The SEQC2 epigenomics quality control (EpiQC) study.

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

Epigenetics of Skeletal Muscle-Associated Genes in the ASB, LRRC, TMEM, and OSBPL Gene Families.

Much remains to be discovered about the intersection of tissue-specific transcription control and the epigenetics of skeletal muscle (SkM), a very complex and dynamic organ. From four gene families, Leucine-Rich Repeat Containing (LRRC), Oxysterol Binding Protein Like (OSBPL), Ankyrin Repeat and Socs Box (ASB), and Transmembrane Protein (TMEM), we chose 21 genes that are preferentially expressed in human SkM relative to 52 other tissue types and analyzed relationships between their tissue-specific epigenetics and expression. We also compared their genetics, proteomics, and descriptions in the literature. For this study, we identified genes with little or no previous descriptions of SkM functionality (ASB4, ASB8, ASB10, ASB12, ASB16, LRRC14B, LRRC20, LRRC30, TMEM52, TMEM233, OSBPL6/ORP6, and OSBPL11/ORP11) and included genes whose SkM functions had been previously addressed (ASB2, ASB5, ASB11, ASB15, LRRC2, LRRC38, LRRC39, TMEM38A/TRIC-A, and TMEM38B/TRIC-B). Some of these genes have associations with SkM or heart disease, cancer, bone disease, or other diseases. Among the transcription-related SkM epigenetic features that we identified were: super-enhancers, promoter DNA hypomethylation, lengthening of constitutive low-methylated promoter regions, and SkM-related enhancers for one gene embedded in a neighboring gene (e.g., ASB8-PFKM, LRRC39-DBT, and LRRC14B-PLEKHG4B gene-pairs). In addition, highly or lowly co-expressed long non-coding RNA (lncRNA) genes probably regulate several of these genes. Our findings give insights into tissue-specific epigenetic patterns and functionality of related genes in a gene family and can elucidate normal and disease-related regulation of gene expression in SkM.

Tissue-specific epigenetics of atherosclerosis-related ANGPT and ANGPTL genes.

AIM: To understand tissue-specific regulation of angiopoietin/angiopoietin-like (ANGPT/ANGPTL) genes (especially the five genes embedded in introns of host genes) and their association with atherosclerosis. METHODS: Transcription and epigenomic databases from various normal tissues were examined in the vicinity of ANGPT1, ANGPT2, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4 and ANGPTL8. RESULTS: We identified tissue-specific enhancer chromatin regions that are likely to regulate transcription of ANGPT/ANGPTL genes and were intragenic, intergenic or host gene-linked. In addition, we found atherosclerosis-linked differentially methylated regions associated with ANGPT2 and with sequences encoding miR-145, a microRNA that targets ANGPT2 mRNA in cancers. CONCLUSION: Our findings implicate enhancers as major contributors to tissue-specific expression of ANGPT/ANGPTL genes, which play critical roles in angiogenesis, atherosclerosis, cancer, and inflammatory and metabolic diseases.

Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers.

BACKGROUND AND AIMS: Atherosclerosis is a widespread and complicated disease involving phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs), the predominant cells in aorta, as well as changes in endothelial cells and infiltrating monocytes. Alterations in DNA methylation are likely to play central roles in these phenotypic changes, just as they do in normal differentiation and cancer. METHODS: We examined genome-wide DNA methylation changes in atherosclerotic aorta using more stringent criteria for differentially methylated regions (DMRs) than in previous studies and compared these DMRs to tissue-specific epigenetic features. RESULTS: We found that disease-linked hypermethylated DMRs account for 85% of the total atherosclerosis-associated DMRs and often overlap aorta-associated enhancer chromatin. These hypermethylated DMRs were associated with functionally different sets of genes compared to atherosclerosis-linked hypomethylated DMRs. The extent and nature of the DMRs could not be explained as direct effects of monocyte/macrophage infiltration. Among the known atherosclerosis- and contractile SMC-related genes that exhibited hypermethylated DMRs at aorta enhancer chromatin were ACTA2 (aorta α2 smooth muscle actin), ELN (elastin), MYOCD (myocardin), C9orf3 (miR-23b and miR-27b host gene), and MYH11 (smooth muscle myosin). Our analyses also suggest a role in atherosclerosis for developmental transcription factor genes having little or no previous association with atherosclerosis, such as NR2F2 (COUP-TFII) and TBX18. CONCLUSIONS: We provide evidence for atherosclerosis-linked DNA methylation changes in aorta SMCs that might help minimize or reverse the standard contractile character of many of these cells by down-modulating aorta SMC-related enhancers, thereby facilitating pro-atherosclerotic phenotypic changes in many SMCs.

