Unveiling the Role of Exosomes in the Pathophysiology of Sepsis: Insights into Organ Dysfunction and Potential Biomarkers.

Extracellular vesicles (EVs) are tools for intercellular communication, mediating molecular transport processes. Emerging studies have revealed that EVs are significantly involved in immune processes, including sepsis. Sepsis, a dysregulated immune response to infection, triggers systemic inflammation and multi-organ dysfunction, posing a life-threatening condition. Although extensive research has been conducted on animals, the complex inflammatory mechanisms that cause sepsis-induced organ failure in humans are still not fully understood. Recent studies have focused on secreted exosomes, which are small extracellular vesicles from various body cells, and have shed light on their involvement in the pathophysiology of sepsis. During sepsis, exosomes undergo changes in content, concentration, and function, which significantly affect the metabolism of endothelia, cardiovascular functions, and coagulation. Investigating the role of exosome content in the pathogenesis of sepsis shows promise for understanding the molecular basis of human sepsis. This review explores the contributions of activated immune cells and diverse body cells' secreted exosomes to vital organ dysfunction in sepsis, providing insights into potential molecular biomarkers for predicting organ failure in septic shock.

Integrative Epigenetic and Molecular Analysis Reveals a Novel Promoter for a New Isoform of the Transcription Factor TEAD4.

TEAD4 is a transcription factor that plays a crucial role in the Hippo pathway by regulating the expression of genes related to proliferation and apoptosis. It is also involved in the maintenance and differentiation of the trophectoderm during pre- and post-implantation embryonic development. An alternative promoter for the TEAD4 gene was identified through epigenetic profile analysis, and a new transcript from the intronic region of TEAD4 was discovered using the 5'RACE method. The transcript of the novel promoter encodes a TEAD4 isoform (TEAD4-ΔN) that lacks the DNA-binding domain but retains the C-terminal protein-protein interaction domain. Gene expression studies, including end-point PCR and Western blotting, showed that full-length TEAD4 was present in all investigated tissues. However, TEAD4-ΔN was only detectable in certain cell types. The TEAD4-ΔN promoter is conserved throughout evolution and demonstrates transcriptional activity in transient-expression experiments. Our study reveals that TEAD4 interacts with the alternative promoter and increases the expression of the truncated isoform. DNA methylation plays a crucial function in the restricted expression of the TEAD4-ΔN isoform in specific tissues, including the umbilical cord and the placenta. The data presented indicate that the DNA-methylation status of the TEAD4-ΔN promoter plays a critical role in regulating organ size, cancer development, and placenta differentiation.

A Promising Way to Overcome Temozolomide Resistance through Inhibition of Protein Neddylation in Glioblastoma Cell Lines.

There is no effective therapy for the lately increased incidence of glioblastoma multiforme (GBM)-the most common primary brain tumor characterized by a high degree of invasiveness and genetic heterogeneity. Currently, DNA alkylating agent temozolomide (TMZ) is the standard chemotherapy. Nevertheless, TMZ resistance is a major problem in the treatment of GBM due to numerous molecular mechanisms related to DNA damage repair, epigenetic alterations, cellular drug efflux, apoptosis-autophagy, and overactive protein neddylation. Low molecular weight inhibitors of NEDD8-activating enzyme (NAE), such as MLN4924, attenuate protein neddylation and present a promising low-toxicity anticancer agent. The aim of our study was to find an effective combination treatment with TMZ and MLN4924 in our TMZ-resistant GBM cell lines and study the effect of these combination treatments on different protein expressions such as O6-methylguanine methyltransferase (MGMT) and p53. The combination treatment successfully decreased cell viability and sensitized TMZ-resistant cells to TMZ, foreshadowing a new treatment strategy for GBM.

Analysis of Gene Expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models.

We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

DNA Methylation-Governed Gene Expression in Autoimmune Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease hallmarked by progressive and irreversible joint destruction. RA pathogenesis is a T cell-regulated and B cell-mediated process in which activated lymphocyte-produced chemokines and cytokines promote leukocyte infiltration that ultimately leads to destruction of the joints. There is an obvious need to discover new drugs for RA treatment that have different biological targets or modes of action than the currently employed therapeutics. Environmental factors such as cigarette smoke, certain diet components, and oral pathogens can significantly affect gene regulation via epigenetic factors. Epigenetics opened a new field for pharmacology, and DNA methylation and histone modification-implicated factors are feasible targets for RA therapy. Exploring RA pathogenesis involved epigenetic factors and mechanisms is crucial for developing more efficient RA therapies. Here we review epigenetic alterations associated with RA pathogenesis including DNA methylation and interacting factors. Additionally, we will summarize the literature revealing the involved molecular structures and interactions. Finally, potential epigenetic factor-based therapies will be discussed that may help in better management of RA in the future.

