Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization.

Naive CD4⁺ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell-fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hr of in vitro polarization toward T helper 1 (Th1) and T helper 2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic-associated SNPs suggests a role for distal regulatory elements in disease etiology.

Activity of recombinant cysteine-rich domain proteins derived from the membrane-bound MUC17/Muc3 family mucins.

BACKGROUND: The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and have cytoprotective properties. Their extracellular regions contain two EGF-like Cys-rich domains (CRD1 and CRD2) connected by an intervening linker segment with SEA module (L), and may function to stimulate intestinal cell restitution. The purpose of this study was to determine the effect of size, recombinant host source, and external tags on mucin CRD1-L-CRD2 protein activity. METHODS: Four recombinant Muc3-CRD proteins and three MUC17-CRD proteins were generated using Escherichiacoli or baculovirus-insect cell systems and tested in colonic cell cultures for activity related to cell migration and apoptosis. RESULTS: N-terminal glutathione-S-transferase (GST) or C-terminal His(8) tags had no effect on either the cell migration or anti-apoptosis activity of Muc3-CRD1-L-CRD2. His-tagged Muc3-CRD1-L-CRD2 proteins with truncated linker regions, or the linker region alone, did not demonstrate biologic activity. The human recombinant MUC17-CRD1-L-CRD2-His(8) was shown to have anti-apoptotic and pro-migratory activity, but did not stimulate cell proliferation. This protein showed similar in vitro biologic activity, whether produced in E. coli or a baculovirus-insect cell system. CONCLUSIONS: Recombinant mucin proteins containing a bivalent display of Cys-rich domains accelerate colon cell migration and inhibit apoptosis, require a full-length intervening Linker-SEA segment for optimal biologic activity, and are functional when synthesized in either E. coli and insect cell systems. GENERAL SIGNIFICANCE: These results indicate that an Escherichiacoli-derived full-length His(8)-tagged human MUC17 CRD1-L-CRD2 recombinant protein is a biologically active candidate for further development as a therapeutic agent.

High-sensitivity O-glycomic analysis of mice deficient in core 2 {beta}1,6-N-acetylglucosaminyltransferases.

Core 2 ß1,6-N-acetylglucosaminyltransferase (C2GnT), which exists in three isoforms, C2GnT1, C2GnT2 and C2GnT3, is one of the key enzymes in the O-glycan biosynthetic pathway. These isoenzymes produce core 2 O-glycans and have been correlated with the biosynthesis of core 4 O-glycans and I-branches. Previously, we have reported mice with single and multiple deficiencies of C2GnT isoenzyme(s) and have evaluated the biological and structural consequences of the loss of core 2 function. We now present more comprehensive O-glycomic analyses of neutral and sialylated glycans expressed in the colon, small intestine, stomach, kidney, thyroid/trachea and thymus of wild-type, C2GnT2 and C2GnT3 single knockouts and the C2GnT1-3 triple knockout mice. Very high-quality data have emerged from our mass spectrometry techniques with the capability of detecting O-glycans up to at least 3500 Da. We were able to unambiguously elucidate the types of O-glycan core, branching location and residue linkages, which allowed us to exhaustively characterize structural changes in the knockout tissues. The C2GnT2 knockout mice suffered a major loss of core 2 O-glycans as well as glycans with I-branches on core 1 antennae especially in the stomach and the colon. In contrast, core 2 O-glycans still dominated the O-glycomic profile of most tissues in the C2GnT3 knockout mice. Analysis of the C2GnT triple knockout mice revealed a complete loss of both core 2 O-glycans and branched core 1 antennae, confirming that the three known isoenzymes are entirely responsible for producing these structures. Unexpectedly, O-linked mannosyl glycans are upregulated in the triple deficient stomach. In addition, our studies have revealed an interesting terminal structure detected on O-glycans of the colon tissues that is similar to the RM2 antigen from glycolipids.

The Risk of QTc Prolongation in Non-Diabetic and Diabetic Patients Taking Tyrosine Kinase Inhibitors (TKIs)- A Patient Safety Project at a Private Oncology Practice.

Objective: To assess the prevalence of QTc prolongation in both non-diabetic and diabetic patients on TKIs. Some TKIs have been reported to cause QTc prolongation, which is prevalent in diabetes. However, there is no Risk Evaluation and Mitigation Strategy using series ECG to monitor those patients. Methods: Patients taking TKIs, with two ECGs recorded between 1 January 2010 and 31 December 2017 were selected from the electronic database. The QTc duration >450 ms was determined as prolonged. Percentage of QTc prolongation on participants were compared using Chi-Square test. Results: This study included 313 patients (age 66.1 ± 0.8 years and 57.5% are female) taking TKIs. In non-Diabetic patients, the prevalence of QTc prolongation is 19.1% (n = 253) before and 34.8% (n = 253) after treatment with TKIs (p < 0.001), respectively. In diabetic patients, the prevalence of QTc prolongation is 21.7% (n = 60) before and 40% (n = 60) after treatment with TKIs (p = 0.03), respectively. In addition, we examined the effect of modifying risk factors for cardiovascular disease (CVD) on the prevalence of QTc prolongation caused by TKIs. In non-diabetic patients, the prevalence of QTc prolongation is 33.3% (n = 57) before and 34.2% (n = 196) after risk factors modification (p = 0.91), respectively. In diabetic patients, the prevalence of QTc prolongation is 50% (n = 24) before and 33.3% (n = 36) after risk factors modification (p = 0.20), respectively. Conclusion: Use of TKIs is associated with a significantly increased risk of QTc prolongation for patients, particularly when patients are diabetic. Modification of risk factors for CVD does not significantly affect the prevalence of QTc prolongation caused by TKIs.