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription.

DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development.

Modeling Methylation Patterns with Long Read Sequencing Data.

Variation in cytosine methylation at CpG dinucleotides is often observed in genomic regions, and analysis typically focuses on estimating the proportion of methylated sites observed in a given region and comparing these levels across samples to determine association with conditions of interest. While sites are tacitly treated as independent, when observed at the level of individual molecules methylation patterns exhibit strong evidence of local spatial dependence. We previously developed a neighboring sites model to account for correlation and clustering behavior observed in two tandem repeat regions in a collection of ovarian carcinomas. We now introduce extensions of the model that account for the effect of distance between sites as well as asymmetric correlation in de novo methylation and demethylation rates. We apply our models to published data from a whole genome bisulfite sequencing experiment using long reads, estimating model parameters for a selection of CpG-dense regions spanning between 21 and 67 sites. Our methods detect evidence of local spatial correlation as a function of site-to-site distance and demonstrate the added value of employing long read sequencing data in epigenetic research.

DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression.

Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1's super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation.

An Ex Vivo Tissue Culture Model for Anti-angiogenic Drug Testing.

Angiogenesis, defined as the growth of new blood vessels from existing ones, plays a key role in development, growth, and tissue repair. Its necessary role in tumor growth and metastasis has led to the creation of a new category of anti-angiogenic cancer therapies. Preclinical development and evaluation of potential drug candidates require models that mimic real microvascular networks. Here, we describe the rat mesentery culture model as a simple ex vivo assay that offers time-lapse imaging of intact microvascular network remodeling and demonstrate its application for anti-angiogenic drug testing.

BRAF Testing in Multifocal Papillary Thyroid Carcinoma.

BACKGROUND: BRAF V600E mutation is associated with poor prognosis in patients with papillary thyroid carcinoma (PTC). PTC is often multifocal, and there are no guidelines on how many tumors to test for BRAF mutation in multifocal PTC. METHODS: Fifty-seven separate formalin-fixed and paraffin-embedded PTCs from twenty-seven patients were manually macrodissected and tested for BRAF mutation using a commercial allele-specific real-time polymerase chain reaction-based assay (Entrogen, Woodland Hills, CA). Data related to histologic characteristics, patient demographics, and clinical outcomes were collected. RESULTS: All mutations detected were BRAF V600E. Seventeen patients (63%) had concordant mutation status in the largest and second-largest tumors (i.e., both were positive or both were negative). The remaining ten patients (37%) had discordant mutation status. Six of the patients with discordant tumors (22% overall) had a BRAF-negative largest tumor and a BRAF-positive second-largest tumor. No histologic feature was found to help predict which cases would be discordant. CONCLUSIONS: Patients with multifocal PTC whose largest tumor is BRAF-negative can have smaller tumors that are BRAF-positive. Therefore, molecular testing of more than just the dominant tumor should be considered. Future studies are warranted to establish whether finding a BRAF mutation in a smaller tumor has clinical significance.

High-Resolution Rapid Diagnostic Imaging of Whole Prostate Biopsies Using Video-Rate Fluorescence Structured Illumination Microscopy.

Rapid assessment of prostate core biopsy pathology at the point-of-procedure could provide benefit in a variety of clinical situations. Even with advanced transrectal ultrasound guidance and saturation biopsy protocols, prostate cancer can be missed in up to half of all initial biopsy procedures. In addition, collection of tumor specimens for downstream histologic, molecular, and genetic analysis is hindered by low tumor yield due to inability to identify prostate cancer grossly. However, current point-of-procedure pathology protocols, such as frozen section analysis (FSA), are destructive and too time- and labor-intensive to be practical or economical. Ex vivo microscopy of the excised specimens, stained with fast-acting fluorescent histology dyes, could be an attractive nondestructive alternative to FSA. In this work, we report the first demonstration of video-rate structured illumination microscopy (VR-SIM) for rapid high-resolution diagnostic imaging of prostate biopsies in realistic point-of-procedure timeframes. Large mosaic images of prostate biopsies stained with acridine orange are rendered in seconds and contain excellent contrast and detail, exhibiting close correlation with corresponding hematoxylin and eosin histology. A clinically relevant review of VR-SIM images of 34 unfixed and uncut prostate core biopsies by two independent pathologists resulted in an area under the receiver operative curve (AUC) of 0.82-0.88, with a sensitivity ranging from 63% to 88% and a specificity ranging from 78% to 89%. When biopsies contained more than 5% tumor content, the sensitivity improved to 75% to 92%. The image quality, speed, minimal complexity, and ease of use of VR-SIM could prove to be features in favor of adoption as an alternative to destructive pathology at the point-of-procedure.

Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes.

Myogenic regulatory factor (MRF) genes, MYOD1, MYOG, MYF6 and MYF5, are critical for the skeletal muscle lineage. Here, we used various epigenome profiles from human myoblasts (Mb), myotubes (Mt), muscle and diverse non-muscle samples to elucidate the involvement of multigene neighborhoods in the regulation of MRF genes. We found more far-distal enhancer chromatin associated with MRF genes in Mb and Mt than previously reported from studies in mice. For the MYF5/MYF6 gene-pair, regions of Mb-associated enhancer chromatin were located throughout the adjacent 236-kb PTPRQ gene even though Mb expressed negligible amounts of PTPRQ mRNA. Some enhancer chromatin regions inside PTPRQ in Mb were also seen in PTPRQ mRNA-expressing non-myogenic cells. This suggests dual-purpose PTPRQ enhancers that upregulate expression of PTPRQ in non-myogenic cells and MYF5/MYF6 in myogenic cells. In contrast, the myogenic enhancer chromatin regions distal to MYOD1 were intergenic and up to 19 kb long. Two of them contain small, known MYOD1 enhancers, and one displayed an unusually high level of 5-hydroxymethylcytosine in a quantitative DNA hydroxymethylation assay. Unexpectedly, three regions of MYOD1-distal enhancer chromatin in Mb and Mt overlapped enhancer chromatin in umbilical vein endothelial cells, which might upregulate a distant gene (PIK3C2A). Lastly, genes surrounding MYOG were preferentially transcribed in Mt, like MYOG itself, and exhibited nearby myogenic enhancer chromatin. These neighboring chromatin regions may be enhancers acting in concert to regulate myogenic expression of multiple adjacent genes. Our findings reveal the very different and complex organization of gene neighborhoods containing closely related transcription factor genes.

An ex vivo model for anti-angiogenic drug testing on intact microvascular networks.

New models of angiogenesis that mimic the complexity of real microvascular networks are needed. Recently, our laboratory demonstrated that cultured rat mesentery tissues contain viable microvascular networks and could be used to probe pericyte-endothelial cell interactions. The objective of this study was to demonstrate the efficacy of the rat mesentery culture model for anti-angiogenic drug testing by time-lapse quantification of network growth. Mesenteric windows were harvested from adult rats, secured in place with an insert, and cultured for 3 days according to 3 experimental groups: 1) 10% serum (angiogenesis control), 2) 10% serum + sunitinib (SU11248), and 3) 10% serum + bevacizumab. Labeling with FITC conjugated BSI-lectin on Day 0 and 3 identified endothelial cells along blood and lymphatic microvascular networks. Comparison between day 0 (before) and 3 (after) in networks stimulated by 10% serum demonstrated a dramatic increase in vascular density and capillary sprouting. Growing networks contained proliferating endothelial cells and NG2+ vascular pericytes. Media supplementation with sunitinib (SU11248) or bevacizumab both inhibited the network angiogenic responses. The comparison of the same networks before and after treatment enabled the identification of tissue specific responses. Our results establish, for the first time, the ability to evaluate an anti-angiogenic drug based on time-lapse imaging on an intact microvascular network in an ex vivo scenario.

Identification of biomarkers for tuberculosis susceptibility via integrated analysis of gene expression and longitudinal clinical data.

Tuberculosis (TB) is an infectious disease caused by the bacteria Mycobacterium tuberculosis (Mtb) that affects millions of people worldwide. The majority of individuals who are exposed to Mtb develop latent infections, in which an immunological response to Mtb antigens is present but there is no clinical evidence of disease. Because currently available tests cannot differentiate latent individuals who are at low risk from those who are highly susceptible to developing active disease, there is considerable interest in the identification of diagnostic biomarkers that can predict reactivation of latent TB. We present results from our analysis of a controlled longitudinal experiment in which a group of rhesus macaques were exposed to a low dose of Mtb to study their progression to latent infection or active disease. Subsets of the animals were then euthanized at scheduled time points, and granulomas taken from their lungs were assayed for gene expression using microarrays. The clinical profiles associated with the animals following Mtb exposure revealed considerable variability, and we developed models for the disease trajectory for each subject using a Bayesian hierarchical B-spline approach. Disease severity estimates were derived from these fitted curves and included as covariates in linear models to identify genes significantly associated with disease progression. Our results demonstrate that the incorporation of clinical data increases the value of information extracted from the expression profiles and contributes to the identification of predictive biomarkers for TB susceptibility.

RNA CoMPASS: a dual approach for pathogen and host transcriptome analysis of RNA-seq datasets.

