The high mobility group protein HMG20A cooperates with the histone reader PHF14 to modulate TGFß and Hippo pathways.

High mobility group (HMG) proteins are chromatin regulators with essential functions in development, cell differentiation and cell proliferation. The protein HMG20A is predicted by the AlphaFold2 software to contain three distinct structural elements, which we have functionally characterized: i) an amino-terminal, intrinsically disordered domain with transactivation activity; ii) an HMG box with higher binding affinity for double-stranded, four-way-junction DNA than for linear DNA; and iii) a long coiled-coil domain. Our proteomic study followed by a deletion analysis and structural modeling demonstrates that HMG20A forms a complex with the histone reader PHF14, via the establishment of a two-stranded alpha-helical coiled-coil structure. siRNA-mediated knockdown of either PHF14 or HMG20A in MDA-MB-231 cells causes similar defects in cell migration, invasion and homotypic cell-cell adhesion ability, but neither affects proliferation. Transcriptomic analyses demonstrate that PHF14 and HMG20A share a large subset of targets. We show that the PHF14-HMG20A complex modulates the Hippo pathway through a direct interaction with the TEAD1 transcription factor. PHF14 or HMG20A deficiency increases epithelial markers, including E-cadherin and the epithelial master regulator TP63 and impaired normal TGFß-trigged epithelial-to-mesenchymal transition. Taken together, these data indicate that PHF14 and HMG20A cooperate in regulating several pathways involved in epithelial-mesenchymal plasticity.

Abdominal wall muscle weakness outcomes after split abdominal flap repair of large congenital diaphragmatic hernias in newborn.

PURPOSE: Split abdominal wall muscle flap (SAWMF) is a technique to repair large defects in congenital diaphragmatic hernia (CDH). A possible objection to this intervention could be any associated abdominal muscle weakness. Our aim is to analyze the evolution of this abdominal muscle wall weakness. METHODS: Retrospective review of CDH repair by SAWMF (internal oblique muscle and transverse) from 2004 to 2023 focusing on the evolution of muscle wall weakness. RESULTS: Eighteen neonates of 148 CDH patients (12,1%) were repaired using SAWMF. Mean gestational age and birth weight were 35.7 ± 3.5 weeks and 2587 ± 816 g. Mean lung-to-head ratio was 1.49 ± 0.28 and 78% liver-up. Seven patients (38%) were prenatally treated by tracheal occlusion. Ninety-four percent of the flaps were used for primary repair and one to repair a recurrence. One patient (5.6%) experienced recurrence. Abdominal muscle wall weakness was present in the form of a bulge. Resolution of weakness at 1, 2 and 3 years was 67%, 89% and 94%, respectively. No patient required treatment for weakness or died. CONCLUSIONS: Abdominal muscular weakness after a split abdominal wall muscle flap repair is not a limitation for its realization since it is asymptomatic and presents a prompt spontaneous resolution. LEVEL OF EVIDENCE: IV.

Differentiation-related epigenomic changes define clinically distinct keratinocyte cancer subclasses.

Keratinocyte cancers (KC) are the most prevalent malignancies in fair-skinned populations, posing a significant medical and economic burden to health systems. KC originate in the epidermis and mainly comprise basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Here, we combined single-cell multi-omics, transcriptomics, and methylomics to investigate the epigenomic dynamics during epidermal differentiation. We identified ~3,800 differentially accessible regions between undifferentiated and differentiated keratinocytes, corresponding to regulatory regions associated with key transcription factors. DNA methylation at these regions defined AK/cSCC subtypes with epidermal stem cell- or keratinocyte-like features. Using cell-type deconvolution tools and integration of bulk and single-cell methylomes, we demonstrate that these subclasses are consistent with distinct cells-of-origin. Further characterization of the phenotypic traits of the subclasses and the study of additional unstratified KC entities uncovered distinct clinical features for the subclasses, linking invasive and metastatic KC cases with undifferentiated cells-of-origin. Our study provides a thorough characterization of the epigenomic dynamics underlying human keratinocyte differentiation and uncovers novel links between KC cells-of-origin and their prognosis.

Transforming growth factor ß1-mediated functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia.

