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Skin homeostasis is indispensable for terrestrial life. The skin, sitting at the interface between the dry external environment and the aqueous interior, acts as a barrier to delimit each from the other. Maintaining this barrier function is critical – if it is compromised, pathogen ingress and water loss may have potentially severe consequences. Skin function is supported by an intricate regulatory network incorporating both protein-coding genes as well as non-coding RNA transcripts. This review will focus on microRNAs, a subset of non-coding RNAs which target specific messenger RNAs for post-transcriptional repression. MicroRNAs regulate every aspect of skin biology, from initial cell fate determination during embryogenesis, to steady state keratinocyte maturation and barrier renewal, as well as coordination of the healing process in response to skin damage. An increased appreciation for the role of microRNAs in skin pathologies not only sheds light on the importance of these small RNAs in healthy skin function, but also provides an expanding repertoire of potential therapeutic targets through which these conditions may be addressed. Mammalian skin appears simple. Its core function is to shield the body from the external environment. Water must be retained to sustain life, whilst harmful pathogens and other environmental insults must be kept out. This simplicity in function belies the array of intricate processes necessary to maintain the skin’s barrier function – multiple series of highly-ordered molecular and cellular events control constant epidermal rejuvenation, as well as rapid repair of any epithelial damage sustained. For internal homeostasis to be maintained, epithelial integrity is essential, and contingent upon precise spatiotemporal control of protein expression in the skin.

Background Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by epigenetic silencing of FMR1 and loss of FMRP expression. Efforts to understand the molecular underpinnings of the disease have been largely performed in rodent or nonisogenic settings. A detailed examination of the impact of FMRP loss on cellular processes and neuronal properties in the context of isogenic human neurons remains lacking. Methods Using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to introduce indels in exon 3 of FMR1, we generated an isogenic human pluripotent stem cell model of FXS that shows complete loss of FMRP expression. We generated neuronal cultures and performed genome-wide transcriptome and proteome profiling followed by functional validation of key dysregulated processes. We further analyzed neurodevelopmental and neuronal properties, including neurite length and neuronal activity, using multielectrode arrays and patch clamp electrophysiology. Results We showed that the transcriptome and proteome profiles of isogenic FMRP-deficient neurons demonstrate perturbations in synaptic transmission, neuron differentiation, cell proliferation and ion transmembrane transporter activity pathways, and autism spectrum disorder–associated gene sets. We uncovered key deficits in FMRP-deficient cells demonstrating abnormal neural rosette formation and neural progenitor cell proliferation. We further showed that FMRP-deficient neurons exhibit a number of additional phenotypic abnormalities, including neurite outgrowth and branching deficits and impaired electrophysiological network activity. These FMRP-deficient related impairments have also been validated in additional FXS patient–derived human-induced pluripotent stem cell neural cells. Conclusions Using isogenic human pluripotent stem cells as a model to investigate the pathophysiology of FXS in human neurons, we reveal key neural abnormalities arising from the loss of FMRP.

Complex multicellular organisms have evolved sophisticated mechanisms to rapidly resolve epithelial injuries. Epithelial integrity is critical to maintaining internal homeostasis. An epithelial breach represents the potential for pathogen ingress and fluid loss, both of which may have severe consequences if not limited. The mammalian wound healing response involves a finely tuned, self-limiting series of cellular and molecular events orchestrated by the transient activation of specific signalling pathways. Accurate regulation of these events is essential; failure to initiate key steps at the right time delays healing and leads to chronic wounds, while aberrant initiation of wound healing processes may produce cell behaviours that promote cancer progression. In this review, we discuss how wound healing pathways co-opted in cancer lose their stringent regulation and become compromised in their reversibility. We hypothesize on how the commandeering of wound healing 'master regulators' is involved in this process, and also highlight the implications of these findings in the treatment of both chronic wounds and cancer.

MicroRNA-138 (miR-138) is a pro-survival oncomiR for glioma stem cells. In malignant gliomas, dysregulated expression of microRNAs, such as miR-138, promotes Tumour initiation and progression. Here, we identify the ancillary role of the CCAAT/enhancer binding protein β (C/EBPβ) as a transcriptional activator of miR-138. We demonstrate that a short 158 bp DNA sequence encoding the precursor of miR-138-2 is essential and sufficient for transcription of miR-138. This short sequence includes the A-box and B-box elements characteristic of RNA Polymerase III (Pol III) promoters, and is also directly bound by C/EBPβ via an embedded ‘C/EBPβ responsive element’ (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBPβ and transcription factor 3C (TFIIIC) interact at the miR-138-2 locus. We propose that this interaction is essential for the recruitment of the RNA Pol III initiation complex and associated transcription of the oncomiR, miR-138 in malignant gliomas.

Exploring the parallels between wound healing and epithelial cancers, Sundaram et al. elucidate the mechanism by which cancer cells hijack the wound healing switch to enhance invasion and metastasis in head and neck squamous cell carcinoma., Epithelial carcinomas are well known to activate a prolonged wound-healing program that promotes malignant transformation. Wound closure requires the activation of keratinocyte migration via a dual-state molecular switch. This switch involves production of either the anti-migratory microRNA miR-198 or the pro-migratory follistatin-like 1 (FSTL1) protein from a single transcript; miR-198 expression in healthy skin is down-regulated in favor of FSTL1 upon wounding, which enhances keratinocyte migration and promotes re-epithelialization. Here, we reveal a defective molecular switch in head and neck squamous cell carcinoma (HNSCC). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIAPH1 and FSTL1. DIAPH1, a miR-198 target, enhances directional migration through sequestration of Arpin, a competitive inhibitor of Arp2/3 complex. FSTL1 blocks Wnt7a-mediated repression of extracellular signal–regulated kinase phosphorylation, enabling production of MMP9, which degrades the extracellular matrix and facilitates metastasis. The prognostic significance of the FSTL1-DIAPH1 gene pair makes it an attractive target for therapeutic intervention.

Breast cancer manifests as a spectrum of subtypes with distinct molecular signatures, and different responses to treatment. Of these subtypes, triple-negative breast cancer (TNBC) has the worst prognoses and limited therapeutic options. Here we report aberrant expression of microRNA-138 (miR-138) in TNBC. Increased miR-138 expression is highly specific to this subtype, correlates with poor prognosis in patients, and is functionally relevant to cancer progression. Our findings establish miR-138 as a specific diagnostic and prognostic biomarker for TNBC. OncomiR-138 is pro-survival; sequence-specific miR-138 inhibition blocks proliferation, promotes apoptosis and inhibits tumour growth in-vivo. miR-138 directly targets a suite of pro-apoptotic and tumour suppressive genes, including tumour suppressor candidate 2 (TUSC2). miR-138 silences TUSC2 by binding to a unique 5′-UTR target-site, which overlaps with the translation start-site of the transcript. Over-expression of TUSC2 mimics the phenotype of miR-138 knockdown and functional rescue experiments confirm that TUSC2 is a direct downstream target of miR-138. Our report of miR-138 as an oncogenic driver in TNBC, positions it as a viable target for oligonucleotide therapeutics and we envision the potential value of using antimiR-138 as an adjuvant therapy to alleviate this therapeutically intractable cancer.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism., Small open reading frame-encoded peptides (SEPs), shorter than 100 amino acids, are involved in many cell biological processes. Here the authors identify 16 nuclear-encoded mitochondrial SEPs, including BRAWNIN, an essential regulator of respiratory chain complex III assembly and ATP production.

Background Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Skin barrier defects contribute to disease initiation and development; however, underlying mechanisms remain elusive. Objective To understand the underlying cause of barrier defect, we investigated aberrant expression of specific microRNAs (miRNAs) in AD. Delineating the molecular mechanism of dysregulated miRNA network, we focused on identification of specific drugs that can modulate miRNA expression and repair the defective barrier in AD. Methods A screen for differentially expressed miRNAs between healthy skin and AD lesional skin resulted in the identification of miR-335 as the most consistently downregulated miRNA in AD. Using in silico prediction combined with experimental validation, we characterized downstream miR-335 targets and elucidated the molecular pathways by which this microRNA maintains epidermal homeostasis in healthy skin. Results miR-335 was identified as a potent inducer of keratinocyte differentiation; it exerts this effect by directly repressing SOX6. By recruiting SMARCA complex components, SOX6 suppresses epidermal differentiation and epigenetically silences critical genes involved in keratinocyte differentiation. In AD lesional skin, miR-335 expression is aberrantly lost. SOX6 is abnormally expressed throughout the epidermis, where it impairs skin barrier development. We demonstrate that miR-335 is epigenetically regulated by histone deacetylases; a screen for suitable histone deacetylase inhibitors identified belinostat as a candidate drug that can restore epidermal miR-335 expression and rescue the defective skin barrier in AD. Conclusion Belinostat is of clinical significance not only as a candidate drug for AD treatment, but also as a potential means of stopping the atopic march and further progression of this systemic allergic disease.

