Maureen A. Sartor

The Circadian Clock in Oral Health and Diseases

Most physiological processes in mammals display circadian rhythms that are driven by the endogenous circadian clock. This clock is comprised of a central component located in the hypothalamic suprachiasmatic nucleus and subordinate clocks in peripheral tissues. Circadian rhythms sustain 24-hour oscillations of a large number of master genes controlling the correct timing and synchronization of diverse physiological and metabolic processes within our bodies. This complex regulatory network provides an important communication link between our brain and several peripheral organs and tissues. At the molecular level, circadian oscillations of gene expression are regulated by a family of transcription factors called “clock genes”. Dysregulation of clock gene expression results in diverse human pathological conditions, including autoimmune diseases and cancer. There is increasing evidence that the circadian clock affects tooth development, salivary gland and oral epithelium homeostasis, and saliva production. This review summarizes current knowledge of the roles of clock genes in the formation and maintenance of oral tissues, and discusses potential links between “oral clocks” and diseases such as head and neck cancer and Sjögren’s syndrome.

ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death

Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER.

MicroRNA-320 Is Involved in the Regulation of Cardiac Ischemia/Reperfusion Injury by Targeting Heat-Shock Protein 20

Background— Recent studies have identified critical roles for microRNAs (miRNAs) in a variety of cellular processes, including regulation of cardiomyocyte death. However, the signature of miRNA expression and possible roles of miRNA in the ischemic heart have been less well studied. Methods and Results— We performed miRNA arrays to detect the expression pattern of miRNAs in murine hearts subjected to ischemia/reperfusion (I/R) in vivo and ex vivo. Surprisingly, we found that only miR-320 expression was significantly decreased in the hearts on I/R in vivo and ex vivo. This was further confirmed by TaqMan real-time polymerase chain reaction. Gain-of-function and loss-of-function approaches were employed in cultured adult rat cardiomyocytes to investigate the functional roles of miR-320. Overexpression of miR-320 enhanced cardiomyocyte death and apoptosis, whereas knockdown was cytoprotective, on simulated I/R. Furthermore, transgenic mice with cardiac-specific overexpression of miR-320 revealed an increased extent of apoptosis and infarction size in the hearts on I/R in vivo and ex vivo relative to the wild-type controls. Conversely, in vivo treatment with antagomir-320 reduced infarction size relative to the administration of mutant antagomir-320 and saline controls. Using TargetScan software and proteomic analysis, we identified heat-shock protein 20 (Hsp20), a known cardioprotective protein, as an important candidate target for miR-320. This was validated experimentally by utilizing a luciferase/GFP reporter activity assay and examining the expression of Hsp20 on miR-320 overexpression and knockdown in cardiomyocytes. Conclusions— Our data demonstrate that miR-320 is involved in the regulation of I/R-induced cardiac injury and dysfunction via antithetical regulation of Hsp20. Thus, miR-320 may constitute a new therapeutic target for ischemic heart diseases.

Metscape 2 bioinformatics tool for the analysis and visualization of metabolomics and gene expression data

MOTIVATION Metabolomics is a rapidly evolving field that holds promise to provide insights into genotype-phenotype relationships in cancers, diabetes and other complex diseases. One of the major informatics challenges is providing tools that link metabolite data with other types of high-throughput molecular data (e.g. transcriptomics, proteomics), and incorporate prior knowledge of pathways and molecular interactions. RESULTS We describe a new, substantially redesigned version of our tool Metscape that allows users to enter experimental data for metabolites, genes and pathways and display them in the context of relevant metabolic networks. Metscape 2 uses an internal relational database that integrates data from KEGG and EHMN databases. The new version of the tool allows users to identify enriched pathways from expression profiling data, build and analyze the networks of genes and metabolites, and visualize changes in the gene/metabolite data. We demonstrate the applications of Metscape to annotate molecular pathways for human and mouse metabolites implicated in the pathogenesis of sepsis-induced acute lung injury, for the analysis of gene expression and metabolite data from pancreatic ductal adenocarcinoma, and for identification of the candidate metabolites involved in cancer and inflammation. AVAILABILITY Metscape is part of the National Institutes of Health-supported National Center for Integrative Biomedical Informatics (NCIBI) suite of tools, freely available at http://metscape.ncibi.org. It can be downloaded from http://cytoscape.org or installed via Cytoscape plugin manager. CONTACT metscape-help@umich.edu; akarnovs@umich.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

LRpath: a logistic regression approach for identifying enriched biological groups in gene expression data.