Mesenchymal stromal cells are involved in the pathogenesis of myelodysplastic syndromes and acute myeloid leukemia, but the underlying mechanisms are incompletely understood. To further characterize the pathological phenotype we performed RNA sequencing of mesenchymal stromal cells from patients with myelodysplastic syndromes and acute myeloid leukemia and found a specific molecular signature of genes commonly deregulated in these disorders. Pathway analysis showed a strong enrichment of genes related to osteogenesis, senescence, inflammation and inhibitory cytokines, thereby reflecting the structural and functional deficits of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia on a molecular level. Further analysis identified transforming growth factor ß1 as the most probable extrinsic trigger factor for this altered gene expression. Following exposure to transforming growth factor ß1, healthy mesenchymal stromal cells developed functional deficits and adopted a phenotype reminiscent of that observed in patient-derived stromal cells. These suppressive effects of transforming growth factor ß1 on stromal cell functionality were abrogated by SD-208, an established inhibitor of transforming growth factor ß receptor signaling. Blockade of transforming growth factor ß signaling by SD-208 also restored the osteogenic differentiation capacity of patient-derived stromal cells, thus confirming the role of transforming growth factor ß1 in the bone marrow microenvironment of patients with myelodysplastic syndromes and acute myeloid leukemia. Our findings establish transforming growth factor ß1 as a relevant trigger causing functional inhibition of mesenchymal stromal cells in myelodysplastic syndromes and acute myeloid leukemia and identify SD-208 as a candidate to revert these effects.

The chromatin remodeller CHD8 is required for E2F-dependent transcription activation of S-phase genes.

The precise regulation of S-phase-specific genes is critical for cell proliferation. How the repressive chromatin configuration mediated by the retinoblastoma protein and repressor E2F factors changes at the G1/S transition to allow transcription activation is unclear. Here we show ChIP-on-chip studies that reveal that the chromatin remodeller CHD8 binds ∼ 2000 transcriptionally active promoters. The spectrum of CHD8 target genes was enriched in E2F-dependent genes. We found that CHD8 binds E2F-dependent promoters at the G1/S transition but not in quiescent cells. Consistently, CHD8 was required for G1/S-specific expression of these genes and for cell cycle re-entry on serum stimulation of quiescent cells. We also show that CHD8 interacts with E2F1 and, importantly, loading of E2F1 and E2F3, but not E2F4, onto S-specific promoters, requires CHD8. However, CHD8 recruiting is independent of these factors. Recruiting of MLL histone methyltransferase complexes to S-specific promoters was also severely impaired in the absence of CHD8. Furthermore, depletion of CHD8 abolished E2F1 overexpression-dependent S-phase stimulation of serum-starved cells, highlighting the essential role of CHD8 in E2F-dependent transcription activation.

Control of neuronal differentiation by sumoylation of BRAF35, a subunit of the LSD1-CoREST histone demethylase complex.

The LSD1-CoREST histone demethylase complex is required to repress neuronal genes in nonneuronal tissues. Here we show that sumoylation of Braf35, one of the subunits of the complex, is required to maintain full repression of neuron-specific genes and for occupancy of the LSD1-CoREST complex at its gene targets. Interestingly, expression of Braf35 was sufficient to prevent neuronal differentiation induced by bHLH neurogenic transcription factors in P19 cells and in neuronal progenitors of the chicken embryo neural tube. Sumoylation of Braf35 is required for this antineurogenic activity. We also show that iBraf, a paralogue of Braf35, forms heterodimers with Braf35. Braf35-iBraf heterodimerization impairs Braf35 interaction with the LSD1-CoREST complex and inhibits Braf35 sumoylation. Consistent with these results, iBraf prevents the antineurogenic activity of Braf35 in vivo. Our data uncover a mechanism of regulation of the LSD1-CoREST complex and provide a molecular explanation for the antagonism between Braf35 and iBraf in neuronal differentiation.

An increase in MECP2 dosage impairs neural tube formation.

Epigenetic mechanisms are fundamental for shaping the activity of the central nervous system (CNS). Methyl-CpG binding protein 2 (MECP2) acts as a bridge between methylated DNA and transcriptional effectors responsible for differentiation programs in neurons. The importance of MECP2 dosage in CNS is evident in Rett Syndrome and MECP2 duplication syndrome, which are neurodevelopmental diseases caused by loss-of-function mutations or duplication of the MECP2 gene, respectively. Although many studies have been performed on Rett syndrome models, little is known about the effects of an increase in MECP2 dosage. Herein, we demonstrate that MECP2 overexpression affects neural tube formation, leading to a decrease in neuroblast proliferation in the neural tube ventricular zone. Furthermore, an increase in MECP2 dose provokes premature differentiation of neural precursors accompanied by greater cell death, resulting in a loss of neuronal populations. Overall, our data indicate that correct MECP2 expression levels are required for proper nervous system development.

Study of the Influence of Boundary Conditions on Corneal Deformation Based on the Finite Element Method of a Corneal Biomechanics Model.