Amelioration of Autoimmune Arthritis in Mice Treated With the DNA Methyltransferase Inhibitor 5'-Azacytidine.

OBJECTIVE: Disease-associated, differentially hypermethylated regions have been reported in rheumatoid arthritis (RA), but no DNA methyltransferase inhibitors have been evaluated in either RA or any animal models of RA. The present study was conducted to evaluate the therapeutic potential of 5'-azacytidine (5'-azaC), a DNA methyltransferase inhibitor, and explore the cellular and gene regulatory networks involved in the context of autoimmune arthritis. METHODS: A disease-associated genome-wide DNA methylation profile was explored by methylated CpG island recovery assay-chromatin immunoprecipitation (ChIP) in arthritic B cells. Mice with proteoglycan-induced arthritis (PGIA) were treated with 5'-azaC. The effect of 5'-azaC on the pathogenesis of PGIA was explored by measuring serum IgM and IgG1 antibody levels using enzyme-linked immunosorbent assay, investigating the efficiency of class-switch recombination (CSR) and Aicda gene expression using real-time quantitative polymerase chain reaction, monitoring germinal center (GC) formation by immunohistochemistry, and determining alterations in B cell subpopulations by flow cytometry. The 5'-azaC-induced regulation of the Aicda gene was explored using RNA interference, ChIP, and luciferase assays. RESULTS: We explored arthritis-associated hypermethylated regions in mouse B cells and demonstrated that DNA demethylation had a beneficial effect on autoimmune arthritis. The 5'-azaC-mediated demethylation of the epigenetically inactivated Ahr gene resulted in suppressed expression of the Aicda gene, reduced CSR, and compromised GC formation. Ultimately, this process led to diminished IgG1 antibody production and amelioration of autoimmune arthritis in mice. CONCLUSION: DNA hypermethylation plays a leading role in the pathogenesis of autoimmune arthritis and its targeted inhibition has therapeutic potential in arthritis management.

Transcription factor Zbtb38 downregulates the expression of anti-inflammatory IL1r2 in mouse model of rheumatoid arthritis.

DNA methylation is a decisive regulator of gene expression. Differentially methylated promoters were described in rheumatoid arthritis (RA), but we do not know how these epimutations can trigger a proinflammatory cytokine milieu. B cell-focused DNA methylome studies identified a group of genes that had undergone disease-associated changes in a murine model of RA. An arthritis-specific epimutation (hypomethylation) was detected in the promoter region of the Zbtb38 gene, which encodes a transcriptional repressor. Gene expression studies revealed that hypomethylation of the Zbtb38 promoter was accompanied by disease-specific repressor expression, and two anti-inflammatory factors interleukin 1 receptor 2 gene (IL1r2) and interleukin-1 receptor antagonist (IL1rn) were among the downregulated genes. We hypothesized that Zbtb38 repressor could induce downregulated expression of these anti-inflammatory genes and that this could significantly contribute to arthritis pathogenesis. Our studies demonstrate that Zbtb38 forms a molecular bridge between an arthritis-associated epimutation (DNA hypomethylation in Zbtb38 promoter) and transcriptional silencing of the IL1r2 gene in B cells. In this way, disease-associated DNA hypomethylation can support autoimmune arthritis by interfering with an anti-inflammatory pathway.

FTY720 (Gilenya) treatment prevents spontaneous autoimmune myocarditis and dilated cardiomyopathy in transgenic HLA-DQ8-BALB/c mice.

Although dilated cardiomyopathy (DCM) is often caused by viral infections, it frequently involves autoimmune mechanisms associated with particular HLA-DR and DQ alleles. Our homozygous HLA-DQ8Ab(0) transgenic mice in the BALB/c background (HLA-DQ8(BALB/c)-Tg) developed early and progressive fatal heart failure from 4 to 5 weeks of age. Clinical signs of the disease included cyanotic eyes, tachycardia with dyspnea (from pale to cyanotic limbs), and terminal whole body edema. Sick mice had extremely dilated hearts, enlarged liver and spleen, and pleural/peritoneal effusion. Histology of the heart showed extensive heart muscle destruction with signs of fibrosis. The autoimmune nature of the disease was shown by high titers of antimyosin antibodies in the sera and IgG deposits in sick heart muscles, as well as focal neutrophil, T cell, and macrophage infiltration of the heart muscle. The sera of the sick mice showed a granular staining pattern on sections of healthy heart muscle. Quantitative analyses of DCM-specific gene expression studies revealed that sets of genes are involved in inflammation, hypoxia, and fibrosis. Treatment with FTY720 (Fingolimod/Gilenya) protected animals from the development of cardiomyopathy. HLA-DQ8(BALB/c)-Tg mice represent a spontaneous autoimmune myocarditis model that may provide a useful tool for studying the autoimmune mechanism of DCM and testing immunosuppressive drugs.

Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA. METHODS: CitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry. FINDINGS: Sera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage. CONCLUSIONS: CitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.

Enigma of IL-17 and Th17 Cells in Rheumatoid Arthritis and in Autoimmune Animal Models of Arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune disorders characterized by the chronic and progressive inflammation of various organs, most notably the synovia of joints leading to joint destruction, a shorter life expectancy, and reduced quality of life. Although we have substantial information about the pathophysiology of the disease with various groups of immune cells and soluble mediators identified to participate in the pathogenesis, several aspects of the altered immune functions and regulation in RA remain controversial. Animal models are especially useful in such scenarios. Recently research focused on IL-17 and IL-17 producing cells in various inflammatory diseases such as in RA and in different rodent models of RA. These studies provided occasionally contradictory results with IL-17 being more prominent in some of the models than in others; the findings of such experimental setups were sometimes inconclusive compared to the human data. The aim of this review is to summarize briefly the recent advancements on the role of IL-17, particularly in the different rodent models of RA.

MIRA-seq for DNA methylation analysis of CpG islands.

AIM: To develop a reliable method for whole genome analysis of DNA methylation. MATERIALS & METHODS: Genome-scale analysis of DNA methylation includes affinity-based approaches such as enrichment using methyl-CpG-binding proteins. One of these methods, the methylated-CpG island recovery assay (MIRA), is based on the high affinity of the MBD2b-MBD3L1 complex for CpG-methylated DNA. Here we provide a detailed description of MIRA and combine it with next generation sequencing platforms (MIRA-seq). RESULTS: We assessed the performance of MIRA-seq and compared the data with whole genome bisulfite sequencing. CONCLUSION: MIRA-seq is a reliable, genome-scale DNA methylation analysis platform for scoring DNA methylation differences at CpG-rich genomic regions. The method is not limited by primer or probe design and is cost effective.

Suppression of proteoglycan-induced autoimmune arthritis by myeloid-derived suppressor cells generated in vitro from murine bone marrow.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA. METHODS: Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined. RESULTS: BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels. CONCLUSIONS: Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.

Epigenetics in the pathogenesis of rheumatoid arthritis.

An increasing number of studies show that besides the inherited genetic architecture (that is, genomic DNA), various environmental factors significantly contribute to the etiology of rheumatoid arthritis. Epigenetic factors react to external stimuli and form bridges between the environment and the genetic information-harboring DNA. Epigenetic mechanisms are implicated in the final interpretation of the encoded genetic information by regulating gene expression, and alterations in their profile influence the activity of the immune system. Overall, epigenetic mechanisms further increase the well-known complexity of rheumatoid arthritis by providing additional subtle contributions to rheumatoid arthritis susceptibility. Although there are controversies regarding the involvement of epigenetic and genetic factors in rheumatoid arthritis etiology, it is becoming obvious that the two systems (genetic and epigenetic) interact with each other and are ultimately responsible for rheumatoid arthritis development. Here, epigenetic factors and mechanisms involved in rheumatoid arthritis are reviewed and new, potential therapeutic targets are discussed.

Differentially expressed epigenome modifiers, including aurora kinases A and B, in immune cells in rheumatoid arthritis in humans and mouse models.

OBJECTIVE: To identify epigenetic factors that are implicated in the pathogenesis of rheumatoid arthritis (RA), and to explore the therapeutic potential of the targeted inhibition of these factors. METHODS: Polymerase chain reaction (PCR) arrays were used to investigate the expression profile of genes that encode key epigenetic regulator enzymes. Mononuclear cells from RA patients and mice were monitored for gene expression changes, in association with arthritis development in murine models of RA. Selected genes were further characterized by quantitative reverse transcription-PCR, Western blot, and flow cytometry methods. The targeted inhibition of the up-regulated enzymes was studied in arthritic mice. RESULTS: A set of genes with arthritis-specific expression was identified by the PCR arrays. Aurora kinases A and B, both of which were highly expressed in arthritic mice and treatment-naive RA patients, were selected for detailed analysis. Elevated aurora kinase expression was accompanied by increased phosphorylation of histone H3, which promotes proliferation of T lymphocytes. Treatment with VX-680, a pan-aurora kinase inhibitor, promoted B cell apoptosis, provided significant protection against disease onset, and attenuated inflammatory reactions in arthritic mice. CONCLUSION: Arthritis development is accompanied by changes in expression of a number of epigenome-modifying enzymes. Drug-induced down-regulation of the aurora kinases, among other targets, seems to be sufficient to treat experimental arthritis. Development of new therapeutics that target aurora kinases can potentially improve RA management.

