Altered Transcriptional Activity Research Articles

N-Terminal Truncated Myb with New Transcriptional Activity Produced Through Use of an Alternative MYB Promoter in Salivary Gland Adenoid Cystic Carcinoma.

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland tumor that frequently displays perineural invasion and is often associated with translocations or overexpression of the MYB oncogene. Detailed analyses of MYB transcripts from ACC patient samples revealed that ACC tumors utilize an alternative MYB promoter, which is rarely used in normal cells or other tumor types. The alternative promoter transcripts produce N-terminally truncated Myb proteins lacking a highly conserved and phosphorylated domain, which includes the pS11 epitope that is frequently used to detect Myb proteins. In RNA-seq assays, Myb isoforms lacking the N-terminal domain displayed unique transcriptional activities, regulating many genes differently than full-length Myb. Thus, a regulatory pathway unique to ACC activates the alternative MYB promoter, leading to the production of a truncated Myb protein with altered transcriptional activities. This could provide new therapeutic opportunities for ACC patients.

Functional variations of the TLR4 gene in association with chronic obstructive pulmonary disease and pulmonary tuberculosis

ObjectiveChronic obstructive pulmonary disease (COPD) and pulmonary tuberculosis (PTB) share a number of common risk factors, including innate immunity-related genetic factors. In the present study, we compared the role of genetic variations of the TLR4 gene in susceptibility to COPD and PTB and illuminated the underlying molecular mechanism of functional single-nucleotide polymorphisms (SNPs).MethodsA population-based case control study was performed in a Chinese Han population and included 152 COPD cases, 1601 PTB cases and 1727 controls. Five SNPs in the TLR4 gene (rs10759932, rs2737190, rs7873784, rs11536889, and rs10983755) were genotyped using TaqMan allelic discrimination technology. We estimated the effects of SNPs using the odds ratio (OR) together with 95% confidence interval (CI). Dual-luciferase reporter vectors expressing different genotypes of SNPs were constructed and transfected into the human HEK 293 T cell line to explore their effects on potential transcription activity.ResultsAfter Bonferroni correction, the genetic polymorphisms of all five SNPs remained significantly associated with COPD, while rs10759932 and rs2737190 were also associated with PTB. Compared with rs10759932-TT, individuals carrying TC (OR: 0.42, 95% CI: 0.28–0.64) or CC (OR: 0.24, 95% CI: 0.09–0.63) had a significantly reduced risk of COPD. However, individuals carrying TC (OR: 1.28, 95% CI: 1.11–1.49) or CC (OR: 1.26, 95% CI: 0.98–1.62) had an increased risk of PTB. The OR (95% CI) for allele rs10759932-C was 0.45 (0.32–0.62) for COPD and 1.18 (1.07–1.32) for PTB. For rs2737190, heterozygous AG was related to a decreased risk of COPD (OR: 0.32, 95% CI: 0.21–0.49) and an increased risk of PTB (OR: 1.30, 95% CI: 1.11–1.52). The dual-luciferase reporter assay showed decreased transcription activity caused by rs10759932-C and rs2737190-G.ConclusionGenetic polymorphisms of rs10759932 and rs2737190 in TLR4 are significantly related to both COPD and PTB but with inverse effects. The altered transcription activity caused by mutations in these two loci may partly explain the observed relationship.

Disease-associated missense variants in ZBTB18 disrupt DNA binding and impair the development of neurons within the embryonic cerebral cortex.

The activities of DNA-binding transcription factors, such as the multi-zinc-finger protein ZBTB18 (also known as RP58, or ZNF238), are essential to coordinate mammalian neurodevelopment, including the birth and radial migration of newborn neurons within the fetal brain. In humans, the majority of disease-associated missense mutations in ZBTB18 lie within the DNA-binding zinc-finger domain and are associated with brain developmental disorder, yet the molecular mechanisms explaining their role in disease remain unclear. To address this, we developed in silico models of ZBTB18, bound to DNA, and discovered that half of the missense variants map to residues (Asn461, Arg464, Glu486) predicted to be essential to sequence-specific DNA contact, whereas others map to residues (Leu434, Tyr447, Arg495) with limited contributions to DNA binding. We studied pathogenic variants to residues with close (N461S) and limited (R495G) DNA contact and found that each bound DNA promiscuously, displayed altered transcriptional regulatory activity in vitro, and influenced the radial migration of newborn neurons in vivo in different ways. Taken together, our results suggest that altered transcriptional regulation could represent an important pathological mechanism for ZBTB18 missense variants in brain developmental disease.

A pathogenic CtBP1 missense mutation causes altered cofactor binding and transcriptional activity.