Implementing in silico corneal biomechanical models for surgery applications can be boosted by developing patient-specific finite element models adapted to clinical requirements and optimized to reduce computational times. This research proposes a novel corneal multizone-based finite element model with octants and circumferential zones of clinical interest for material definition. The proposed model was applied to four patient-specific physiological geometries of keratoconus-affected corneas. Free-stress geometries were calculated by two iterative methods, the displacements and prestress methods, and the influence of two boundary conditions: embedded and pivoting. The results showed that the displacements, stress and strain fields differed for the stress-free geometry but were similar and strongly depended on the boundary conditions for the estimated physiological geometry when considering both iterative methods. The comparison between the embedded and pivoting boundary conditions showed bigger differences in the posterior limbus zone, which remained closer in the central zone. The computational calculation times for the stress-free geometries were evaluated. The results revealed that the computational time was prolonged with disease severity, and the displacements method was faster in all the analyzed cases. Computational times can be reduced with multicore parallel calculation, which offers the possibility of applying patient-specific finite element models in clinical applications.

Analysis of population heterogeneity in CHO cells by genome-wide DNA methylation analysis and by multi-modal single-cell sequencing.

CHO cells are major hosts for the industrial production of therapeutic proteins and their production stability is of considerable economic significance. It is widely known that CHO cells can rapidly acquire genetic alterations, which affects their genetic homogeneity over time. However, the role of non-genetic mechanisms, including epigenetic mechanisms such as DNA methylation, remains poorly understood. We have now used whole-genome bisulfite sequencing to establish single-base methylation maps of eight independent CHO cell lines. Our results identify CpG islands and low-methylated regions as conserved elements with dynamic DNA methylation. Interestingly, methylation patterns were found to cluster clearly along the three main branches of CHO evolution, with no directional changes over short culture periods. Furthermore, multi-ome single-cell sequencing of 9,833 nuclei from three independent cultures revealed dynamic subpopulation structures characterized by robust expression differences in pathways related to protein production. Our findings thus provide novel insights into the epigenetic landscape and heterogeneity of CHO cells and support the development of epigenetic biomarkers that trace the emergence of subpopulations in CHO cultures.

Functionally distinct cancer-associated fibroblast subpopulations establish a tumor promoting environment in squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a serious public health problem due to its high incidence and metastatic potential. It may progress from actinic keratosis (AK), a precancerous lesion, or the in situ carcinoma, Bowen's disease (BD). During this progression, malignant keratinocytes activate dermal fibroblasts into tumor promoting cancer-associated fibroblasts (CAFs), whose origin and emergence remain largely unknown. Here, we generate and analyze >115,000 single-cell transcriptomes from healthy skin, BD and cSCC of male donors. Our results reveal immunoregulatory and matrix-remodeling CAF subtypes that may derive from pro-inflammatory and mesenchymal fibroblasts, respectively. These CAF subtypes are largely absent in AK and interact with different cell types to establish a pro-tumorigenic microenvironment. These findings are cSCC-specific and could not be recapitulated in basal cell carcinomas. Our study provides important insights into the potential origin and functionalities of dermal CAFs that will be highly beneficial for the specific targeting of the cSCC microenvironment.

Reinvestigating the clinical relevance of the m6A writer METTL3 in urothelial carcinoma of the bladder.

METTL3 is the major writer of N6-Methyladenosine (m6A) and has been associated with controversial roles in cancer. This is best illustrated in urothelial carcinoma of the bladder (UCB), where METTL3 was described to have both oncogenic and tumor-suppressive functions. Here, we reinvestigated the role of METTL3 in UCB. METTL3 knockout reduced the oncogenic phenotype and m6A levels of UCB cell lines. However, complete depletion of METTL3/m6A was not achieved due to selection of cells expressing alternative METTL3 isoforms. Systematic vulnerability and inhibitor response analyses suggested that uroepithelial cells depend on METTL3 for viability. Furthermore, expression and survival analyses of clinical data revealed a complex role for METTL3 in UCB, with decreased m6A mRNA levels in UCB tumors. Our results suggest that METTL3 expression may be a suitable diagnostic UCB biomarker, as the enzyme promotes UCB formation. However, the suitability of the enzyme as a therapeutic target should be evaluated carefully.

Base editing repairs an SGCA mutation in human primary muscle stem cells.

Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early-onset, severe, and rapidly progressive form of muscular dystrophy affecting both male and female patients. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and heart. We isolated human muscle stem cells from 2 donors, with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of 2 coregulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with > 90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base-edited patient cells regenerated muscle and contributed to the Pax7+ satellite cell compartment in vivo in mouse xenografts. Here, we provide the first evidence to our knowledge that autologous gene-repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.

