Uncoupling histone modification crosstalk by engineering lysine demethylase LSD1.

Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation, but its functional significance in cells has been difficult to discern. Previous enzymatic studies revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine-specific demethylase 1 (LSD1). In the present study, we engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation. K562 cells with the Y391K LSD1 CRISPR knockin show decreased expression of a set of genes associated with cellular adhesion and myeloid leukocyte activation. Chromatin profiling revealed that the cis-regulatory regions of these silenced genes display a higher level of H3 Lys14 acetylation, and edited K562 cells show diminished H3 mono-methyl Lys4 near these silenced genes, consistent with a role for enhanced LSD1 demethylase activity. These findings illuminate the functional consequences of disconnecting histone modification crosstalk for a key epigenetic enzyme.

Oligonucleotide Therapies for Facioscapulohumeral Muscular Dystrophy: Current Preclinical Landscape.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy, characterized by progressive and asymmetric muscle atrophy, primarily affecting muscles of the face, shoulder girdle, and upper arms before affecting muscles of the lower extremities with age and greater disease severity. FSHD is a disabling condition, and patients may also present with various extramuscular symptoms. FSHD is caused by the aberrant expression of double homeobox 4 (DUX4) in skeletal muscle, arising from compromised epigenetic repression of the D4Z4 array. DUX4 encodes the DUX4 protein, a transcription factor that activates myotoxic gene programs to produce the FSHD pathology. Therefore, sequence-specific oligonucleotides aimed at reducing DUX4 levels in patients is a compelling therapeutic approach, and one that has received considerable research interest over the last decade. This review aims to describe the current preclinical landscape of oligonucleotide therapies for FSHD. This includes outlining the mechanism of action of each therapy and summarizing the preclinical results obtained regarding their efficacy in cellular and/or murine disease models. The scope of this review is limited to oligonucleotide-based therapies that inhibit the DUX4 gene, mRNA, or protein in a way that does not involve gene editing.

Conserved dual-mode gene regulation programs in higher eukaryotes.

Recent genomic data analyses have revealed important underlying logics in eukaryotic gene regulation, such as CpG islands (CGIs)-dependent dual-mode gene regulation. In mammals, genes lacking CGIs at their promoters are generally regulated by interconversion between euchromatin and heterochromatin, while genes associated with CGIs constitutively remain as euchromatin. Whether a similar mode of gene regulation exists in non-mammalian species has been unknown. Here, through comparative epigenomic analyses, we demonstrate that the dual-mode gene regulation program is common in various eukaryotes, even in the species lacking CGIs. In cases of vertebrates or plants, we find that genes associated with high methylation level promoters are inactivated by forming heterochromatin and expressed in a context-dependent manner. In contrast, the genes with low methylation level promoters are broadly expressed and remain as euchromatin even when repressed by Polycomb proteins. Furthermore, we show that invertebrate animals lacking DNA methylation, such as fruit flies and nematodes, also have divergence in gene types: some genes are regulated by Polycomb proteins, while others are regulated by heterochromatin formation. Altogether, our study establishes gene type divergence and the resulting dual-mode gene regulation as fundamental features shared in a broad range of higher eukaryotic species.

Arid3a is essential to execution of the first cell fate decision via direct embryonic and extraembryonic transcriptional regulation.

Despite their origin from the inner cell mass, embryonic stem (ES) cells undergo differentiation to the trophectoderm (TE) lineage by repression of the ES cell master regulator Oct4 or activation of the TE master regulator Caudal-type homeobox 2 (Cdx2). In contrast to the in-depth studies of ES cell self-renewal and pluripotency, few TE-specific regulators have been identified, thereby limiting our understanding of mechanisms underlying the first cell fate decision. Here we show that up-regulation and nuclear entry of AT-rich interactive domain 3a (Arid3a) drives TE-like transcriptional programs in ES cells, maintains trophoblast stem (TS) cell self-renewal, and promotes further trophoblastic differentiation both upstream and independent of Cdx2. Accordingly, Arid3a(-/-) mouse post-implantation placental development is severely impaired, resulting in early embryonic death. We provide evidence that Arid3a directly activates TE-specific and trophoblast lineage-specific genes while directly repressing pluripotency genes via differential regulation of epigenetic acetylation or deacetylation. Our results identify Arid3a as a critical regulator of TE and placental development through execution of the commitment and differentiation phases of the first cell fate decision.

Implications of CpG islands on chromosomal architectures and modes of global gene regulation.

CpG islands (CGIs) have long been implicated in the regulation of vertebrate gene expression. However, the involvement of CGIs in chromosomal architectures and associated gene expression regulations has not yet been thoroughly explored. By combining large-scale integrative data analyses and experimental validations, we show that CGIs clearly reconcile two competing models explaining nuclear gene localizations. We first identify CGI-containing (CGI+) and CGI-less (CGI-) genes are non-randomly clustered within the genome, which reflects CGI-dependent spatial gene segregation in the nucleus and corresponding gene regulatory modes. Regardless of their transcriptional activities, CGI+ genes are mainly located at the nuclear center and encounter frequent long-range chromosomal interactions. Meanwhile, nuclear peripheral CGI- genes forming heterochromatin are activated and internalized into the nuclear center by local enhancer-promoter interactions. Our findings demonstrate the crucial implications of CGIs on chromosomal architectures and gene positioning, linking the critical importance of CGIs in determining distinct mechanisms of global gene regulation in three-dimensional space in the nucleus.

Spontaneous Regression of a Mandibular Plasmacytoma in a Juvenile Dog: A Case Report.

A 4 mo old female Finnish lapphund presented for further investigation of a swelling of the right rostral mandible. A computed tomography scan showed the swelling to be an expansile and osteolytic mandibular lesion. Histopathology revealed a poorly differentiated, moderately well-demarcated, unencapsulated, highly infiltrative round cell neoplasm, and immunohistochemistry was supportive of a plasmacytoma. Performance of a rostral partial mandibulectomy was initially discussed with the owners, but the lesion improved spontaneously both clinically and on repeated computed tomography scanning before surgery could be performed. It subsequently almost completely resolved 6 mo after diagnosis. Hypotheses for spontaneous regression of the lesion are discussed and the human literature is briefly reviewed.

Mechanisms of transcription factor-mediated direct reprogramming of mouse embryonic stem cells to trophoblast stem-like cells.

Direct reprogramming can be achieved by forced expression of master transcription factors. Yet how such factors mediate repression of initial cell-type-specific genes while activating target cell-type-specific genes is unclear. Through embryonic stem (ES) to trophoblast stem (TS)-like cell reprogramming by introducing individual TS cell-specific 'CAG' factors (Cdx2, Arid3a and Gata3), we interrogate their chromosomal target occupancies, modulation of global transcription and chromatin accessibility at the initial stage of reprogramming. From the studies, we uncover a sequential, two-step mechanism of cellular reprogramming in which repression of pre-existing ES cell-associated gene expression program is followed by activation of TS cell-specific genes by CAG factors. Therefore, we reveal that CAG factors function as both decommission and pioneer factors during ES to TS-like cell fate conversion.

Differences in Rate of Complete Excision of Basal Cell Carcinoma by Dermatologists, Plastic Surgeons and General Practitioners: A Large Cross-Sectional Study.

BACKGROUND: Due to the increasing incidence of basal cell carcinoma (BCC) and rising health care costs, health care insurance companies seek ways to shift skin surgery for BCC from secondary to primary care. OBJECTIVES: To study the differences in complete excision of BCC by general practitioners (GPs), dermatologists, and plastic surgeons. METHODS: A retrospective cross-sectional study of pathology records of 2,986 standard excisions of primary BCCs performed by a GP, dermatologist, or plastic surgeon in the area of Southwest Netherlands between 2008 and 2014. To compare the risk of an incomplete BCC excision between the specialties, the odds ratio (OR) was used adjusted for patient age, sex, tumor site, size, and histological subtype. RESULTS: BCCs were completely excised by GPs in 70%, which was lower than the 93% by dermatologists and 83% by plastic surgeons (p < 0.001). Compared to the dermatologist, BCCs which were excised by a GP were 6 times higher at risk of an incomplete excision (adjusted OR 6, 95% CI 5-8) and 2 times higher at risk when excised by a plastic surgeon (adjusted OR 2, 95% CI 2-3). CONCLUSION: BCCs were more often completely excised by dermatologists than by GPs and plastic surgeons. Dermatologists probably perform better because of their extensive training and high experience in BCC care. To minimize incomplete BCC excision, GPs should receive specific training before the shift of BCC care from secondary to primary care is justifiable.

Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking.

Planar cell polarity (PCP) controls the orientation of cells within tissues and the polarized outgrowth of cellular appendages. So far, six PCP core proteins including the transmembrane proteins Frizzled (Fz), Strabismus (Stbm) and Flamingo (Fmi) have been identified. These proteins form asymmetric PCP domains at apical junctions of epithelial cells. Here, we demonstrate that VhaPRR, an accessory subunit of the proton pump V-ATPase, directly interacts with the protocadherin Fmi through its extracellular domain. It also shows a striking co-localization with PCP proteins during all pupal wing stages in Drosophila. This localization depends on intact PCP domains. Reversely, VhaPRR is required for stable PCP domains, identifying it as a novel PCP core protein. VhaPRR performs an additional role in vesicular acidification as well as endolysosomal sorting and degradation. Membrane proteins, such as E-Cadherin and the Notch receptor, accumulate at the surface and in intracellular vesicles of cells mutant for VhaPRR. This trafficking defect is shared by other V-ATPase subunits. By contrast, the V-ATPase does not seem to have a direct role in PCP regulation. Together, our results suggest two roles for VhaPRR, one for PCP and another in endosomal trafficking. This dual function establishes VhaPRR as a key factor in epithelial morphogenesis.

p16 immunostaining in keratinocytic neoplasia in organ transplant recipients: Bowen's disease shows a characteristic pattern.

BACKGROUND: For selecting therapy, it is important to distinguish different types of keratinocytic neoplasia. It is sometimes difficult to make histopathologic diagnosis, especially in organ transplant recipients (OTR) who develop numerous lesions. METHODS: To investigate p16 immunostaining in different types of keratinocytic neoplasia in OTR, we studied 59 actinic keratoses (AK), 51 Bowen' s disease (BD), 63 squamous cell carcinomas (SCC), 16 benign keratotic lesions (BKL) from 31 OTR patients and 25 controls (eczema and psoriasis). Tissue sections were stained for H&E and p16. We scored intensity, proportion and distribution of p16 positive lesional cells. RESULTS: In 19% of AK, 92% of BD, 35% of SCC and 12% of BKL more than 15% of lesional cells were p16-positive. In 16% of AK, 80% of BD, 18% of SCC and 13% of BKL strong p16 staining was observed. BKL, AK and SCC showed focal and patchy staining, BD showed diffuse pattern with strong staining of all atypical cells. Sparing of the basal layer was predominantly seen in BD. No control specimen showed p16-overexpression. CONCLUSIONS: p16 immunostaining shows a characteristic pattern in BD, but not in AK, SCC and BKL. It appears useful in recognizing BD, but not in differentiating between other keratinocytic neoplasia.

Multi-layered global gene regulation in mouse embryonic stem cells.

Embryonic stem (ES) cells derived from the inner cell mass of developing embryos have tremendous potential in regenerative medicine due to their unique properties: ES cells can be maintained for a prolonged time without changes in their cellular characteristics in vitro (self-renewal), while sustaining the capacity to give rise to all cell types of adult organisms (pluripotency). In addition to the development of protocols to manipulate ES cells for therapeutic applications, understanding how such unique properties are maintained has been one of the key questions in stem cell research. During the past decade, advances in high-throughput technologies have enabled us to systematically monitor multiple layers of gene regulatory mechanisms in ES cells. In this review, we briefly summarize recent findings on global gene regulatory modes in ES cells, mainly focusing on the regulatory factors responsible for transcriptional and epigenetic regulations as well as their modular regulatory patterns throughout the genome.

Clinical Trial and In Vitro Study for the Role of Cartilage and Synovia in Acute Articular Infection.

OBJECTIVE: Osteoarthritis is a long-term complication of acute articular infections. However, the roles of cartilage and synovia in this process are not yet fully understood. METHODS: Patients with acute joint infections were enrolled in a prospective clinical trial and the cytokine composition of effusions compared in patients with arthroplasty (n = 8) or with intact joints (n = 67). Cytokines and cell function were also analyzed using a human in vitro model of joint infection. RESULTS: Synovial IL-1ß levels were significantly higher in patients with arthroplasty (p = 0.004). Higher IL-1ß concentrations were also found in the in vitro model without chondrocytes (p < 0.05). The anti-inflammatory cytokines IL-4 and IL-10 were consistently expressed in vivo and in vitro, showing no association with the presence of cartilage or chondrocytes. In contrast, FasL levels increased steadily in vitro, reaching higher levels without chondrocytes (p < 0.05). Likewise, the viability of synovial fibroblasts (SFB) during infection was higher in the presence of chondrocytes. The cartilage-metabolism markers aggrecan and bFGF were at higher concentrations in intact joints, but also synthesized by SFB. CONCLUSIONS: Our data suggest an anti-inflammatory effect of cartilage associated with the SFBs' increased resistance to infections, which displayed the ability to effectively synthesize cartilage metabolites.The trial is registered with DRKS 00003536, MISSinG.

The impact of MEIS1 TALE homeodomain transcription factor knockdown on glioma stem cell growth.

Myeloid ecotropic virus insertion site 1 (MEIS1) is a HOX co-factor necessary for organ development and normal hematopoiesis. Recently, MEIS1 has been linked to the development and progression of various cancers. However, its role in gliomagenesis particularly on glioma stem cells (GSCs) remains unclear. Here, we demonstrate that MEIS1 is highly upregulated in GSCs compared to normal, and glioma cells and to its differentiated counterparts. Inhibition of MEIS1 expression by shRNA significantly reduced GSC growth in both in vitro and in vivo experiments. On the other hand, integrated transcriptomics analyses of glioma datasets revealed that MEIS1 expression is correlated to cell cycle-related genes. Clinical data analysis revealed that MEIS1 expression is elevated in high-grade gliomas, and patients with high MEIS1 levels have poorer overall survival outcomes. The findings suggest that MEIS1 is a prognostic biomarker for glioma patients and a possible target for developing novel therapeutic strategies against GBM.

Histopathological features of subcutaneous and cutaneous mast cell tumors in dogs.

BACKGROUND: Mast cell tumors (MCTs) are the most common malignant skin neoplasms in dogs. In the past, the distinction between cutaneous MCTs (cMCTs), originating from the dermis, and subcutaneous MCTs (scMCTs), originating from the subcutaneous tissue, was not made. Histopathological differentiation, including grading, is important for prognosis. However, the Patnaik and Kiupel grading systems were proposed for cMCTs only. The objective of our study was to describe and compare the signalment of dogs with scMCTs and cMCTs and histopathological features, anticipating similarities in both groups. Data of dogs histologically diagnosed with scMCTs or cMCTs between September 2020 and July 2023 were analyzed retrospectively. Signalment, tumor location, histopathological features, completeness of removal and lymph node status were recorded. RESULTS: Data on 305 scMCTs and 1291 cMCTs were collected. Breed distribution was different between scMCTs and cMCTs (P < 0.0001). Mitotic count (MC) was not different between scMCTs (1.63) and cMCTs (1.58) (P = 0.8490). Compared to cMCTs, scMCTs more often had anisokaryosis, bizarre nuclei and multinucleation. Kiupel high grade was more often assigned to scMCTs (51/292, 17.5%) than cMCTs (154/1291, 11.9%) (P = 0.009). The odds of MCTs being assigned a high grade in scMCT was 1.578 higher than in cMCTs (95% confidence interval [1.116-2.232]). CONCLUSIONS: Histopathological differences between scMCTs and cMCTs were observed. A Kiupel high grade was more often assigned to scMCTs than cMCTs. Whether these histopathological findings correlate with clinical outcome has to be established in additional studies.

The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma.

Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through nonmutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated. Here, we evaluated the corepressor for element 1-silencing transcription factor (CoREST) epigenetic repressor complex and the recently developed bivalent inhibitor corin within the context of melanoma phenotype plasticity and therapeutic resistance. We found that CoREST was a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells led to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin revealed specific effects on histone marks connected to epithelial-mesenchymal transition-associated (EMT-associated) transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor-resistant (BRAFi-R) melanomas with corin promoted resensitization to BRAFi therapy. DUSP1 was consistently downregulated in BRAFi-R melanomas, which was reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity was recapitulated by the p38 MAPK inhibitor BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial for patients with BRAFi-resistant melanoma.

Elevated α-1,2-mannosidase MAN1C1 in glioma stem cells and its implications for immunological changes and prognosis in glioma patients.

Glioblastoma multiforme (GBM) is the most aggressive type of primary brain tumor, and the presence of glioma stem cells (GSCs) has been linked to its resistance to treatments and recurrence. Additionally, aberrant glycosylation has been implicated in the aggressiveness of cancers. However, the influence and underlying mechanism of N-glycosylation on the GSC phenotype and GBM malignancy remain elusive. Here, we performed an in-silico analysis approach on publicly available datasets to examine the function of N-glycosylation-related genes in GSCs and gliomas, accompanied by a qRT-PCR validation experiment. We found that high α-1,2-mannosidase MAN1C1 is associated with immunological functions and worse survival of glioma patients. Differential gene expression analysis and qRT-PCR validation revealed that MAN1C1 is highly expressed in GSCs. Furthermore, higher MAN1C1 expression predicts worse outcomes in glioma patients. Also, MAN1C1 expression is increased in the perinecrotic region of GBM and is associated with immunological and inflammatory functions, a hallmark of the GBM mesenchymal subtype. Further analysis confirmed that MAN1C1 expression is closely associated with infiltrating immune cells and disrupted immune response in the GBM microenvironment. These suggest that MAN1C1 is a potential biomarker for gliomas and may be important as an immunotherapeutic target for GBM.

Cell surface Nestin is a biomarker for glioma stem cells.

Cancer stem cells (CSCs) are the most aggressive cell type in many malignancies. Cell surface proteins are generally used to isolate and characterize CSCs. Therefore, the identification of CSC-specific cell surface markers is very important for the diagnosis and treatment of malignancies. We found that Nestin (a type VI intermediate filament protein), like the glioma stem cell (GSC) markers CD133 and CD15, exhibited different levels of expression in primary human glioblastoma specimens. Similar to our previous finding that cytoplasmic Nestin is expressed as a cell surface form in mouse GSCs, the cell surface form of Nestin was also expressed at different levels in human GSCs. We isolated cell surface Nestin-positive cell populations from human GSCs by fluorescence-activated cell sorting FACS analysis, and observed that these populations exhibited robust CSC properties, such as increased tumorsphere-forming ability and tumorsphere size. Mechanistically, we found that DAPT, a γ-secretase (a multi-subunit protease complex) inhibitor, reduced the proportion of cell surface Nestin-positive cells in human GSCs in a time- and dose-dependent manner, without significant changes in total Nestin expression, implying that a post-translational modification was involved in the generation of cell surface Nestin. Taken together, our data provides the first evidence that cell surface Nestin may serve as a promising GSC marker for the isolation and characterization of heterogeneous GSCs in glioblastomas.

Interferon regulatory factor 7 regulates glioma stem cells via interleukin-6 and Notch signalling.

Inflammatory microenvironment signalling plays a crucial role in tumour progression (i.e. cancer cell proliferation, survival, angiogenesis and metastasis) in many types of human malignancies. However, the role of inflammation in brain tumour pathology remains poorly understood. Here, we report that interferon regulatory factor 7 is a crucial regulator of brain tumour progression and heterogeneity. Ectopic expression of interferon regulatory factor 7 in glioma cells promotes tumorigenicity, angiogenesis, microglia recruitment and cancer stemness in vivo and in vitro through induction of interleukin 6, C-X-C motif chemokine 1 and C-C motif chemokine 2. In particular, interferon regulatory factor 7-driven interleukin 6 plays a pivotal role in maintaining glioma stem cell properties via Janus kinase/signal transducer and activator of transcription-mediated activation of Jagged-Notch signalling in glioma cells and glioma stem cells derived from glioma patients. Accordingly, the short hairpin RNA-mediated depletion of interferon regulatory factor 7 in glioma stem cells markedly suppressed interleukin 6-Janus kinase/signal transducer and activator of transcription-mediated Jagged-Notch-signalling pathway, leading to decreases in glioma stem cell marker expression, tumoursphere-forming ability, and tumorigenicity. Furthermore, in a mouse model of wound healing, depletion of interferon regulatory factor 7 suppressed tumour progression and decreased cellular heterogeneity. Finally, interferon regulatory factor 7 was overexpressed in patients with high-grade gliomas, suggesting its potential as an independent prognostic marker for glioma progression. Taken together, our findings indicate that interferon regulatory factor 7-mediated inflammatory signalling acts as a major driver of brain tumour progression and cellular heterogeneity via induction of glioma stem cell genesis and angiogenesis.

Suspected Primary Intracranial Melanoma with Widespread Distant Metastases in a Cat.

An 8-year-old female Domestic Shorthair presented with signs of intracranial disease. Magnetic resonance imaging (MRI) of the head showed an extra-axial space-occupying mass within the cranial vault with a similar intensity lesion within the overlying temporalis muscle. Postmortem examination found masses within the head, lung, liver, spleen, and kidney consistent with malignant melanoma. Intracranial melanoma is rarely reported in cats and is typically only seen as a metastatic lesion associated with an ocular mass. Melanomas can be readily recognised on MRI as they are one of the few lesions which are hyperintense on T1-weighted images.