We previously reported a pathogenic de novo p.R342W mutation in the transcriptional corepressor CTBP1 in four independent patients with neurodevelopmental disabilities [1]. Here, we report the clinical phenotypes of seven additional individuals with the same recurrent de novo CTBP1 mutation. Within this cohort, we identified consistent CtBP1-related phenotypes of intellectual disability, ataxia, hypotonia, and tooth enamel defects present in most patients. The R342W mutation in CtBP1 is located within a region implicated in a high affinity-binding cleft for CtBP-interacting proteins. Unbiased proteomic analysis demonstrated reduced interaction of several chromatin-modifying factors with the CtBP1 W342 mutant. Genome-wide transcriptome analysis in human glioblastoma cell lines expressing -CtBP1 R342 (wt) or W342 mutation revealed changes in the expression profiles of genes controlling multiple cellular processes. Patient-derived dermal fibroblasts were found to be more sensitive to apoptosis during acute glucose deprivation compared to controls. Glucose deprivation strongly activated the BH3-only pro-apoptotic gene NOXA, suggesting a link between enhanced cell death and NOXA expression in patient fibroblasts. Our results suggest that context-dependent relief of transcriptional repression of the CtBP1 mutant W342 allele may contribute to deregulation of apoptosis in target tissues of patients leading to neurodevelopmental phenotypes.

Effects of single and mixture exposure of cadmium and copper in apoptosis and immune related genes at transcriptional level on the midge Chironomus riparius Meigen (Diptera, Chironomidae)

Metals and heavy metals are natural contaminants with an increasing presence in aquatic ecosystems as a result of human activities. Although they are mixed in the water, research is usually focused on analyzing them in isolation, so there is a lack of knowledge about their combined effects. The aim of this work was to assess the damage produced by mixtures of cadmium and copper, two frequent metals used in industry, in the harlequin midge Chironomus riparius (Diptera). The effects of acute doses of cadmium and copper were evaluated in fourth instar larvae by analyzing the mRNA levels of six genes related to apoptosis (DRONC, IAP1), immune system (PO1, Defensin), stress (Gp93), and copper homeostasis (Ctr1). DRONC, Ctr1, and IAP1 transcripts are described here for first time in this species. Individual fourth instar larvae were submitted to 10 μM, 1 μM and 0.1 μM of CdCl2 or CuCl2, and mixture. The employed individuals came from different egg masses. Real-time PCR analysis showed a complex pattern of alterations in transcriptional activity for two genes, DRONC and Gp93, while the rest of them did not show any statistically significant differences. The effector caspase DRONC showed upregulation with the highest concentration tested of the mixture. In case of gp93, chaperone involved in regulation of immune response, differences in expression levels were found with 1 and 10 μM Cu and 0.1 and 10 μM of mixtures, compared to control samples. These results suggest that mixtures affect the transcriptional activity differently and produce changes in apoptosis and stress processes, although it is also possible that Gp93 alteration could be related to the immune system since it is homologous to human protein Gp96, which has been related with Toll-like receptors. In conclusion, cadmium and copper mixtures can affect the population by affecting the ability of larvae to respond to the infection and the apoptosis, an important process in the metamorphosis of insects.

SAT-326 INPP4B Suppresses Prostate Inflammation And Protects Mice Fed With High-fat Diet From The Development Of Prostate Intraepithelial Neoplasia

High fat diet leads to obesity, chronic inflammation, accelerated prostate cancer progression, and decreased survival. INPP4B, a dual specificity phosphatase, is a tumor suppressor which opposes Akt and PKC signaling pathways activated in response to obesity and prostate cancer progression. Previous research demonstrated that overexpression of PKC in mouse prostate leads to prostate intraepithelial neoplasia (PIN). Akt pathway stimulates prostate cancer progression in mice and is activated in all advanced prostate cancers in men. Using INPP4B knockout mouse model we tested whether INPP4B regulates prostate physiology in mice on high fat diet. The WT and Inpp4b-/- male mice were fed a low-fat diet (LFD) (11% fat) or high-fat diet (HFD) (58% fat) for 12 weeks. The mouse prostate was dissected into the anterior prostate (AP), dorsolateral prostate (DLP) and ventral prostate (VP). We observed that INPP4B and androgen receptor (AR) were expressed primarily in DLP and VP. INPP4B expression was decreased in the DLP of HFD fed mice. In the prostate of mice fed a LFD, we observed similar histological architecture in both WT and Inpp4b-/- mice. All the Inpp4b-/- mice fed a HFD developed PIN, whereas WT mice did not. Cells with polymorphic nuclei were abundant in PIN loci. The staining of α-SMA indicated that prostate epithelial cells invaded the stroma of HFD Inpp4b-/- mice. Consistent with the previous findings, HFD activated PKC, and the loss of INPP4B increased the levels of phospho-PKC βII and ζ in HFD mouse DLP. We previously reported that INPP4B loss changes AR transcriptional output in prostate cancer cells and normal mouse prostate. In HFD group, loss of INPP4B did not affect the level of AR in any of the three prostate lobes but led to an altered AR-dependent transcriptional activity. We show that the pro-inflammatory cytokines Il6, Tnfα and Il1β are expressed at low or undetectable levels in prostates of mice fed LFD. However, all three mediators were strikingly elevated in DLP of HFD Inpp4b-/- mice, where most of the PIN was also observed. Using an IL6 ELISA, we confirmed increases of IL6 protein levels in the prostates of the Inpp4b-/- HFD group. Consistent with elevated levels of inflammatory cytokines in prostates of Inpp4b-/- mice we detected an increase of macrophage specific biomarkers in the DLP of HFD groups, indicating HFD promotes macrophage infiltration in both WT and Inpp4b-/- mice. Importantly, no changes in PTEN were detected at both RNA and protein levels in Inpp4b-/- mice, indicating that increased inflammation and PIN development is due to the loss of INPP4B. Our data suggests that INPP4B suppresses oncogenic signaling pathways and opposes inflammation and neoplastic transformation in prostates of mice maintained on a high fat diet.

