Genome-wide analysis identifies differential promoter methylation of Leprel2, Foxf1, Mmp25, Igfbp6, and Peg12 in murine tendinopathy.

We have used a murine Achilles tendinopathy model to investigate whether tissue changes (such as collagen disorganization, chondroid metaplasia, and loss of tensile properties) which are broadly characteristic of human tendinopathies, are accompanied by changes in the expression of chromatin-modifying enzymes and the methylation status of promoter regions of tendon cell DNA. Tendinopathy was induced by two intra-tendinous TGF-ß1 injections followed by cage activity or treadmill running for up to 28 days. Activation of DNA methyltransferases occurred at 3 days after the TGF-ß1 injections and also at 14 days, but only with treadmill activity. Genome-wide Methyl Mini-Seq™ analysis identified 19 genes with differentially methylated promoters, five of which perform functions with an apparent direct relevance to tendinopathy (Leprel2, Foxf1, Mmp25, Igfbp6, and Peg12). The functions of the genes identified included collagen fiber assembly and pericellular interactions, therefore their perturbation could play a role in the characteristic disorganization of fibers in affected tendons. We postulate that a study of the functional genomics of these genes in animal and human tendon could further delineate the pathogenesis of this multi-factorial complex disease. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:947-955, 2017.

Genome-wide analysis of DNA methylation and gene expression defines molecular characteristics of Crohn's disease-associated fibrosis.

BACKGROUND: Fibrosis of the intestine is a common and poorly understood complication of Crohn's disease (CD) characterized by excessive deposition of extracellular matrix and accompanied by narrowing and obstruction of the gut lumen. Defining the molecular characteristics of this fibrotic disorder is a vital step in the development of specific prediction, prevention, and treatment strategies. Previous epigenetic studies indicate that alterations in DNA methylation could explain the mechanism by which mesenchymal cells adopt the requisite pro-fibrotic phenotype that promotes fibrosis progression. However, to date, genome-wide analysis of the DNA methylome of any type of human fibrosis is lacking. We employed an unbiased approach using deep sequencing to define the DNA methylome and transcriptome of purified fibrotic human intestinal fibroblasts (HIF) from the colons of patients with fibrostenotic CD. RESULTS: When compared with normal fibroblasts, we found that the majority of differential DNA methylation was within introns and intergenic regions and not associated with CpG islands. Only a low percentage occurred in the promoters and exons of genes. Integration of the DNA methylome and transcriptome identified regions in three genes that inversely correlated with gene expression: wingless-type mouse mammary tumor virus integration site family, member 2B (WNT2B) and two eicosanoid synthesis pathway enzymes (prostacyclin synthase and prostaglandin D2 synthase). These findings were independently validated by RT-PCR and bisulfite sequencing. Network analysis of the data also identified candidate molecular interactions relevant to fibrosis pathology. CONCLUSIONS: Our definition of a genome-wide fibrosis-specific DNA methylome provides new gene networks and epigenetic states by which to understand mechanisms of pathological gene expression that lead to fibrosis. Our data also provide a basis for development of new fibrosis-specific therapies, as genes dysregulated in fibrotic Crohn's disease, following functional validation, can serve as new therapeutic targets.

The epithelial danger signal IL-1α is a potent activator of fibroblasts and reactivator of intestinal inflammation.

Intestinal epithelial cell (IEC) death is typical of inflammatory bowel disease (IBD). We investigated: i) whether IEC-released necrotic cell products (proinflammatory mediators) amplify mucosal inflammation, ii) the capacity of necrotic cell lysates from HT29 cells or human IECs to induce human intestinal fibroblasts' (HIF) production of IL-6 and IL-8, and iii) whether IL-1α, released by injured colonocytes, exacerbated experimental IBD. Necrotic cell lysates potently induced HIF IL-6 and IL-8 production independent of Toll-like receptors 2 and 4, receptor for advanced glycation end-products, high-mobility group box 1, uric acid, IL-33, or inflammasome activation. IL-1α was the key IEC-derived necrotic cell product involved in HIF cytokine production. IL-1α-positive cells were identified in the epithelium in human IBD and dextran sulfate sodium (DSS)-induced colitis. IL-1α was detected in the stool of colitic mice before IL-1ß. IL-1α enemas reactivated inflammation after DSS colitis recovery, induced IL-1 receptor expression in subepithelial fibroblasts, and activated de novo inflammation even in mice without overt colitis, after the administration of low-dose DSS. IL-1α amplifies gut inflammation by inducing cytokine production by mesenchymal cells. IL-1α-mediated IEC-fibroblast interaction may be involved in amplifying and perpetuating inflammation, even without obvious intestinal damage. IL-1α may be a target for treating early IBD or preventing the reactivation of IBD.

Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters.

BACKGROUND & AIMS: Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The Drosophila chromosome-associated protein D3 (dCAP-D3) regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS: Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS: CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, the products of which heterodimerize to form an amino acid transporter in HT-29 cells after bacterial infection; levels of SLC7A5-SLC3A2 were increased in tissues from patients with UC compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5-SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3-deficient cells. CONCLUSIONS: CAP-D3 down-regulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with active UC; strategies to increase its levels might restore mucosal homeostasis to patients with active UC.

Epigenetics: the fine-tuner in inflammatory bowel disease?

PURPOSE OF REVIEW: Epigenetic studies are transforming our understanding of a variety of complex pathological conditions including cancer, autoimmune, and inflammatory diseases. A selection of the major recent advances in this area will be reviewed, focusing on the important emerging themes that are relevant to these diseases including inflammatory bowel disease (IBD). RECENT FINDINGS: The main current themes that will be addressed on the role of epigenetics in disease pathogenesis include current understanding of the nature and function of histone modifications and DNA methylation; the connection between epigenetics and metabolic pathways; new studies on the mechanism of heritability of epigenetic changes; the role of stochastic noise and the expanding research on chromatin readers and their potential as selective therapeutic targets. The recent contribution of epigenetic modifications in defining the molecular basis of IBD and how such changes may act as fine-tuners of gene expression in these intestinal disorders are also discussed. SUMMARY: Published evidence over the last 12-18 months indicates that targeting epigenetic factors can be efficacious in cancer and inflammatory disease. All the indications are that future research will continue to reveal new epigenetic targets and mechanisms that will advance the prospects for selective epigenetic therapy for IBD and other complex diseases.

Cytokine-induced chromatin modifications of the type I collagen alpha 2 gene during intestinal endothelial-to-mesenchymal transition.

BACKGROUND: Fibrosis of the intestine is currently an irreversible complication of inflammatory bowel disease; yet, little is understood of the underlying pathogenesis and antifibrotic strategies remain elusive. To develop effective therapies, knowledge of the mechanism of transcription and excessive deposition of type I collagen, a hallmark of fibrosis, is needed. We have shown previously that endothelial-to-mesenchymal transition (EndoMT) contributes to the pool of intestinal fibrotic cells and that a cytokine cocktail (interleukin 1-ß, tumor necrosis factor α, and transforming growth factor ß) induces collagen I alpha 2 (COL1A2) mRNA and protein. METHODS: Chromatin immunoprecipitation assays on pure cultures of human intestinal mucosal endothelial cells undergoing EndoMT were performed with antibodies to specific histone modifications and RNA polymerase II. Reverse transcriptase-PCR was used to quantify the levels of Col1A2 and endothelial-specific von Willebrand factor (vWF) mRNA. RESULTS: We showed that cytokines induce selective chromatin modifications (histone 4 hyperacetylation, and hypermethylation of histone 3) and phosphorylated RNA polymerase II at the COL1A2 promoter. Hypoacetylated and hypomethylated histone 3 was detected on the repressed vWF gene. Prolonged exposure to cytokines (16 days) retained hyperacetylation of select lysines in H4 on the COL1A2 promoter. Removal of cytokines after 16 days and continued culture for 10 days showed persistent hyperacetylation at lysine 16 in histone H4. CONCLUSIONS: This is the first study to show that COL1A2 gene expression is associated with cytokine-induced, temporally ordered, and persistent chromatin modifications and suggests that these are important determinants of gene expression in EndoMT and intestinal fibrosis.

Size-based clinical response evaluation is insufficient to assess clinical response of sarcomas treated with isolated limb perfusion with TNF-α and melphalan.

BACKGROUND: The clinical assessment of the response of sarcomas to preoperative treatment is usually defined using size-based evaluation standards. For nonresectable sarcomas, hyperthermic isolated limb perfusion with TNF-α and melphalan (TM-ILP) yields high response rates. Based on our experience, we assume that anatomic radiological response criteria are insufficient to assess the degree of regression after TM-ILP. METHODS: The clinical response of 35 sarcomas to TM-ILP was assessed by unidimensional, bidimensional, and tridimensional size-based anatomical criteria, and responders were identified according to the established thresholds. The same tumors were investigated for pathological response according to the Salzer-Kuntschik regression scale (>90% devitalization) and reviewed for cystic degeneration, hemorrhage, and predominant necrotic or fibrosclerotic regression phenotype. RESULTS: None of the clinical response criteria were able to reliably identify the pathologic responders. The extent of size changes showed no association with the pathological degree of regression. The number of clinical responders was low compared with the number of pathological responders (RECIST N = 1, WHO N = 3, volumetry N = 3, pathology N = 19). The occurrence of hemorrhage and/or cystic degeneration was more frequently observed in predominant necrotic sarcomas and was associated with an increase in tumor size after TM-ILP. Furthermore, we identified the fibrosclerotic phenotype of regression to be more significantly strongly associated with posttherapeutic shrinkage than necrosis. CONCLUSIONS: Size-based clinical response evaluation is insufficient to assess clinical response in TM-ILP-treated sarcomas. The size changes of tumors after therapy reflect the type of regression rather than the extent of destruction.