Non-MHC risk alleles in rheumatoid arthritis and in the syntenic chromosome regions of corresponding animal models.

Rheumatoid arthritis (RA) is a polygenic autoimmune disease primarily affecting the synovial joints. Numerous animal models show similarities to RA in humans; some of them not only mimic the clinical phenotypes but also demonstrate the involvement of homologous genomic regions in RA. This paper compares corresponding non-MHC genomic regions identified in rodent and human genome-wide association studies (GWAS). To date, over 30 non-MHC RA-associated loci have been identified in humans, and over 100 arthritis-associated loci have been identified in rodent models of RA. The genomic regions associated with the disease are designated by the name(s) of the gene having the most frequent and consistent RA-associated SNPs or a function suggesting their involvement in inflammatory or autoimmune processes. Animal studies on rats and mice preferentially have used single sequence length polymorphism (SSLP) markers to identify disease-associated qualitative and quantitative trait loci (QTLs) in the genome of F2 hybrids of arthritis-susceptible and arthritis-resistant rodent strains. Mouse GWAS appear to be far ahead of rat studies, and significantly more mouse QTLs correspond to human RA risk alleles.

Suppression of dendritic cell maturation and T cell proliferation by synovial fluid myeloid cells from mice with autoimmune arthritis.

OBJECTIVE: To determine whether myeloid cells (such as granulocytes) present in the synovial fluid (SF) of arthritic joints have an impact on adaptive immunity. Specifically, we investigated the effects of SF cells harvested from the joints of mice with proteoglycan-induced arthritis (PGIA), on dendritic cell (DC) maturation and antigen-specific T cell proliferation. METHODS: We monitored DC maturation (MHCII and CD86 expression) by flow cytometry upon coculture of DCs with SF cells or spleen myeloid cells from mice with PGIA. The effects of these myeloid cells on T cell proliferation were studied using T cells purified from PG-specific T cell receptor (TCR)-transgenic (Tg) mice. Phenotype analysis of myeloid cells was performed by immunostaining, reverse transcription-polymerase chain reaction, Western blotting, and biochemical assays. RESULTS: Inflammatory SF cells significantly suppressed the maturation of DCs upon coculture. PG-TCR-Tg mouse T cells cultured with antigen-loaded DCs showed dramatic decreases in proliferation in the presence of SF cells. Spleen myeloid cells from arthritic mice did not have suppressive effects. SF cells were unable to suppress CD3/CD28-stimulated proliferation of the same T cells, suggesting a DC-dependent mechanism. SF cells exhibited all of the characteristics of myeloid-derived suppressor cells (MDSCs) and exerted suppression primarily through the production of nitric oxide and reactive oxygen species by granulocyte-like cells. CONCLUSION: SF in the joints of mice with PGIA contains a population of granulocytic MDSCs that potently suppress DC maturation and T cell proliferation. These MDSCs have the potential to limit the expansion of autoreactive T cells, thus breaking the vicious cycle of autoimmunity and inflammation.

DNA methylation biomarkers for lung cancer.

Changes in DNA methylation patterns are an important characteristic of human cancer including lung cancer. In particular, hypermethylation of CpG islands is a signature of malignant progression. Methylated CpG islands are promising diagnostic markers for the early detection of cancer. However, the full extent and sequence context of DNA hypermethylation in lung cancer has remained unknown. We have used the methylated CpG island recovery assay and high-resolution microarray analysis to find hypermethylated CpG islands in squamous cell carcinomas (SCC) and adenocarcinomas of the lung. Each tumor contained several hundred hypermethylated CpG islands. In an initial microarray screen, 36 CpG islands were methylated in five of five (=100%) of the SCC tumors tested and 52 CpG islands were methylated in at least 75% of the adenocarcinomas tested (n=8). Using sodium-bisulfite-based approaches, 12 CpG islands (associated with the BARHL2, EVX2, IRX2, MEIS1, MSX1, NR2E1, OC2, OSR1, OTX1, PAX6, TFAP2A, and ZNF577 genes) were confirmed to be methylated in 85% to 100% of the squamous cell carcinomas and 11 CpG islands (associated with the CHAD, DLX4, GRIK2, KCNG3, NR2E1, OSR1, OTX1, OTX2, PROX1, RUNX1, and VAX1 genes) were methylated in >80% of the adenocarcinomas. From the list of genes that were methylated in lung adenocarcinomas, we identified the gene FAT4 and found that this gene was methylated in 39% of the tumors. FAT4 is the closest mammalian homologue of the Drosophila tumor suppressor Fat which is an important component of the Hippo growth control pathway. Many of these newly discovered methylated CpG islands hold promise for becoming biomarkers for the early detection of lung cancer.

5-Hydroxymethylcytosine is strongly depleted in human cancers but its levels do not correlate with IDH1 mutations.

The base 5-hydroxymethylcytosine (5hmC) was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. Here, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) and 5-methyl-2'-deoxycytidine (5mdC) in genomic DNA, we investigated whether levels of 5hmC can distinguish normal tissue from tumor tissue. In squamous cell lung cancers, levels of 5hmdC were depleted substantially with up to 5-fold reduction compared with normal lung tissue. In brain tumors, 5hmdC showed an even more drastic reduction with levels up to more than 30-fold lower than in normal brain, but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1. Furthermore, immunohistochemical analysis indicated that 5hmC is remarkably depleted in many types of human cancer. Importantly, an inverse relationship between 5hmC levels and cell proliferation was observed with lack of 5hmC in proliferating cells. The data therefore suggest that 5hmdC is strongly depleted in human malignant tumors, a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue. In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.

Alteration in the gene encoding protein tyrosine phosphatase nonreceptor type 6 (PTPN6/SHP1) may contribute to neutrophilic dermatoses.

We have found a B2 repeat insertion in the gene encoding protein tyrosine phosphatase nonreceptor type 6 (PTPN6) in a mouse that developed a skin disorder with clinical and histopathological features resembling those seen in human neutrophilic dermatoses. Neutrophilic dermatoses are a group of complex heterogeneous autoinflammatory diseases that all demonstrate excessive neutrophil infiltration of the skin. Therefore, we tested the cDNA and genomic DNA sequences of PTPN6 from patients with Sweet's syndrome (SW) and pyoderma gangrenosum and found numerous novel splice variants in different combinations. Isoforms resulting from deletions of exons 2, 5, 11, and 15 and retention of intron 1 or 5 were the most common in a patients with a familial case of SW, who had a neonatal onset of an inflammatory disorder with skin lesions and a biopsy specimen consistent with SW. These isoforms were associated with a heterozygous E441G mutation and a heterozygous 1.7-kbp deletion in the promoter region of the PTPN6 gene. Although full-length PTPN6 was detected in all other patients with either pyoderma gangrenosum or SW, it was always associated with splice variants: a partial deletion of exon 4 with the complete deletion of exon 5, alterations that were not detected in healthy controls. The defect in transcriptional regulation of the hematopoietic PTPN6 appears to be involved in the pathogenesis of certain subsets of the heterogeneous group of neutrophilic dermatoses.

Chromosome-wide analysis of parental allele-specific chromatin and DNA methylation.

To reveal the extent of domain-wide epigenetic features at imprinted gene clusters, we performed a high-resolution allele-specific chromatin analysis of over 100 megabases along the maternally or paternally duplicated distal chromosome 7 (Chr7) and Chr15 in mouse embryo fibroblasts (MEFs). We found that reciprocal allele-specific features are limited to imprinted genes and their differentially methylated regions (DMRs), whereas broad local enrichment of H3K27me3 (BLOC) is a domain-wide feature at imprinted clusters. We uncovered novel allele-specific features of BLOCs. A maternally biased BLOC was found along the H19-Igf2 domain. A paternal allele-specific gap was found along Kcnq1ot1, interrupting a biallelic BLOC in the Kcnq1-Cdkn1c domain. We report novel allele-specific chromatin marks at the Peg13 and Slc38a4 DMRs, Cdkn1c upstream region, and Inpp5f_v2 DMR and paternal allele-specific CTCF binding at the Peg13 DMR. Additionally, we derived an imprinted gene predictor algorithm based on our allele-specific chromatin mapping data. The binary predictor H3K9ac and CTCF or H3K4me3 in one allele and H3K9me3 in the reciprocal allele, using a sliding-window approach, recognized with precision the parental allele specificity of known imprinted genes, H19, Igf2, Igf2as, Cdkn1c, Kcnq1ot1, and Inpp5f_v2 on Chr7 and Peg13 and Slc38a4 on Chr15. Chromatin features, therefore, can unequivocally identify genes with imprinted expression.