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The transcription factor p63 is a master regulator of epidermal development. Mutations in p63 give rise to human developmental diseases that often manifest epidermal defects. In this review, we summarize major p63 isoforms identified so far and p63 mutation-associated human diseases that show epidermal defects. We discuss key roles of p63 in epidermal keratinocyte proliferation and differentiation, emphasizing its master regulatory control of the gene expression pattern and epigenetic landscape that define epidermal fate. We subsequently review the essential function of p63 during epidermal commitment and transdifferentiation towards epithelial lineages, highlighting the notion that p63 is the guardian of the epithelial lineage. Finally, we discuss current therapeutic development strategies for p63 mutation-associated diseases. Our review proposes future directions for dissecting p63-controlled mechanisms in normal and diseased epidermal development and for developing therapeutic options.

Kleefstra syndrome (KS) is a neurodevelopmental disorder caused by mutations in the histone methyltransferase EHMT1 . To study the impact of decreased EHMT1 function in human cells, we generated excitatory cortical neurons from induced pluripotent stem (iPS) cells derived from KS patients. Neuronal networks of patient-derived cells exhibit network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes are mediated by upregulation of NMDA receptor (NMDAR) subunit 1 correlating with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. In mice EHMT1 deficiency leads to similar neuronal network impairments with increased NMDAR function. Finally, we rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Summarized, we demonstrate a direct link between EHMT1 deficiency and NMDAR hyperfunction in human neurons, providing a potential basis for more targeted therapeutic approaches for KS. Kleefstra syndrome is a neurodevelopmental disorder associated with hapoinsufficiency of the histone methyltransferase EHMT1 . Here the authors show using induced pluripotent cells-derived neurons from patients that network dysfunction occurs and is due to dysfunction of the NMDA receptor.

Significance p63, the sister homolog of p53, is a master regulator of epithelial stem cell (SC) biology. p63 is indeed intimately implicated in the maintenance of the self-renewal capacity of stratified epithelia and their derivatives, including the mammary gland. Although the physiological role of p63 in normal mammary SCs is now acknowledged, proof of its implications in breast cancer SCs remains elusive. Here, we find that mammary cancer stem cells (CSCs) possess increased levels of p63 expression compared with normal progenitors. p63 promotes self-renewal and expansion of mammary CSCs and breast tumor growth in vivo. Additionally, this study provides a link between p63 and the Sonic Hedgehog signaling pathway in the regulation of breast cancer stemness. The predominant p63 isoform, ΔNp63, is a master regulator of normal epithelial stem cell (SC) maintenance. However, in vivo evidence of the regulation of cancer stem cell (CSC) properties by p63 is still limited. Here, we exploit the transgenic MMTV-ErbB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2) mouse model of carcinogenesis to dissect the role of p63 in the regulation of mammary CSC self-renewal and breast tumorigenesis. ErbB2 tumor cells enriched for SC-like properties display increased levels of ΔNp63 expression compared with normal mammary progenitors. Down-regulation of p63 in ErbB2 mammospheres markedly restricts self-renewal and expansion of CSCs, and this action is fully independent of p53. Furthermore, transplantation of ErbB2 progenitors expressing shRNAs against p63 into the mammary fat pads of syngeneic mice delays tumor growth in vivo. p63 knockdown in ErbB2 progenitors diminishes the expression of genes encoding components of the Sonic Hedgehog (Hh) signaling pathway, a driver of mammary SC self-renewal. Remarkably, p63 regulates the expression of Sonic Hedgehog ( Shh ), GLI family zinc finger 2 ( Gli2 ), and Patched1 ( Ptch1 ) genes by directly binding to their gene regulatory regions, and eventually contributes to pathway activation. Collectively, these studies highlight the importance of p63 in maintaining the self-renewal potential of mammary CSCs via a positive modulation of the Hh signaling pathway.

The transcription factor p63 regulates epidermal genes and the enhancer landscape in skin keratinocytes. Its molecular function in controlling the chromatin structure is, however, not yet completely understood. Here, we integrated multi-omics profiles, including the transcriptome, transcription factor DNA-binding and chromatin accessibility, in skin keratinocytes isolated from EEC syndrome patients carrying p63 mutations, to examine the role of p63 in shaping the chromatin architecture. We found decreased chromatin accessibility in p63- and CTCF-bound open chromatin regions that potentially contributed to gene deregulation in mutant keratinocytes. Cooperation of p63 and CTCF seemed to assist chromatin interactions between p63-bound enhancers and gene promoters in skin keratinocytes. Our study suggests an intriguing model where cell type-specific transcription factors such as p63 cooperate with the genome organizer CTCF in the three-dimensional chromatin space to regulate the transcription program important for the proper cell identity.