Epigenetics: Concepts and relevance to IBD pathogenesis.

The purpose of this review is to introduce the exciting field of epigenetics and to describe how it could explain the mechanisms by which environmental changes induce pathological gene expression and determine cell phenotype and function in IBD. We outline how epigenetics research in the context of a variety of clinical conditions, but mainly in cancer, has begun to define the role of multiple combinations of modifications to chromatin, diverse families of enzymes, and non-coding RNAs in determining transcriptional outcomes. These findings are applicable to understanding the context-specific events that underlie the expression of genes in diseases like IBD and have the potential to reveal new targets for improved IBD therapy. The current status of epigenetics-based therapies is also summarized.

New S-adenosyl-L-methionine analogues: synthesis and reactivity studies.

Two new and complementary synthetic strategies for 5'-N-chloroethylamino-5'-deoxyadenosines are presented. Additionally, the reaction kinetics of their conversion into aziridines under typical enzyme assay conditions is reported using time-resolved NMR spectroscopy. A stable photocaged derivative of 5'-N-chloroethylamino-5'-deoxyadenosine has also been synthesized, and its stability and activation in aqueous solution at physiological pH have been examined.

The prognostic significance of steroid receptor co-regulators in breast cancer: co-repressor NCOR2/SMRT is an independent indicator of poor outcome.

BACKGROUND: Advances in understanding the molecular basis of breast cancer has necessitated a definition of improved indicators of prognosis that are central to the underlying cancer biology and that reflect the heterogeneous nature of the disease. This study investigates the pattern of expression of the steroid receptor co-regulators NCOA1/SRC1, NCOA3/RAC3, NCOR2/SMRT, and CBP/p300 in breast cancer. The aims were to identify whether their expression was related to patient outcome, their relationships to known prognostic factors and to provide a basis for further research into the mechanistic significance of such associations. METHODS: The protein levels of steroid receptor co-regulators were assessed by immunohistochemistry in a large well-characterised series of breast carcinomas prepared as tissue microarrays. Relationships between these targets, other clinicopathological variables and patients' outcome were examined. RESULTS: NCOR2/SMRT was an independent prognostic indicator of overall patient survival (OS) and disease free interval (DFI) and was significantly correlated with distant metastases and local recurrence whereas tumours expressing NCOA1/SRC1 had a significantly longer OS and DFI. There were also significant correlations between co-regulator expression of NCOA1/SRC1, CBP/p300 and NCOA3/RAC3, which were associated with lower tumour grade. NCOA1/SRC1 was also correlated with smaller tumour size. Furthermore, the co-activators had a significant association with steroid receptors, particularly ERalpha. CONCLUSIONS: NCOR2/SMRT is associated with poor patient outcome, independent of other prognostic factors. In contrast, steroid receptor co-activator expression is generally associated with a good prognosis. Further investigations are needed to establish the mechanisms of these links between the steroid receptor co-regulator system and patient outcome.

The relationship between intranuclear mobility of the NF-kappaB subunit p65 and its DNA binding affinity.

It has been hypothesized that the main determinant of the intranuclear mobility of transcription factors is their ability to bind DNA. In the present study, we have extensively tested the relationship between the intranuclear mobility of the NF-kappaB subunit p65 and binding to its consensus target sequence. The affinity of p65 for this binding site is altered by mutation of specific acetylation sites, so these mutants provide a model system to study the relationship between specific DNA binding affinity and intranuclear mobility. DNA binding affinity was measured in vitro using an enzyme-linked immunosorbent assay-based method, and intranuclear mobility was measured using the fluorescence recovery after photobleaching technique on yellow fluorescent protein-tagged p65 constructs. A negative correlation was observed between DNA binding affinity and intranuclear mobility of p65 acetylation site mutants. However, moving the yellow fluorescent protein tag from the C terminus of p65 to the N terminus resulted in an increased mobility but did not significantly affect DNA binding affinity. Thus, all changes in DNA binding affinity produce alterations in mobility, but not vice versa. Finally, a positive correlation was observed between mobility and the randomness of the intranuclear distribution of p65. Our data are in line with a model in which the intranuclear mobility and distribution of a transcription factor are determined by its affinity for specific DNA sequences, which may be altered by protein-protein interactions.