MOTIVATION The elucidation of biological pathways enriched with differentially expressed genes has become an integral part of the analysis and interpretation of microarray data. Several statistical methods are commonly used in this context, but the question of the optimal approach has still not been resolved. RESULTS We present a logistic regression-based method (LRpath) for identifying predefined sets of biologically related genes enriched with (or depleted of) differentially expressed transcripts in microarray experiments. We functionally relate the odds of gene set membership with the significance of differential expression, and calculate adjusted P-values as a measure of statistical significance. The new approach is compared with Fishers exact test and other relevant methods in a simulation study and in the analysis of two breast cancer datasets. Overall results were concordant between the simulation study and the experimental data analysis, and provide useful information to investigators seeking to choose the appropriate method. LRpath displayed robust behavior and improved statistical power compared with tested alternatives. It is applicable in experiments involving two or more sample types, and accepts significance statistics of the investigators choice as input.

ConceptGen: a gene set enrichment and gene set relation mapping tool

MOTIVATION The elucidation of biological concepts enriched with differentially expressed genes has become an integral part of the analysis and interpretation of genomic data. Of additional importance is the ability to explore networks of relationships among previously defined biological concepts from diverse information sources, and to explore results visually from multiple perspectives. Accomplishing these tasks requires a unified framework for agglomeration of data from various genomic resources, novel visualizations, and user functionality. RESULTS We have developed ConceptGen, a web-based gene set enrichment and gene set relation mapping tool that is streamlined and simple to use. ConceptGen offers over 20,000 concepts comprising 14 different types of biological knowledge, including data not currently available in any other gene set enrichment or gene set relation mapping tool. We demonstrate the functionalities of ConceptGen using gene expression data modeling TGF-beta-induced epithelial-mesenchymal transition and metabolomics data comparing metastatic versus localized prostate cancers.

Genome-Wide DNA Methylation Differences Between Late-Onset Alzheimer's Disease and Cognitively Normal Controls in Human Frontal Cortex

Evidence supports a role for epigenetic mechanisms in the pathogenesis of late-onset Alzheimers disease (LOAD), but little has been done on a genome-wide scale to identify potential sites involved in disease. This study investigates human postmortem frontal cortex genome-wide DNA methylation profiles between 12 LOAD and 12 cognitively normal age- and gender-matched subjects. Quantitative DNA methylation is determined at 27,578 CpG sites spanning 14,475 genes via the Illumina Infinium HumanMethylation27 BeadArray. Data are analyzed using parallel linear models adjusting for age and gender with empirical Bayes standard error methods. Gene-specific technical and functional validation is performed on an additional 13 matched pair samples, encompassing a wider age range. Analysis reveals 948 CpG sites representing 918 unique genes as potentially associated with LOAD disease status pending confirmation in additional study populations. Across these 948 sites the subtle mean methylation difference between cases and controls is 2.9%. The CpG site with a minimum false discovery rate located in the promoter of the gene Transmembrane Protein 59 (TMEM59) is 7.3% hypomethylated in cases. Methylation at this site is functionally associated with tissue RNA and protein levels of the TMEM59 gene product. The TMEM59 gene identified from our discovery approach was recently implicated in amyloid-β protein precursor post-translational processing, supporting a role for epigenetic change in LOAD pathology. This study demonstrates widespread, modest discordant DNA methylation in LOAD-diseased tissue independent from DNA methylation changes with age. Identification of epigenetic biomarkers of LOAD risk may allow for the development of novel diagnostic and therapeutic targets.

Conditional Activation of RET/PTC3 and BRAFV600E in Thyroid Cells Is Associated with Gene Expression Profiles that Predict a Preferential Role of BRAF in Extracellular Matrix Remodeling