Transgenerational Transmission of Radiation-Induced Expression Patterns of Arabidopsis Thaliana (L.) Heynh. Rad51 and Rad1 Genes

Transcription rates of the genes AtKu70, AtRAD51, AtRad1, involved in maintaining Arabidopsis thaliana genome stability, in relation to the modification of phenotypic characteristics in irradiated plants and their progeny after the action of acute and fractionated X-ray radiation were studied. Differences in the transcription rate were measured by densitometric analysis of cDNA, synthesized by reverse transcription at the template of mRNAs, extracted from fresh leaves after 2 hours irradiation treatment. The doses 3 Gy, 12 Gy, 15 Gy and 21 Gy with 1.48 Gy/s specific dose rate were applied. Significant correlation between phenotype modifications in F0 and F1 generations, between phenotype traits and caretaker genes activity in irradiated F0 plants were shown. Also preservation of changes in the pattern of AtRad1 and AtRAD51 but not AtKu70 expression in F1 plant leaves had been revealed. Changes in F1 compared with F0 generation do not correspond to the extrapolation of dependence between the phenotypic modifications and DNA repair genes transcription rate in the leaves of irradiated plants. Based on the obtained data it could be suggested that the altered transcriptional activity of AtRAD51 and AtRad1 reflects the transfer of DNA lesions from parent to offspring.

Abstract 5833: Escape form adaptive drug tolerance through OGT and TET1 mediated H3K4me3 remodeling in MAPKi resistant melanoma

Abstract Background Acquired drug resistance in BRAF mutant melanoma is the main cause for disease relapse. We previously described a slow cycling induced drug tolerant state upon continuous BRAF/MEK treatment preceding permanent resistance with generic changes in histone methylation. Rationale Genetic alterations linked to acquired BRAF inhibitor resistance are absent in about 40% of relapsed melanoma patients suggesting the involvement of epigenetic alterations in the development of acquired drug resistance. We investigated epigenetic remodeling in BRAF mutant melanoma upon BRAF/MEK inhibition. Methods An in-vitro model of time lapse dependent transition to acquired drug resistance using mutant BRAF melanoma was used to investigate epigenetic changes following chronic drug exposure. Histone methylation patters were investigated using ChIP-seq, followed by target gene promoter ChIP-PCR and functional verification. Findings were confirmed by gene silencing, combined treatment regimes in vivo, in PDX tumors and clinical data sets. Results A state dependent response to chronic drug treatment was observed. Long term treatment of more than 45 days enables the cells to escape the slow cycling state which results in proliferating cellular clusters (drug-tolerant persister colonies) with stem-like characteristics that regain global H3K4me3. Persister colonies are then giving rise to fast proliferating BRAF/MEK inhibitor resistant cells. H3K4me3 ChIP-seq of colonies compared to parental cells revealed differential marking at promotor regions of several target genes involved in MAPKi resistance, including ARAF, BRAF, and CRAF. H3K4me3 remodeling corresponded to increased gene expression and susceptibility to pan-RAF inhibitors, suggesting an H3K4me3 mediated increase of ARAF and CRAF as a mechanism of BRAF/MEK inhibitor resistance. Two enzymes, OGT and TET1 that are both linked to H3K4me3 regulation in embryonic stem cells are highly upregulated in persister colonies and tumor tissue of PDXs from BRAF mutant melanoma patients under MEK1/2 inhibition. A shift in OGT nuclear localization and O-linked glycosylation patterns was observed in colonies compared to parental cells suggestive of altered transcriptional and protein activity. OGT ChIP-PCR of colonies compared to parental cells confirmed a set of genes with exclusively H3K4me3 marking in colonies. shRNA mediated knockdown of OGT and TET1 blocked H3K4me3 increase in IDTC colonies, prevented colony formation and delayed tumor relapse in a BRAF mutant xenograft mouse model. High TET1 mRNA expression is linked to significantly shorter survival in TGCA data. Conclusion OGT and TET1 mediated epigenetic remodeling through H3K4me3 with upregulation of MAPKi resistant genes is responsible for the emergence of permanent resistance. Both enzymes are promising targets to combat treatment failure and prolong overall survival. Citation Format: Dinoop Ravindran Menon, Heinz Hammerlindl, Abdullah Al Emran, Joachim Torrano, Sabrina Hammerlindl, Gao Zhang, Rajasekharan Somasundaram, Richard A. Sturm, Nikolas K. Haass, Keith Flaherty, Meenhard Herlyn, Helmut Schaider. Escape form adaptive drug tolerance through OGT and TET1 mediated H3K4me3 remodeling in MAPKi resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5833.