Prevalence of QT prolongation and associated LVEF changes in diabetic patients over a four-year retrospective time period.

Aim: To evaluate the prevalence and longitudinal changes of prolonged QTc in DM patients admitted to our community hospital, and to determine, if any, its correlation with changes of left ventricular ejection fraction (LVEF). Methods: A retrospective chart review of patients with Type 1 (T1DM) and Type 2 (T2DM) with at least two admissions during a four-year period was performed to identify QTc interval, and LVEF, as measured on transthoracic echocardiogram. Changes in QTc and LVEF between patient hospital admissions were compared. Results: A prolonged QTc interval was found in 66.7% (n = 24) of type 1 and 51.3% (n = 154) type 2 diabetic patients. The QTc interval is progressively increased in both type 1 and type 2 diabetes during follow-up, although it did not reach statistical significance. A total of 62% patients (23 out 37 patients) had a reduction of LVEF during follow-up. Conclusion and Discussion: High prevalence of QTc prolongation was confirmed in hospitalized patients with in both T1DM and T2DM. Significant reduction of LVEF correlated with QTc prolongation over a mean of 17.3 months in T2DM patients, and may have implications for interventions. Abbreviations CHF: Congestive heart failure LVEF: Left ventricular ejection fraction.

An assessment of histone-modification antibody quality.

We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).

Human intestinal MUC17 mucin augments intestinal cell restitution and enhances healing of experimental colitis.

UNLABELLED: The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and are thought to be cytoprotective. The extracellular regions of these mucins contain EGF-like Cys-rich segments (CRD1 and CRD2) connected by an intervening linker domain (L). The purpose of this study was to determine the functional activity of human MUC17 membrane-bound mucin. METHODS: Endogenous MUC17 was inhibited in LS174T colon cells by stable transfection of a small hairpin RNA targeting MUC17 (LSsi cells). The effect of recombinant MUC17-CRD1-L-CRD2 protein on migration, apoptosis, and experimental colitis was determined. RESULTS: Reduced MUC17 expression in LSsi cells was associated with visibly reduced cell aggregation, reduced cell-cell adherence, and reduced cell migration, but no change in tumorigenicity. LSsi cells also demonstrated a 3.7-fold increase in apoptosis rates compared with control cells following treatment with etoposide. Exposure of colonic cell lines to exogenous recombinant MUC17-CRD1-L-CRD2 protein significantly increased cell migration and inhibited apoptosis. As a marker of biologic activity, MUC17-CRD1-L-CRD2 proteins stimulate ERK phosphorylation in colonic cell lines; and inhibition of ERK phosphorylation reduced the anti-apoptosis and migratory effect of MUC17-CRD1-L-CRD2. Finally, mice treated with MUC17-CRD1-L-CRD2 protein given per rectum demonstrated accelerated healing in acetic acid and dextran sodium sulfate induced colitis in vivo. These data indicate that both native MUC17 and the exogenous recombinant cysteine-rich domain of MUC17 play a role in diverse cellular mechanisms related to cell restitution, and suggest a potential role for MUC17-CRD1-L-CRD2 recombinant protein in the treatment of mucosal inflammatory diseases.

Distinct epigenomic landscapes of pluripotent and lineage-committed human cells.

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells, yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes, but how much epigenomes differ remains unclear. Here, we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts, we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks, which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally, we observe novel, context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment.

Glycosyltransferase function in core 2-type protein O glycosylation.

Three glycosyltransferases have been identified in mammals that can initiate core 2 protein O glycosylation. Core 2 O-glycans are abundant among glycoproteins but, to date, few functions for these structures have been identified. To investigate the biological roles of core 2 O-glycans, we produced and characterized mice deficient in one or more of the three known glycosyltransferases that generate core 2 O-glycans (C2GnT1, C2GnT2, and C2GnT3). A role for C2GnT1 in selectin ligand formation has been described. We now report that C2GnT2 deficiency impaired the mucosal barrier and increased susceptibility to colitis. C2GnT2 deficiency also reduced immunoglobulin abundance and resulted in the loss of all core 4 O-glycan biosynthetic activity. In contrast, the absence of C2GnT3 altered behavior linked to reduced thyroxine levels in circulation. Remarkably, elimination of all three C2GnTs was permissive of viability and fertility. Core 2 O-glycan structures were reduced among tissues from individual C2GnT deficiencies and completely absent from triply deficient mice. C2GnT deficiency also induced alterations in I-branching, core 1 O-glycan formation, and O mannosylation. Although the absence of C2GnT and C4GnT activities is tolerable in vivo, core 2 O glycosylation exerts a significant influence on O-glycan biosynthesis and is important in multiple physiological processes.