Background Early hematoma expansion (HE) occurs in 20 to 40% of spontaneous intracerebral hemorrhage (ICH) patients and is a primary determinant of early deterioration and poor prognosis. Previous studies have shown that inflammation is a major pathological feature of ICH, and the neutrophil-to-platelet ratio (NPR) is a marker of systemic inflammation. Therefore, we aimed to assess the association between the NPR and HE in ICH patients. Methods We retrospectively collected and analyzed data from ICH patients who received treatment at our institution from January 2018 to November 2019. The NPR was calculated from the admission blood test. Brain computed tomography (CT) scans were performed at admission and repeated within 24 h. Hematoma growth was defined as relative growth > 33% or absolute growth > 6 ml. Results A total of 317 patients were enrolled in our study. Multivariate logistic regression analysis indicated that the NPR was an independent predictor of HE [odds ratio (OR) = 1.742; 95% CI: 1.508–2.012, p  < 0.001]. Receiver operating characteristic (ROC) curve analysis revealed that the NPR could predict HE, with an area under the curve of 0.838 (95% CI, 0.788–0.888, p  < 0.001). The best predictive cut-off of the NPR for HE was 5.47 (sensitivity, 75.3%; specificity, 77.6%). Conclusions A high NPR was associated with an increased risk of HE in patients with ICH.

Background The CCTC-binding factor (CTCF) protein is involved in genome organization, including mediating three-dimensional chromatin interactions. Human patient lymphocytes with mutations in a single copy of the CTCF gene have reduced expression of enhancer-associated genes involved in response to stimuli. We hypothesize that CTCF interactions stabilize enhancer-promoter chromatin interaction domains, facilitating increased expression of genes in response to stimuli. Here we systematically investigate this model using computational analyses. Results We use CTCF ChIA-PET data from the ENCODE project to show that CTCF-associated chromatin loops have a tendency to enclose regions of enhancer-regulated stimulus responsive genes, insulating them from neighboring regions of constitutively expressed housekeeping genes. To facilitate cell type-specific CTCF loop identification, we develop an algorithm to predict CTCF loops from ChIP-seq data alone by exploiting the CTCF motif directionality in loop anchors. We apply this algorithm to a hundred ENCODE cell line datasets, confirming the universality of our observations as well as identifying a general distinction between primary and immortal cells in loop-enclosed gene content. Finally, we combine the existing evidence to propose a model for the formation of CTCF loops in which partner sites are brought together by chromatin template reeling through stationary RNA polymerases, consistent with the transcription factory hypothesis. Conclusions We provide computational evidence that CTCF-mediated chromatin interactions enclose domains of stimulus responsive enhancer-regulated genes, insulating them from nearby housekeeping genes.

Kleefstra syndrome, caused by haploinsufficiency of euchromatin histone methyltransferase 1 (EHMT1), is characterized by intellectual disability (ID), autism spectrum disorder (ASD), characteristic facial dysmorphisms, and other variable clinical features. In addition to EHMT1 mutations, de novo variants were reported in four additional genes (MBD5, SMARCB1, NR1I3, and KMT2C), in single individuals with clinical characteristics overlapping Kleefstra syndrome. Here, we present a novel cohort of five patients with de novo loss of function mutations affecting the histone methyltransferase KMT2C. Our clinical data delineates the KMT2C phenotypic spectrum and reinforces the phenotypic overlap with Kleefstra syndrome and other related ID disorders. To elucidate the common molecular basis of the neuropathology associated with mutations in KMT2C and EHMT1, we characterized the role of the Drosophila KMT2C ortholog, trithorax related (trr), in the nervous system. Similar to the Drosophila EHMT1 ortholog, G9a, trr is required in the mushroom body for short term memory. Trr ChIP-seq identified 3371 binding sites, mainly in the promoter of genes involved in neuronal processes. Transcriptional profiling of pan-neuronal trr knockdown and G9a null mutant fly heads identified 613 and 1123 misregulated genes, respectively. These gene sets show a significant overlap and are associated with nearly identical gene ontology enrichments. The majority of the observed biological convergence is derived from predicted indirect target genes. However, trr and G9a also have common direct targets, including the Drosophila ortholog of Arc (Arc1), a key regulator of synaptic plasticity. Our data highlight the clinical and molecular convergence between the KMT2 and EHMT protein families, which may contribute to a molecular network underlying a larger group of ID/ASD-related disorders.

Pseudouridine (Ψ) is an abundant RNA chemical modification that plays critical biological functions. Current Ψ detection methods are limited in identifying Ψs at base-resolution in U-rich sequence contexts, where Ψ occurs frequently. Here we report “Mut-Ψ-seq” that utilizes the classic N-cyclohexyl N′-(2-morpholinoethyl)carbodiimide (CMC) agent and an evolved reverse transcriptase (“RT-1306”) for Ψ mapping at base-resolution. CMC selectively labels Ψs in RNA forming the CMC-Ψ adduct and we show that RT-1306 presents promoted read-through and mutation against the CMC-Ψ. We report a high-confidence list of Ψ sites in polyA-enriched RNAs from HEK-293T cells identified by orthogonal chemical treatments (CMC and bisulfite). The mutation signatures resolve the position of Ψ in UU-containing sequences, revealing diverse occurrence of Ψs in such sequences. This work provides methods and datasets for biological research of Ψ, and expands the toolkit for epitranscriptomic studies by combining the reverse transcriptase engineering and selective chemical labeling strategies. Mut-Ψ-seq deploys an evolved reverse transcriptase and the CMC chemistry to resolve the ambiguity for the occurrence of Ψ in UU sequence contexts and reports a high-confident list of Ψ sites in the human transcriptome by combining orthogonal chemistry methods.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2 , a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies.