Ectodermal Research Articles

Rare Presentation of Alopecia Universalis Congenita and Twenty-nail Dystrophy in Siblings.

Congenital alopecia universalis is one of the rarest anomaly which involves skin and appendages. The inheritance pattern can be autosomal recessive, X-linked recessive, or autosomal dominant. However, the most common is autosomal recessive form and it is the most severe phenotype. Twenty-nail dystrophy refers to the condition in which all the twenty nails are affected in the form of excessive ridging and nail plate roughness leading to unsightly lustureless nails. We report a rare case of two siblings with alopecia universalis congenita with twenty-nail dystrophy. To the best of our knowledge, this case is the first case to be reported with such association in both siblings. This case reports highlights the fact that alopecia areata is an autoimmune disease with a genetic predisposition as in our case both siblings had alopecia universalis and nail dystrophy. There was no evidence of any other ectodermal dyplasia and had normal teeth and seat glands. The skin biopsy ruled out congenital atrichia and was suggestive of alopecia areata.

A Novel Role of Silibinin as a Putative Epigenetic Modulator in Human Prostate Carcinoma.

Silibinin, extracted from milk thistle (Silybum marianum L.), has exhibited considerable preclinical activity against prostate carcinoma. Its antitumor and chemopreventive activities have been associated with diverse effects on cell cycle, apoptosis, and receptor-dependent mitogenic signaling pathways. Here we hypothesized that silibinin’s pleiotropic effects may reflect its interference with epigenetic mechanisms in human prostate cancer cells. More specifically, we have demonstrated that silibinin reduces gene expression levels of the Polycomb Repressive Complex 2 (PRC2) members Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste Homolog 12 (SUZ12), and Embryonic Ectoderm Development (EED) in DU145 and PC3 human prostate cancer cells, as evidenced by Real Time Polymerase Chain Reaction (RT-PCR). Furthermore immunoblot and immunofluorescence analysis revealed that silibinin-mediated reduction of EZH2 levels was accompanied by an increase in trimethylation of histone H3 on lysine (Κ)-27 residue (H3K27me3) levels and that such response was, in part, dependent on decreased expression levels of phosphorylated Akt (ser473) (pAkt) and phosphorylated EZH2 (ser21) (pEZH2). Additionally silibinin exerted other epigenetic effects involving an increase in total DNA methyltransferase (DNMT) activity while it decreased histone deacetylases 1-2 (HDACs1-2) expression levels. We conclude that silibinin induces epigenetic alterations in human prostate cancer cells, suggesting that subsequent disruptions of central processes in chromatin conformation may account for some of its diverse anticancer effects.

Nuf and Rip11 requirement for polarity determinant recycling during Drosophila development

ABSTRACTA tight relationship between apico-basal polarity and trafficking is essential for epithelial physiology and tissue homeostasis. Recent studies have described how some Rab GTPases, key components of the intracellular traffic machinery, contribute to the establishment of cell polarity in vertebrates. We have demonstrated a novel connection between cell polarity and trafficking: in Drosophila epithelia, the apical determinant aPKC is recycled via Rab11-Nuf-recycling endosomes to maintain cell polarity. Furthermore, the phosphorylation of Nuf by aPKC allows aPKC to control the sub-cellular localization of Nuf and its own membrane accumulation. Here we review these data and show the different contribution of the 2 Drosophila Rab11 adaptor proteins, Nuf and Rip11, to the maintenance of Drosophila embryonic ectoderm polarity.

Propagation of trimethylated H3K27 regulated by polycomb protein EED is required for embryogenesis, hematopoietic maintenance, and tumor suppression

Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

Maintenance of the functional integrity of mouse hematopoiesis by EED and promotion of leukemogenesis by EED haploinsufficiency.

Polycomb repressive complex 2 (PRC2) participates in transcriptional repression through methylation of histone H3K27. The WD-repeat protein embryonic ectoderm development (EED) is a non-catalytic but an essential component of PRC2 and its mutations were identified in hematopoietic malignancies. To clarify the role(s) of EED in adult hematopoiesis and leukemogenesis, we generated Eed conditional knockout (EedΔ/Δ) mice. EedΔ/Δ mice died in a short period with rapid decrease of hematopoietic cells. Hematopoietic stem/progenitor cells (HSPCs) were markedly decreased with impaired bone marrow (BM) repopulation ability. Cell cycle analysis of HSPCs demonstrated increased S-phase fraction coupled with suppressed G0/G1 entry. Genes encoding cell adhesion molecules are significantly enriched in EedΔ/Δ HSPCs, and consistently, EedΔ/Δ HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other, and these conflicting events would lead to abnormal differentiation and functional defect of EedΔ/Δ HSPCs. In addition, Eed haploinsufficiency induced hematopoietic dysplasia, and Eed heterozygous mice were susceptible to malignant transformation and developed leukemia in cooperation with Evi1 overexpression. Our results demonstrated differentiation stage-specific and dose-dependent roles of EED in normal hematopoiesis and leukemogenesis.

Abstract 1841: Dissecting gene expression programs that define tumor aggression and patient outcome in pancreatic cancer

Abstract By the year 2020, pancreatic cancer (PDAC) is projected to be the second leading cause of cancer deaths in the United States. Current systemic therapies offer limited survival advantages in subgroups of patients, suggesting underlying biological differences in this deadly cancer. Our lab has recently identified keratin-17 (K17) gene expression as a prognostic and predictive biomarker in PDAC. Furthermore, we found that K17 acts as an oncoprotein by promoting degradation of tumor suppressor p27KIP1, while knockdown of K17 limits tumor size and sensitizes cancer cells to chemotherapy agents. Together, these findings suggest that targeting K17 expression in PDACs may increase patient survival and response to treatment. The factors that lead to increased K17 gene expression in only a subset of PDAC cases, however, are unknown. K17 is expressed in embryonic ectoderm and aberrantly induced in keratinocytes from psoriasis patients through paracrine signaling from the microenvironment. To determine how K17 gene expression is upregulated in PDAC, we tested the effects of two major contributing factors in PDAC pathogenesis: driver mutations and inflammatory mediators present in tumor microenvironment. Using in vitro and in vivo loss- and gain-of function approaches, we modulated oncogene expression in and performed acute cytokine treatments on cells to test for changes in K17 gene expression. K17 expression was evaluated by qRT-PCR and western blot. We found that forced expression of oncogenes like KRASG12D, Q61K or TP53R248W significantly increase K17 expression, yet these cell-autonomous factors are not sufficient to explain K17 gene expression in PDACs. In addition, we found that pro-inflammatory cytokines, typically released by helper T cells, induce a strong increase in K17 gene expression. Together these findings suggest that K17 gene expression across otherwise clinically identical PDAC cases may be explained by early transformation events and maintained by positive feedback loops associated with patient specific adaptive immune response. Ongoing studies are seeking to test the combined effects of genetic and paracrine events in K17 expression, and understand the signaling pathways and transcription factors downstream of these inducing factors. Ultimately, insight into the mechanisms that mediate K17 overexpression in PDACs could help guide future research to develop K17 as a potential therapeutic target. Citation Format: Danielle J. Fassler, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. Dissecting gene expression programs that define tumor aggression and patient outcome in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1841.

Deletion of Polycomb Repressive Complex 2 From Mouse Intestine Causes Loss of Stem Cells

The polycomb repressive complex 2 (PRC2) regulates differentiation by contributing to repression of gene expression and thereby stabilizing the fate of stem cells and their progeny. PRC2 helps to maintain adult stem cell populations, but little is known about its functions in intestinal stem cells. We studied phenotypes of mice with intestine-specific deletion of the PRC2 proteins embryonic ectoderm development (EED) (a subunit required for PRC2 function) and enhancer of zeste homolog 2 (EZH2) (a histone methyltransferase). We performed studies of AhCre;EedLoxP/LoxP (EED knockout) mice and AhCre;Ezh2LoxP/LoxP (EZH2 knockout) mice, which have intestine-specific disruption in EED and EZH2, respectively. Small intestinal crypts were isolated and subsequently cultured to grow organoids. Intestines and organoids were analyzed by immunohistochemical, in situ hybridization, RNA sequence, and chromatin immunoprecipitation methods. Intestines of EED knockout mice had massive crypt degeneration and lower numbers of proliferating cells compared with wild-type control mice. Cdkn2a became derepressed and we detected increased levels of P21. We did not observe any differences between EZH2 knockout and control mice. Intestinal crypts from EED knockout mice had signs of aberrant differentiation of uncommitted crypt cells-these differentiated toward the secretory cell lineage. Furthermore, crypts from EED-knockout mice had impaired Wnt signaling and concomitant loss of intestinal stem cells, this phenotype was not reversed upon ectopic stimulation of Wnt and Notch signaling in organoids. Analysis of gene expression patterns from intestinal tissues of EED knockout mice showed dysregulation of several genes involved in Wnt signaling. Wnt signaling was regulated directly by PRC2. In intestinal tissues of mice, PRC2 maintains small intestinal stem cells by promoting proliferation and preventing differentiation in the intestinal stem cell compartment. PRC2 controls gene expression in multiple signaling pathways that regulate intestinal homeostasis. Sequencing data are available in the genomics data repository GEO under reference series GSE81578; RNA sequencing data are available under subseries GSE81576; and ChIP sequencing data are available under subseries GSE81577.

HIFI-α activation underlies a functional switch in the paradoxical role of Ezh2/PRC2 in breast cancer

Despite the established oncogenic function of Polycomb repressive complex 2 (PRC2) in human cancers, its role as a tumor suppressor is also evident; however, the mechanism underlying the regulation of the paradoxical functions of PRC2 in tumorigenesis is poorly understood. Here we show that hypoxia-inducible factor 1, α-subunit (HIFI-α) is a crucial modulator of PRC2 and enhancer of zeste 2 (EZH2) function in breast cancer. Interrogating the genomic expression of breast cancer indicates high HIF1A activity correlated with high EZH2 expression but low PRC2 activity in triple-negative breast cancer compared with other cancer subtypes. In the absence of HIFIA activation, PRC2 represses the expression of matrix metalloproteinase genes (MMPs) and invasion, whereas a discrete Ezh2 complexed with Forkhead box M1 (FoxM1) acts to promote the expression of MMPs. HIF1-α induction upon hypoxia results in PRC2 inactivation by selective suppression of the expression of suppressor of zeste 12 protein homolog (SUZ12) and embryonic ectoderm development (EED), leading to a functional switch toward Ezh2/FoxM1-dependent induction of the expression of MMPs and invasion. Our study suggests a tumor-suppressive function of PRC2, which is restricted by HIF1-α, and an oncogenic function of Ezh2, which cooperates with FoxM1 to promote invasion in triple-negative breast cancer.

Christ-Seimmens-Touraine Syndrome

Ectodermal dysplasia is a large heterogenous group of inherited disorders, defined by primary defect in development of two or more tissue derived from embryonic ectoderm. The tissues primarily involved are skin, its appendages (hair, eccrine glands, sebaceous gland & nails) and teeth. Ectodermal dysplasias are congenital, diffuse & nonprogressive. The genetic defects responsible for approximately 62 of the ectodermal dysplasias have been identified. The most common are X-linked hypohidrotic ectodermal dysplasia (Christ-Seimmens-Touraine Syndrome) which is caused by mutation of EDA gene which encodes the ectodysplasin protein, a soluble ligand that activates the NF-kappaB and JNK/c-fos/c-jun signaling pathways. Ectodysplasin is important in promoting cell survival, growth, and differentiation. Using specialized techniques, including confocal imaging, phototrichogram analysis, and pilocarp ineiontophoresis, a complete absence of eccrine ducts, reduction in hair follicle units & hair follicle density and decreased growth rate of terminal hairs has been demonstrated in patients with XL-HED. In present case we have found classical features of X-linked hypohidrotic Ectodermal dysplasia also known as Christ-Seimmens-Touraine Syndrome.

Polycomb repressive complex's evolutionary conserved function: the role of EZH2 status and cellular background.

When assembled in multiprotein polycomb repressive complexes (PRCs), highly evolutionary conserved polycomb group (PcG) proteins epigenetically control gene activity. Although the composition of PRCs may vary considerably, it is well established that the embryonic ectoderm development (EED) 1, suppressor of zeste (SUZ) 12, and methyltransferase enhancer of zeste (EZH2)-containing complex, PRC2, which is abundant in highly proliferative cells (including cancer cells), establishes a repressive methylation mark on histone 3 (H3K27me3). From the perspective of molecular cancer pathogenesis, this effect, when directed towards a promoter of tumor suppressor genes, represents pro-tumorigenic effect. This mode of action was shown in several cancer models. However, EZH2 function extends beyond this scenario. The highly specific cellular background, related to the origin of cell and numerous external stimuli during a given time-window, may be the trigger for EZH2 interaction with other proteins, not necessarily histones. This is particularly relevant for cancer.This review provides a critical overview of the evolutional importance of PRC and discusses several important aspects of EZH2 functioning within PRC. The review also deals with mutational studies on EZH2. Due to the existence of several protein (and messenger RNA (mRNA)) isoforms, these mutations were stratified, using the protein sequence which is considered canonical. This approach showed that there is an urgent need for the uniformed positioning of currently known EZH2 mutations (somatic—in tumors, as well as germline mutations in the Weaver’s syndrome).Finally, we discuss EZH2 function with respect to amount of trimethylated H3K27, in a specific cellular milieu, through presenting the most recent data related to EZH2-H3K27m3 relationship in cancer. All these points are significant in considering EZH2 as a therapeutic target.

EZH2 mutations and promoter hypermethylation in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and young adults. The polycomb repressive complex 2 (PRC2) has been identified as one of the most frequently mutated epigenetic protein complexes in hematologic cancers. PRC2 acts as an epigenetic repressor through histone H3 lysine 27 trimethylation (H3K27me3), catalyzed by the histone methyltransferase enhancer of zeste homolog 2 protein (EZH2). To study the prevalence and clinical impact of PRC2 aberrations in an unselected childhood ALL cohort (n=152), we performed PRC2 mutational screenings by Sanger sequencing and promoter methylation analyses by quantitative pyrosequencing for the three PRC2 core component genes EZH2, suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED). Targeted deep next-generation sequencing of 30 frequently mutated genes in leukemia was performed to search for cooperating mutations in patients harboring PRC2 aberrations. Finally, the functional consequence of EZH2 promoter hypermethylation on H3K27me3 was studied by Western blot analyses of primary cells. Loss-of-function EZH2 mutations were detected in 2/152 (1.3%) patients with common-ALL and early T-cell precursor (ETP)-ALL, respectively. In one patient, targeted deep sequencing identified cooperating mutations in ASXL1 and TET2. EZH2 promoter hypermethylation was found in one patient with ETP-ALL which led to reduced H3K27me3. In comparison with healthy children, the EZH2 promoter was significantly higher methylated in T-ALL patients. No mutations or promoter methylation changes were identified for SUZ12 or EED genes, respectively. Although PRC2 aberrations seem to be rare in childhood ALL, our findings indicate that EZH2 aberrations might contribute to the disease in specific cases. Hereby, EZH2 promoter hypermethylation might have functionally similar consequences as loss-of-function mutations.

Neural transcription factors bias cleavage stage blastomeres to give rise to neural ectoderm.

The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. genesis 54:334-349, 2016. © 2016 Wiley Periodicals, Inc.

OVOL2 Maintains the Transcriptional Program of Human Corneal Epithelium by Suppressing Epithelial-to-Mesenchymal Transition.

In development, embryonic ectoderm differentiates into neuroectoderm and surface ectoderm using poorly understood mechanisms. Here, we show that the transcription factor OVOL2 maintains the transcriptional program of human corneal epithelium cells (CECs), a derivative of the surface ectoderm, and that OVOL2 may regulate the differential transcriptional programs of the two lineages. A functional screen identified OVOL2 as a repressor of mesenchymal genes to maintain CECs. Transduction of OVOL2 with several other transcription factors induced the transcriptional program of CECs in fibroblasts. Moreover, neuroectoderm derivatives were found to express mesenchymal genes, and OVOL2 alone could induce the transcriptional program of CECs in neural progenitors by repressing these genes while activating epithelial genes. Our data suggest that the difference between the transcriptional programs of some neuroectoderm- and surface ectoderm-derivative cells may be regulated in part by a reciprocallyrepressive mechanism between epithelial and mesenchymal genes, as seen in epithelial-to-mesenchymal transition.

Effects of bone marrow mesenchymal stem cells transplantation on tumor necrosis factor-α and interleukin-1β in preterm rats with hypoxic-ischemic brain damage

Objective To determine the changes in tumor necrosis factor-α (TNF-α) and interleukin-lβ (IL-1β) expression after bone marrow mesenchymal stem cells (BMSCs) transplantation in a rat pup model of hypoxic-ischemic brain damage (HIBD) and discuss the anti-inflammatory mechanism of BMSCs transplantation in the repair of HIBD. Methods Three-day-old Sprague-Dawley rat pups were randomly divided into three groups: the transplantation group, in which a model of HIBD was established by ligation of left common carotid artery and hypoxia for two hours followed by the injection of 2 μl BMSCs (2×105 cells) into the lateral ventricle; the HIBD model group, in which HIBD model was established and 2 μl phosphate saline buffer was injected into the lateral ventricle; and the sham-operation group, in which no intervention was given. Histological changes in the brain were detected by HE staining and the number of cells positive for ectodermal dysplasia-1 (ED-1) staining, which is a specific marker for activated microglia, was detected by immunohistochemistry. The protein and mRNA levels of TNF-α and IL-1β were determined by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. One-way analysis of variance and LSD test were applied for statistical analysis. Results HE staining showed that cellular edema and necrocytosis was not observed in cerebral white matter on the 7th post-transplantation day in the transplantation group, but observed in the HIBD model group. In the sham-operation group, cerebral white matter was normal. The number of ED-1 positive cells in the transplantation group (26.3±2.5) was significantly lower than that in the HIBD model group (33.0±4.0), but higher than that in the sham-operation group (2.3±0.6) (LSD test, all P<0.05). The contents of TNF-α and IL-1β both in the transplantation group and the HIBD model group increased and peaked 24 h after transplantation, then gradually decreased, but did not reach normal levels (sham-operation group) on the 7th day. The contents of TNF-α and IL-1β in brain tissue in the HIBD model group [TNF-α: (3.03±0.10), (5.57±0.19), (7.78±0.19), (4.39±0.20), (2.70±019) μg/L; IL-1β: (293.1±7.9), (369.8±17.5), (303.6±23.9), (226.7±21.6), (183.9±33.4) ng/L] were significantly higher than those in the transplantation group [TNF-α: (2.84±0.20), (3.80±0.14), (4.63±0.17), (3.56±0.03), (1.99±0.17) μg/L; IL-1β: (267.6±14.5), (323.5±26.9), (211.2±24.9), (140.8±7.4), (100.2±8.3) ng/L] at 6, 12, 24, 48 h and 7 days after BMSCs transplantation, respectively. The contents of TNF-α and IL-1β in the sham-operation group [TNF-α: (1.03±0.02), (1.13±0.03), (1.05±0.02), (1.09±0.02), (1.07±0.02) μg/L; IL-1β: (63.6±13.0), (64.0±11.3), (60.8±10.0), (67.9±13.5), (66.2±11.7) ng/L] were significantly lower than those in the transplantation group and HIBD model group (LSD test, all P<0.05). TNF-α and IL-1β mRNA at 24 h after transplantation in the HIBD model group (TNF-α: 2.69±0.43; IL-1β: 3.07±0.38) were significantly higher than those in the transplantation group (TNF-α: 1.61±0.29; IL-1β: 1.08±0.11) and those in the sham-operation group (TNF-α: 0.94±0.16; IL-1β: 1.08±0.11) (LSD test, all P<0.05). Conclusions BMSCs may play an important role in the recovery of preterm HIBD and the mechanism may involve the inhibition of microglia activation and down-regulation of the expression of inflammatory factors. Key words: Hypoxia-ischemia, brain; Mesenchymal stem cell transplantation; Tumor necrosis factor-alpha; Interleukin-1beta

Construction of HaCaT cell lines stably expressing the human GJB6 gene by using a Tet-On lentiviral vector and their identification

Objective To construct HaCaT cell lines stably expressing the wild type human GJB6 gene or its mutant by using a Tet-On lentiviral vector, and to lay an experimental foundation for studies on pathogenesis of hidrotic ectodermal dysplasia. Methods The wild-type human GJB6 gene and its mutant (A88V) were amplified by PCR, and then inserted into the Tet - on lentivirus plasmid to construct recombinant lentivirus vectors. The recombinants were identified by gene sequencing and enzymatic digestion. Cultured HaCaT cells were classified into three groups to be transfected with a negative control lentiviral vector (NC group), the lentivirus vector expressing the wild-type human GJB6 gene (WT group), or the lentivirus vector expressing the mutant human GJB6 gene (MU group). Puromycin was used to select HaCaT cell clones stably expressing the GJB6 gene which encodes the connexin 30 (Cx30) protein. The selected HaCaT cell clones were cultured with or without tetracycline for 48 hours, thereafter, real-time PCR (RT-PCR) was performed to detect GJB6 gene mRNA expression, Western-blot analysis to measure expressions of Cx30 and FLAG-tag proteins, and cell counting kit 8 (CCK8) assay to evaluate cellular proliferative activity. Results Enzymatic digestion and gene sequencing showed that recombinant lentivirus plasmids were successfully constructed. RT-PCR showed evidently increased mRNA expression of the GJB6 gene in stably transfected HaCaT cells. Moreover, the expression abundance of the GJB6 gene was 112.369 times higher in the WT group induced by tetracycline than in that without tetracycline treatment (P 0.05). Conclusion HaCaT cell lines which stably express the wild - type GJB6 gene or its mutant (A88V) are successfully constructed. Key words: Ectodermal dysplasia; Connexins; GJB6 gene; HaCaT cells; Tet-on

Loss of H3K27me3 Expression Is a Highly Sensitive Marker for Sporadic and Radiation-induced MPNST.

Most malignant peripheral nerve sheath tumors (MPNSTs) exhibit combined inactivation of NF1, CDKN2A, and polycomb repressive complex 2 component genes (Embryonic Ectoderm Development [EED] and Suppressor of Zeste 12 [SUZ12]). Mutations in EED and SUZ12 induce loss of trimethylation at lysine 27 of histone 3 (H3K27me3), with subsequent aberrant transcriptional activation of polycomb repressive complex 2-repressed homeobox master regulators. These findings prompted us to investigate the performance of an anti-H3K27me3 monoclonal antibody clone C36B11 as an immunohistochemical marker for MPNSTs. We assessed the C36B11 reactivity pattern in a pathologically and genetically well-characterized cohort of 68 MPNSTs, spanning various clinical presentations, such as type 1 neurofibromatosis (NF1), radiotherapy, and sporadic MPNSTs. We found that 69% (n=47) of all MPNSTs demonstrated loss of H3K27me3 expression, with 42 (61%) showing complete loss and 5 (7%) showing partial loss, whereas 31% (n=21) retained H3K27me3 expression. Among the NF1-related high-grade MPNSTs, 60% demonstrated loss of expression. In contrast, the majority of both sporadic (95%) and radiotherapy-related (91%) MPNSTs showed loss of H3K27me3 expression. Two of the 3 low-grade MPNSTs and all neurofibromas showed retained expression. Furthermore, all 5 epithelioid MPNSTs retained H3K27me3 labeling. The specificity of H3K27me3 loss as a marker for MPNSTs was studied by testing a large spectrum of lesions included in MPNST differential diagnosis, such as spindle/desmoplastic melanomas, synovial sarcomas, myoepithelial tumors, and other mesenchymal neoplasms, all of which retained expression of H3K27me3. We conclude that immunohistochemical analysis of H3K27me3 has good sensitivity and robust specificity for the diagnosis of MPNST, particularly outside of NF1 clinical history, which represents the most challenging diagnostic setting.

Dissecting the Roles of Polycomb Repressive Complex 2 Subunits in the Control of Skin Development

Polycomb repressive complex 2 (PRC2) is an essential regulator of cell physiology. Although there have been numerous studies on PRC2 function in somatic tissue development and stem cell control, these have focused on the loss of a single PRC2 subunit. Recent studies, however, have shown that PRC2 subunits may function independently of the PRC2 complex. To investigate the function of PRC2 in the control of skin development, we generated and analyzed three conditional knockout mouse lines, in which the essential PRC2 subunits embryonic ectoderm development (EED), suppressor of zeste 12 homolog (Suz12), and enhancer of zeste homologs 1 and 2 (Ezh1/2) are conditionally ablated in the embryonic epidermal progenitors that give rise to the epidermis, hair follicles, and Merkel cells. Our studies showed that the observed loss-of-function phenotypes are shared between the three knockouts, indicating that in the skin epithelium, EED, Suz12, and Ezh1/2 function largely as subunits of the PRC2 complex. Interestingly, the absence of PRC2 results in dramatically different phenotypes across the different skin lineages: premature acquisition of a functional epidermal barrier, formation of ectopic Merkel cells, and defective postnatal development of hair follicles. The strikingly different roles of PRC2 in the formation of three lineages exemplify the complex outcomes that the lack of PRC2 can have in a somatic stem cell system.

Activation of the polycomb repressive complex pathway in the bone marrow resident cells of diffuse large B-cell lymphoma patients

The present study investigated the activation of polycomb repressive complex 2 (PRC2) pathway proteins in the resident cells within the bone marrow hematopoietic microenvironment of diffuse large B-cell lymphoma (DLBCL) patients. PRC2 proteins (enhancer of zeste homolog 2, suppressor of zeste 12 homolog, and embryonic ectoderm development), histone methylation mark (H3K27me3), and c-MYC activation were evaluated in pretreatment bone marrow from 208 DLBLC patients. Positive expression of the PRC2, H3K27me3, and c-MYC in the bone marrow resident cells was more frequent in cases with bone marrow involvement of tumor. The expression among PRC2, H3K27me3 mark, and c-MYC was closely correlated. Positive PRC2 expression in bone marrow resident cells was significantly associated with inferior progression-free survival (PFS) and overall survival (OS) and determined to be an independent prognostic factor of inferior PFS and OS. In conclusion, the PRC pathway was frequently activated in bone marrow resident cells of DLBCL patients, and PRC activation was tumor-related and associated with poor clinical outcomes.

Unilateral duplication of parotid duct – a rare cadaveric case report

The parotid gland is drained by the parotid duct which normally measures 50 mm in length and 3 mm in width. The parotid duct emerges at the anterior border of the gland and runs horizontally across the masseter muscle to pierce the buccinator and open into the vestibule of the mouth. The occurrence of double parotid duct is 7% and there are only very few literature references. The parotid gland develops as an epithelial bud arising from the oral ectoderm and invaginating into the underlying mesenchyme. The proximal part canalises to form the duct and the distal end differentiates to form the acini. An early division of the parotid duct with acini from both ducts intermingling with each other results in the formation of double parotid duct. The cytodifferentiation and morphogenesis of parotid gland are influenced by cell specific gene expression and cell-matrix interactions that produce collagen responsible for branching and hyaluronidase responsible for acini formation. The awareness of presence of a double parotid duct is significant in ductal endoscopic procedures and in performing surgery in the parotid region.

A rare case of anhidrotic ectodermal dysplasia in a six-year-old boy

Introduction: Ectodermal dysplasia is a rare, non-progressive, genetic disorder resulting from abnormal development of two or more tissues at a time which are derived from the embryonic ectoderm. It classically manifests with skin, eccrine gland, nail and hair changes, with an incidence of 1 in 1, 00,000 births. More than 170 different syndromes have been identified. X