Papillary thyroid cancers (PTC) are associated with nonoverlapping mutations of genes coding for mitogen-activated protein kinase signaling effectors (i.e., the TK receptors RET or NTRK and the signaling proteins RAS and BRAF). We examined the pattern of gene expression after activation of these oncoproteins in thyroid PCCL3 cells, with the goal of identifying pathways or gene subsets that may account for the phenotypic differences observed in human cancers. We hybridized cDNA from cells treated with or without doxycycline to induce expression of BRAF(V600E), RET/PTC3, or RET/PTC3 with small interfering RNA-mediated knockdown of BRAF, respectively, to slides arrayed with a rat 70-mer oligonucleotide library consisting of 27,342 oligos. Among the RET/PTC3-induced genes, 2,552 did not require BRAF as they were similarly regulated by RET/PTC3 with or without BRAF knockdown and not by expression of BRAF(V600E). Immune response and IFN-related genes were highly represented in this group. About 24% of RET/PTC3-regulated genes were BRAF dependent, as they were similarly modified by RET/PTC3 and BRAF(V600E) but not in cells expressing RET/PTC3 with knockdown of BRAF. A gene cluster coding for components of the mitochondrial electron transport chain pathway was down-regulated in this group, potentially altering regulation of cell viability. Metalloproteinases were also preferentially induced by BRAF, particularly matrix metalloproteinase 3 (MMP3), MMP9, and MMP13. Accordingly, conditional expression of BRAF was associated with markedly increased invasion into Matrigel compared with cells expressing RET/PTC3. The preferential induction of MMPs by BRAF could explain in part the more invasive behavior of thyroid cancers with BRAF mutations.

Progression of BRAF -induced thyroid cancer is associated with epithelial–mesenchymal transition requiring concomitant MAP kinase and TGFβ signaling

Mice with thyroid-specific expression of oncogenic BRAF (Tg-Braf) develop papillary thyroid cancers (PTCs) that are locally invasive and have well-defined foci of poorly differentiated thyroid carcinoma (PDTC). To investigate the PTC–PDTC progression, we performed a microarray analysis using RNA from paired samples of PDTC and PTC collected from the same animals by laser capture microdissection. Analysis of eight paired samples revealed a profound deregulation of genes involved in cell adhesion and intracellular junctions, with changes consistent with an epithelial–mesenchymal transition (EMT). This was confirmed by immunohistochemistry, as vimentin expression was increased and E-cadherin lost in PDTC compared with adjacent PTC. Moreover, PDTC stained positively for phospho-Smad2, suggesting a role for transforming growth factor (TGF)β in mediating this process. Accordingly, TGFβ-induced EMT in primary cultures of thyroid cells from Tg-Braf mice, whereas wild-type thyroid cells retained their epithelial features. TGFβ-induced Smad2 phosphorylation, transcriptional activity and induction of EMT required mitogen-activated protein kinase (MAPK) pathway activation in Tg-Braf thyrocytes. Hence, tumor initiation by oncogenic BRAF renders thyroid cells susceptible to TGFβ-induced EMT, through a MAPK-dependent process.

Genome-wide methylation and expression differences in HPV(+) and HPV(-) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis

Oncogenic human papillomaviruses (HPV) are associated with nearly all cervical cancers and are increasingly important in the etiology of oropharyngeal tumors. HPV-associated head and neck squamous cell carcinomas (HNSCC) have distinct risk profiles and appreciate a prognostic advantage compared to HPV-negative HNSCC. Promoter hypermethylation is widely recognized as a mechanism in the progression of HNSCC, but the extent to which this mechanism is consistent between HPV(+) and HPV(-) tumors is unknown. To investigate the epigenetic regulation of gene expression in HPV-induced and carcinogen-induced cancers, we examined genome-wide DNA methylation and gene expression in HPV(+) and HPV(-) SCC cell lines. We used two platforms: the Illumina Infinium Methylation BeadArray and tiling arrays, and confirmed illustrative examples with pyrosequencing and quantitative PCR. These analyses indicate that HPV(+) cell lines have higher DNA methylation in genic and LINE-1 regions than HPV(-) cell lines. Differentially methylated loci between HPV(+) and HPV(-) cell lines significantly correlated with HPV-typed HNSCC primary tumor DNA methylation levels. Novel findings include higher promoter methylation of polycomb repressive complex 2 target genes in HPV(+) cells compared to HPV(-) cells and increased expression of DNMT3A in HPV(+) cells. Additionally, CDKN2A and KRT8 were identified as interaction hubs among genes with higher methylation and lower expression in HPV(-) cells. Conversely, RUNX2, IRS-1 and CCNA1 were major hubs with higher methylation and lower expression in HPV(+) cells. Distinct HPV(+) and HPV(-) epigenetic profiles should provide clues to novel targets for development of individualized therapeutic strategies.