Background Commitment of pluripotent stem cells into differentiated cells and associated gene expression necessitate specific epigenetic mechanisms that modify the DNA and corresponding histone proteins to render the chromatin in an open or closed state. This in turn dictates the associated genetic machinery, including transcription factors, acknowledging the cellular signals provided. Activating histone methyltransferases represent crucial enzymes in the epigenetic machinery that cause transcription initiation by delivering the methyl mark on histone proteins. A number of studies have evidenced the vital role of one such histone modifier, DOT1L, in transcriptional regulation. Involvement of DOT1L in differentiating pluripotent human embryonic stem (hES) cells into the cardiac lineage has not yet been investigated. Methods The study was conducted on in-house derived (KIND1) and commercially available (HES3) human embryonic stem cell lines. Chromatin immunoprecipitation (ChIP) was performed followed by sequencing to uncover the cardiac genes harboring the DOT1L specific mark H3K79me2. Following this, dual immunofluorescence was employed to show the DOT1L co-occupancy along with the cardiac progenitor specific marker. DOT1L was knocked down by siRNA to further confirm its role during cardiac differentiation. Results ChIP sequencing revealed a significant number of peaks characterizing H3K79me2 occupancy in the proximity of the transcription start site. This included genes like MYOF, NR2F2, NKX2.5, and HAND1 in cardiac progenitors and cardiomyocytes, and POU5F1 and NANOG in pluripotent hES cells. Consistent with this observation, we also show that DOT1L co-localizes with the master cardiac transcription factor NKX2.5, suggesting its direct involvement during gene activation. Knockdown of DOT1L did not alter the pluripotency of hES cells, but it led to the disruption of cardiac differentiation observed morphologically as well as at transcript and protein levels. Conclusions Collectively, our data suggests the crucial role of H3K79me2 methyltransferase DOT1L for activation of NKX2.5 during the cardiac differentiation of hES cells. Electronic supplementary material The online version of this article (10.1186/s13287-018-0810-8) contains supplementary material, which is available to authorized users.

The repression of telomerase activity during cellular differentiation promotes replicative aging and functions as a physiological barrier for tumorigenesis in long-lived mammals, including humans. However, the underlying mechanisms remain largely unclear. Here we describe how miR-615-3p represses hTERT expression. mir-615-3p is located in an intron of the HOXC5 gene, a member of the highly conserved homeobox family of transcription factors controlling embryogenesis and development. Unexpectedly, we found that HoxC5 also represses hTERT expression by disrupting the long-range interaction between hTERT promoter and its distal enhancer. The 3′UTR of hTERT and its upstream enhancer region are well conserved in long-lived primates. Both mir-615-3p and HOXC5 are activated upon differentiation, which constitute a feed-forward loop that coordinates transcriptional and post-transcriptional repression of hTERT during cellular differentiation. Deregulation of HOXC5 and mir-615-3p expression may contribute to the activation of hTERT in human cancers., The expression of telomerase catalytic subunit hTERT is frequently upregulated in many cancers. Here, the authors show HoxC5 and miR-615-3p can negatively regulate hTERT to impede tumorigenesis by targeting the newly evolved cis-regulatory genomic elements of hTERT.

Human embryonic (hES) stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development. In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential ChIP revealed the binding of NR2F2 with polycomb group member EZH2 and pluripotent factor OCT4 indicating its crucial involvement in cardiac differentiation. The study provides a detailed insight into genetic and epigenetic changes associated with hES cells differentiation into cardiac cells and a role for NR2F2 is deciphered for the first time to down-regulate OCT-4 via EZH2 during cardiac differentiation.

In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including nanog, pou5f3 and sox19b, which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

Kindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin α5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) Published version