Aggressive PDACs Show Hypomethylation of Repetitive Elements and the Execution of an Intrinsic IFN Program Linked to a Ductal Cell of Origin.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, which challenges the molecular analyses of bulk tumor samples. Here we FACS-purified epithelial cells from human PDAC and normal pancreas and derived their genome-wide transcriptome and DNA methylome landscapes. Clustering based on DNA methylation revealed two distinct PDAC groups displaying different methylation patterns at regions encoding repeat elements. Methylationlow tumors are characterized by higher expression of endogenous retroviral transcripts and double-stranded RNA sensors, which lead to a cell-intrinsic activation of an interferon signature (IFNsign). This results in a protumorigenic microenvironment and poor patient outcome. Methylationlow/IFNsignhigh and Methylationhigh/IFNsignlow PDAC cells preserve lineage traits, respective of normal ductal or acinar pancreatic cells. Moreover, ductal-derived Kras G12D/Trp53 -/- mouse PDACs show higher expression of IFNsign compared with acinar-derived counterparts. Collectively, our data point to two different origins and etiologies of human PDACs, with the aggressive Methylationlow/IFNsignhigh subtype potentially targetable by agents blocking intrinsic IFN signaling. SIGNIFICANCE: The mutational landscapes of PDAC alone cannot explain the observed interpatient heterogeneity. We identified two PDAC subtypes characterized by differential DNA methylation, preserving traits from normal ductal/acinar cells associated with IFN signaling. Our work suggests that epigenetic traits and the cell of origin contribute to PDAC heterogeneity.This article is highlighted in the In This Issue feature, p. 521.

DNA Methylation in Epidermal Differentiation, Aging, and Cancer.

The formation and maintenance of the epidermis depend on epidermal stem cell differentiation and must be tightly regulated. Epigenetic mechanisms such as DNA methylation allow the precise gene expression cascade needed during cellular differentiation. However, these mechanisms become deregulated during aging and tumorigenesis, where cellular function and identity become compromised. Here we provide a review of this rapidly developing field. We discuss recent discoveries related to epidermal homeostasis, aging, and cancer, including the functional role of DNA methyltransferases, the methylation clock, and the determination of tumor cells-of-origin. Finally, we focus on future advances, greatly influenced by single-cell sequencing technologies.

Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming.

Fibroblasts are an essential cell population for human skin architecture and function. While fibroblast heterogeneity is well established, this phenomenon has not been analyzed systematically yet. We have used single-cell RNA sequencing to analyze the transcriptomes of more than 5,000 fibroblasts from a sun-protected area in healthy human donors. Our results define four main subpopulations that can be spatially localized and show differential secretory, mesenchymal and pro-inflammatory functional annotations. Importantly, we found that this fibroblast 'priming' becomes reduced with age. We also show that aging causes a substantial reduction in the predicted interactions between dermal fibroblasts and other skin cells, including undifferentiated keratinocytes at the dermal-epidermal junction. Our work thus provides evidence for a functional specialization of human dermal fibroblasts and identifies the partial loss of cellular identity as an important age-related change in the human dermis. These findings have important implications for understanding human skin aging and its associated phenotypes.

The first GHEP-ISFG collaborative exercise on forensic applications of massively parallel sequencing.

One of the main goals of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. The GHEP-ISFG supports several Working Commissions which develop different scientific activities. One of them, the Working Commission on "Massively Parallel Sequencing (MPS): Forensic Applications", organized its first collaborative exercise on forensic applications of MPS technology in 2019. The aim of this exercise was to assess the concordance between the MPS results and those obtained with conventional technologies (capillary electrophoresis and Sanger sequencing), as well as to compare the results obtained within the different MPS platforms and/or the different kits/panels and analysis software packages (commercial and open-access) available on the market. The seven participating laboratories analyzed some samples of the annual GHEP-ISFG proficiency test (EIADN No. 27 (2019)), using Ion Torrent™ or MiSeq FGx® platforms. Six of them sent autosomal STR sequence data, five laboratories performed MPS analysis of individual identification SNPs, four laboratories reported MPS data of Y-chromosomal STRs, and X-chromosomal STRs, three laboratories performed MPS analysis of ancestry informative SNPs and phenotype informative SNPs, two labs performed MPS analysis of the mitochondrial DNA control region, and only one lab produced MPS data of lineage informative SNPs. Autosomal STR sequencing results were highly concordant to the consensus obtained by capillary electrophoresis in the EIADN No. 27 (2019) exercise. Furthermore, in general, a high level of concordance was observed between the results of the participating laboratories, regardless of the platform used. The main discordances were due to errors during the analysis process or from sequence data obtained with low depth of coverage. In this paper we highlight some issues that still arise, such as standardization of the nomenclature for STRs analyzed by sequencing with MPS, the universal uptake of a nomenclature framework by the analysis software, and well established validation and accreditation of the new MPS platforms for use in routine forensic case-work.

