

Background/Aims: The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene was first found to be activated in colorectal neoplasia. Now, it also has been found to be upregulated in many other solid tumors. Whether CRNDE affects tumorigenesis remains unknown. Methods: We conducted bioinformatics, real-time polymerase chain reaction (PCR), Western blot analysis, cell proliferation assay, colony formation assay, wound healing assay, cell migration and invasion assays, RNA immunoprecipitation, and reporter vector construction and luciferase assays. Results: CRNDE was upregulated in hepatocellular carcinoma (HCC). The overexpression of CRNDE promoted HCC cellular proliferation, migration, and invasion in intro and in vivo, and acted as an oncogene in HCC progression. Furthermore, CRNDE impaired miR-136-5P expression in a RISC manner, and a reciprocal repression feedback loop was possible between CRNDE and miR-136-5P. We found that the neighboring mRNA of CRNDE was IRX5, and IRX5 increased the tumorigenicity of HCC cells. IRX5 was a potential downstream target gene of miR-136-5P. MiR-136 regulated IRX5 by interacting with its 3'UTR. In addition, miR-136-5P was involved in the CRNDE-regulated expression of IRX5. Conclusion: CRNDE acted as a tumor oncogene by exhibiting oncogenic properties of human HCC and revealed a novel CRNDE-miR-136-5P-IRX5 regulatory network in HCC. CRNDE may be considered to be a potential target for HCC therapies based on its ability to upregulate IRX5, and it deserves further investigation. [ABSTRACT FROM AUTHOR]

The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3-14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2'-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27kip1), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle check-point control and apoptosis. [ABSTRACT FROM AUTHOR]

Background: The long noncoding RNA (lncRNA) colorectal neoplasia differentially expressed - h (CRNDE-h) plays important roles in the early stages of human development and cancer progression. We investigated the expression and clinical significance of lncRNA CRNDE-h in colorectal cancer (CRC). Methods: The expression level of lncRNA CRNDE-h was analyzed in 142 CRC tissues and 142 paired adjacent nontumorous tissues, along with 21 inflammatory bowel diseases, 69 hyperplastic polyp, and 73 colorectal adenoma samples, using quantitative real-time polymerase chain reaction. The association between lncRNA CRNDE-h, and Iroquois homeobox protein 5 (IRX5) mRNA was examined in the same 142 CRC tissues. Results: We found that lncRNA CRNDE-h level was elevated in the CRC and adenoma groups compared with the other groups (all at P<0.001). In CRC, upregulation of lncRNA CRNDE-h was significantly correlated with large tumor size, positive regional lymph node metastasis, and distant metastasis (all at P<0.05). Area under the curve for lncRNA CRNDE-h showed diagnostic capability for distinguishing CRC from other groups. Patients with CRC with high lncRNA CRNDE-h expression level had poorer overall survival than those with low lncRNA CRNDE-h expression (log-rank test, P<0.001). Further, multivariable Cox regression analysis suggested that increased expression of lncRNA CRNDE-h was an independent prognostic indicator for CRC (hazard ratio [HR]=2.173; 95% confidence interval [CI], 1.282-3.684, P=0.004). Furthermore, lncRNA CRNDE-h expression was positively correlated with IRX5 mRNA in CRC tissues. Conclusions: Our data offers convincing evidence for the first time that lncRNA CRNDE-h is associated with adverse clinical characteristics and poor prognosis, which suggests that it might play an important role in CRC development and progression and might have clinical potential as a useful prognostic predictor. [ABSTRACT FROM AUTHOR]

Abstract: Sonic Hedgehog (Shh) signaling helps pattern the vertebrate neural tube, in part by regulating the dorsal/ventral expression of a number of homeodomain containing transcription factors. These Hh responsive genes have been divided into two classes, with Class II genes being activated by Hh signaling and Class I genes being repressed by Hh signaling. While the transcriptional response to varying Hh levels is well defined in chick and mouse, it is only partially described in zebrafish, despite the fact that zebrafish has emerged as a powerful genetic system for the study of neural patterning. To better characterize the Hh response in the zebrafish neural tube, we cloned the zebrafish Class II Hh target genes nkx2.9 and nkx6.2. We then analyzed the expression of a number of Class I and Class II Hh responsive genes in wild type, Hh mutant, and Hh over-expressing zebrafish embryos. We show that expression of Class I and Class II genes is highly conserved in the vertebrate neural tube. Further, ventral-most Class II gene expression was completely lost in all Hh pathway mutants analyzed, indicating high levels of Hh signaling are blocked in all of these mutants. In contrast, more dorsally expressed genes were variably affected in different Hh pathway mutants, indicating mid-levels of Hh signaling are differentially affected. This comprehensive expression study provides an important tool for the characterization of Hh signaling in zebrafish and provides a sensitive assay for determining the degree to which newly identified zebrafish mutants affect Hh signaling. [Copyright &y& Elsevier]