High-throughput RNA sequencing (RNA-seq) has become an instrumental assay for the analysis of multiple aspects of an organism's transcriptome. Further, the analysis of a biological specimen's associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naïve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt's lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs (which have a germinal center-like phenotype) from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at http://rnacompass.sourceforge.net/.

Notch signaling genes: myogenic DNA hypomethylation and 5-hydroxymethylcytosine.

Notch intercellular signaling is critical for diverse developmental pathways and for homeostasis in various types of stem cells and progenitor cells. Because Notch gene products need to be precisely regulated spatially and temporally, epigenetics is likely to help control expression of Notch signaling genes. Reduced representation bisulfite sequencing (RRBS) indicated significant hypomethylation in myoblasts, myotubes, and skeletal muscle vs. many nonmuscle samples at intragenic or intergenic regions of the following Notch receptor or ligand genes: NOTCH1, NOTCH2, JAG2, and DLL1. An enzymatic assay of sites in or near these genes revealed unusually high enrichment of 5-hydroxymethylcytosine (up to 81%) in skeletal muscle, heart, and cerebellum. Epigenetics studies and gene expression profiles suggest that hypomethylation and/or hydroxymethylation help control expression of these genes in heart, brain, myoblasts, myotubes, and within skeletal muscle myofibers. Such regulation could promote cell renewal, cell maintenance, homeostasis, and a poised state for repair of tissue damage.

Assessing needs and assets for building a regional network infrastructure to reduce cancer related health disparities.

Significant cancer health disparities exist in the United States and Puerto Rico. While numerous initiatives have been implemented to reduce cancer disparities, regional coordination of these efforts between institutions is often limited. To address cancer health disparities nation-wide, a series of regional transdisciplinary networks through the Geographic Management Program (GMaP) and the Minority Biospecimen/Biobanking Geographic Management Program (BMaP) were established in six regions across the country. This paper describes the development of the Region 3 GMaP/BMaP network composed of over 100 investigators from nine institutions in five Southeastern states and Puerto Rico to develop a state-of-the-art network for cancer health disparities research and training. We describe a series of partnership activities that led to the formation of the infrastructure for this network, recount the participatory processes utilized to develop and implement a needs and assets assessment and implementation plan, and describe our approach to data collection. Completion, by all nine institutions, of the needs and assets assessment resulted in several beneficial outcomes for Region 3 GMaP/BMaP. This network entails ongoing commitment from the institutions and institutional leaders, continuous participatory and engagement activities, and effective coordination and communication centered on team science goals.

Differences in gastric carcinoma microenvironment stratify according to EBV infection intensity: implications for possible immune adjuvant therapy.

Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC - high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

Cell-surface expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) in heterogeneous cultures of marrow-derived mesenchymal stem cells.

Cellular heterogeneity of mesenchymal stem cells (MSCs) impedes their use in regenerative medicine. The objective of this research is to identify potential biomarkers for the enrichment of progenitors from heterogeneous MSC cultures. To this end, the present study examines variation in expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) on the surface of MSCs derived from human bone marrow in response to culture conditions and among cell populations. Multipotent cells isolated from heterogeneous MSC cultures exhibit a greater than three-fold increase in surface expression for NG2 and greater than two-fold increase for CD146 as compared with parental and lineage-committed MSCs. For both antigens, surface expression is downregulated by greater than or equal to six-fold when MSCs become confluent. During serial passage, maximum surface expression of NG2 and CD146 is associated with minimum doubling time. Upregulation of NG2 and CD146 during loss of adipogenic potential at early passage suggests some limits to their utility as potency markers. A potential relationship between proliferation and antigen expression was explored by sorting heterogeneous MSCs into rapidly and slowly dividing groups. Fluorescence-activated cell sorting revealed that rapidly dividing MSCs display lower scatter and 50% higher NG2 surface expression than slowly dividing cells, but CD146 expression is comparable in both groups. Heterogeneous MSCs were sorted based on scatter properties and surface expression of NG2 and CD146 into high (HI) and low (LO) groups. Sc(LO)NG2(HI) and Sc(LO)NG2(HI)CD146(HI) MSCs have the highest proliferative potential of the sorted groups, with colony-forming efficiencies that are 1.5-2.2 times the value for the parental controls. The Sc(LO) gate enriches for rapidly dividing cells. Addition of the NG2(HI) gate increases cell survival to 1.5 times the parental control. Further addition of the CD146(HI) gate does not significantly improve cell division or survival. The combination of low scatter and high NG2 surface expression is a promising selection criterion to enrich a proliferative phenotype from heterogeneous MSCs during ex vivo expansion, with potentially numerous applications.

TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production.

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

Early de novo DNA methylation and prolonged demethylation in the muscle lineage.

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.