Epigenetic deregulation of lamina-associated domains in Hutchinson-Gilford progeria syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid disease characterized by the early onset of age-related phenotypes including arthritis, loss of body fat and hair, and atherosclerosis. Cells from affected individuals express a mutant version of the nuclear envelope protein lamin A (termed progerin) and have previously been shown to exhibit prominent histone modification changes. METHODS: Here, we analyze the possibility that epigenetic deregulation of lamina-associated domains (LADs) is involved in the molecular pathology of HGPS. To do so, we studied chromatin accessibility (Assay for Transposase-accessible Chromatin (ATAC)-see/-seq), DNA methylation profiles (Infinium MethylationEPIC BeadChips), and transcriptomes (RNA-seq) of nine primary HGPS fibroblast cell lines and six additional controls, two parental and four age-matched healthy fibroblast cell lines. RESULTS: Our ATAC-see/-seq data demonstrate that primary dermal fibroblasts from HGPS patients exhibit chromatin accessibility changes that are enriched in LADs. Infinium MethylationEPIC BeadChip profiling further reveals that DNA methylation alterations observed in HGPS fibroblasts are similarly enriched in LADs and different from those occurring during healthy aging and Werner syndrome (WS), another premature aging disease. Moreover, HGPS patients can be stratified into two different subgroups according to their DNA methylation profiles. Finally, we show that the epigenetic deregulation of LADs is associated with HGPS-specific gene expression changes. CONCLUSIONS: Taken together, our results strongly implicate epigenetic deregulation of LADs as an important and previously unrecognized feature of HGPS, which contributes to disease-specific gene expression. Therefore, they not only add a new layer to the study of epigenetic changes in the progeroid syndrome, but also advance our understanding of the disease's pathology at the cellular level.

Impact of DLK1-DIO3 imprinted cluster hypomethylation in smoker patients with lung cancer.

DNA methylation is important for gene expression and genome stability, and its disruption is thought to play a key role in the initiation and progression of cancer and other diseases. The DLK1-DIO3 cluster has been shown to be imprinted in humans, and some of its components are relevant to diverse pathological processes. The purpose of this study was to assess the methylation patterns of the DLK1-DIO3 cluster in patients with lung cancer to study its relevance in the pathogenesis of this disease. We found a characteristic methylation pattern of this cluster in smoking associated lung cancer, as compared to normal lung tissue. This methylation profile is not patent however in lung cancer of never smokers nor in lung tissue of COPD patients. We found 3 deregulated protein-coding genes at this locus: one was hypermethylated (DIO3) and two were hypomethylated (DLK1 and RTL1). Statistically significant differences were also detected in two different families of SNORDs, two miRNA clusters and four lncRNAs (MEG3, MEG8, MEG9 and LINC00524). These findings were validated using data from the cancer genome atlas (TCGA) database. We have then showed an inverse correlation between DNA methylation and expression levels in 5 randomly selected genes. Several targets of miRNAs included in the DLK1-DIO3 cluster have been experimentally verified as tumor suppressors. All of these results suggest that the dysmethylation of the imprinted DLK1-DIO3 cluster could have a relevant role in the pathogenesis of lung cancer in current and former smokers and may be used for diagnostic and/or therapeutic purposes.

Cell-of-Origin DNA Methylation Signatures Are Maintained during Colorectal Carcinogenesis.

Colorectal adenomas are precursor lesions of colorectal cancers and represent clonal amplifications of single cells from colonic crypts. DNA methylation patterns specify cell-type identity during cellular differentiation and, therefore, provide opportunities for the molecular analysis of tumors. We have now analyzed DNA methylation patterns in colorectal adenomas and identified three biologically defined subclasses that describe different intestinal crypt differentiation stages. Importantly, colorectal carcinomas could be classified into the same methylation subtypes, reflecting their shared cell types of origin with adenomas. Further data analysis also revealed significantly reduced overall survival for one of the subtypes. Our results provide a concept for understanding the methylation patterns observed in colorectal cancer and provide opportunities for tumor subclassification and patient stratification.