Abstract: In the mouse retina, at least ten distinct types of bipolar interneurons are involved in the transmission of visual signals from photoreceptors to ganglion cells. How bipolar interneuron diversity is generated during retinal development is poorly understood. Here, we show that Irx5, a member of the Iroquois homeobox gene family, is expressed in developing bipolar cells starting at postnatal day 5 and is localized to a subset of cone bipolar cells in the mature mouse retina. In Irx5-deficient mice, defects were observed in the expression of some, but not all, immunohistological markers that define mature Type 2 and Type 3 OFF cone bipolar cells, indicating a role for Irx5 in bipolar cell differentiation. The differentiation of these two bipolar cell types has previously been shown to require the homeodomain-CVC transcription factor, Vsx1. However, the defects observed in Irx5-deficient retinas do not coincide with a reduction of Vsx1 expression, and conversely, the expression of Irx5 in cone bipolar cells does not require the presence of a functional Vsx1 allele. These results indicate that there are at least two distinct genetic pathways (Irx5-dependent and Vsx1-dependent) regulating the development of Type 2 and Type 3 cone bipolar cells. [Copyright &y& Elsevier]

Previous studies have established that transmural gradients of the fast transient outward K + current ( I to,f ) correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high I to,f (Irx5-KO) or lacking I to,f 4.2-KO) or both. Irx5-KO mice exhibited decreased global LV contractility in association with elevated Irx5 (Irx5-KO) or lacking Kcnd2 (K V 4.2-KO) or both. Irx5-KO mice exhibited decreased global LV contractility in association with elevated I to,f , as well as reduced cell shortening and Ca 2+ transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of Irx5 ablation on ECC-related genes was to increase I to,f gene expression (i.e., Kcnd2 and Kcnip2 ) in the ENDO, but not the EPI. By contrast, K V 4.2-KO mice showed selective increases in cell shortening and Ca 2+ transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both Irx5 and Kcnd2 displayed elevated ventricular contractility, comparable to K V Irx5 is a vital transcription factor that establishes the transmural heterogeneity of ventricular myocyte contractility, thereby ensuring proper contractile function in the healthy heart. Regional differences in excitation-contraction coupling in the ventricular myocardium are primarily mediated through the inverse relationship between Irx5 and the fast transient outward K Irx5 -dependent modulation of I to,f in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the Irx5 -dependent I to,f gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart. NEW & NOTEWORTHY Irx5 is a vital transcription factor that establishes the transmural heterogeneity of ventricular myocyte contractility, thereby ensuring proper contractile function in the healthy heart. Regional differences in excitation-contraction coupling in the ventricular myocardium are primarily mediated through the inverse relationship between Irx5 and the fast transient outward K + current ( I to,f ) across the ventricular wall.

The author discusses functional circuitry of fat mass and obesity associated gene (FTO) locus and adipocyte browning in humans.

We report on 2 cousins, a girl and a boy, born to first-cousin Lebanese parents with Hamamy syndrome, exhibiting developmental delay, intellectual disability, severe telecanthus, abnormal ears, dentinogenesis imperfecta, and bone fragility. Whole-exome sequencing studies performed on the 2 affected individuals and one obligate carrier revealed the presence of a homozygous c.503G>A (p.Arg168His) missense mutation in IRX5 in both sibs, not reported in any other family. Review of the literature and differential diagnoses are discussed., Competing Interests: The authors have no conflicts of interest to report., (Copyright © 2021 by S. Karger AG, Basel.)

Aims: Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function., Methods and Results: Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression., Conclusion: Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Healthy development of ovarian follicles depends on appropriate interactions and function between oocytes and their surrounding granulosa cells. Previously, we showed that double knockout of Irx3 and Irx5 (Irx3/5 DKO) in mice resulted in abnormal follicle morphology and follicle death. Further, female mouse models of individual Irx3 or Irx5 knockouts were both subfertile but with distinct defects. Notably, the expression profile of each gene suggests independent roles for each; first, they are colocalized in pre-granulosa cells during development that then progresses to include oocyte expression during germline nest breakdown and primordial follicle formation. Thereafter, their expression patterns diverge between oocytes and granulosa cells coinciding with the formulation and maturation of intimate oocyte-granulosa cell interactions. The objective of this study was to investigate the contributions of Irx5 and somatic cell-specific expression of Irx3 during ovarian development. Our results show that Irx3 and Irx5 contribute to female fertility through different mechanisms and that Irx3 expression in somatic cells is important for oocyte quality and survival. Based on evaluation of a series of genetically modified mouse models, we conclude that IRX3 and IRX5 collaborate in the same cells and then in neighboring cells to foster a healthy and responsive follicle. Long after these two factors have extinguished, their legacy enables these intercellular connections to mature and respond to extracellular signals to promote follicle maturation and ovulation., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3-14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2'-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27(kip1)), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis., (Copyright © 2016 the American Physiological Society.)

CRNDE is the gene symbol for Colorectal Neoplasia Differentially Expressed (non-protein-coding), a long non-coding RNA (lncRNA) gene that expresses multiple splice variants and displays a very tissue-specific pattern of expression. CRNDE was initially identified as a lncRNA whose expression is highly elevated in colorectal cancer, but it is also upregulated in many other solid tumors and in leukemias. Indeed, CRNDE is the most upregulated lncRNA in gliomas and here, as in other cancers, it is associated with a "stemness" signature. CRNDE is expressed in specific regions within the human and mouse brain; the mouse ortholog is high in induced pluripotent stem cells and increases further during neuronal differentiation. We suggest that CRNDE is a multifunctional lncRNA whose different splice forms provide specific functional scaffolds for regulatory complexes, such as the polycomb repressive complex 2 (PRC2) and CoREST chromatin-modifying complexes, which CRNDE helps pilot to target genes.