Rosiglitazone rescues human neural stem cells from amyloid-beta induced ER stress via PPARγ dependent signaling

Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated nuclear receptors known to regulate many crucial physiological and pathological conditions. Indeed, altered PPARγ transcriptional activity contributes to metabolic syndromes (obesity and hyperglycemia associated with type 2 diabetes mellitus), stroke and neurodegenerative diseases. Various studies suggest that PPARγ agonists influence neuronal deficits in Alzheimer's Disease (AD) patients and rodent models of AD. Expression of amyloid-beta (Aβ), a neuropathological marker associated with the pathogenesis of AD neuronal impairment, is inversely correlated with the activation of PPARγ-dependent neuroprotective responses. Nevertheless, molecular mechanisms by which the effects of PPARγ agonists in AD remain to be clarified. Here, we explore the PPARγ signaling pathways and networks that protect against Aβ-induced endoplasmic reticulum (ER) stress (e.g., caspase 4, Bip, CHOP, ASK1 and ER calcium), cell death (e.g., viability and cytochrome c) and mitochondrial deficiency (e.g., maximal respiratory function, COX activity, and mitochondrial membrane potential) events in the human neural stem cells (hNSCs) treated with Aβ. Co-treatment with GW9662 (an antagonist of PPARγ) effectively blocked these protective effects by rosiglitazone, providing strong evidence that PPARγ-dependent signaling rescues hNSCs from Aβ-mediated toxicity. Together, our data suggest activation of PPARγ pathway might be critical to protecting against AD-related ER stress, ER disequilibrium and mitochondrial deficiency. These findings also improve our understanding of the role of PPARγ in hNSCs, and may aid in the development and implementation of new therapeutic strategies for the treatment of AD.

Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection.

Ehrlichia chaffeensis has a group of well-characterized type I secreted tandem repeat protein (TRP) effectors that have moonlighting capabilities. TRPs modulate various cellular processes, reprogram host gene transcription as nucleomodulins, function as ubiquitin ligases, and directly activate conserved host cell signaling pathways to promote E. chaffeensis infection. One TRP-interacting host target is polycomb group ring finger protein 5 (PCGF5), a member of the polycomb group (PcG) protein family and a component of the polycomb repressive complex 1 (PRC1). The current study demonstrates that during early infection, PCGF5 strongly colocalizes with TRP120 in the nucleus and later dramatically redistributes to the ehrlichial vacuole along with other PCGF isoforms. Ectopic expression and immunoprecipitation of TRP120 confirmed the interaction of TRP120 with multiple different PCGF isoforms. At 48 h postinfection, a dramatic redistribution of PCGF isoforms from the nucleus to the ehrlichial vacuole was observed, which also temporally coincided with proteasomal degradation of PCGF isoforms and TRP120 expression on the vacuole. A decrease in PRC1-mediated repressive chromatin mark and an altered transcriptional activity in PRC1-associated Hox genes primarily from HOXB and HOXC clusters were observed along with the degradation of PCGF isoforms, suggesting disruption of the PRC1 in E. chaffeensis-infected cells. Notably, small interfering RNA (siRNA)-mediated knockdown of PCGF isoforms resulted in significantly increased E. chaffeensis infection. This study demonstrates a novel strategy in which E. chaffeensis manipulates PRC complexes through interactions between TRP120 and PCGF isoforms to promote infection.

Genome-Wide Transcriptome Analysis of Estrogen Receptor-Positive and Human Epithelial Growth Factor Receptor 2-Positive Breast Cancers by Ribonucleic Acid Sequencing

Aim: The aim is to identify complex pathogenesis of breast cancer subtypes and disclose the whole landscape of altered transcriptional activities in these cancers. Methods: We downloaded raw expression data from public database, and performed transcriptome analysis of 8 estrogen receptor-positive (ER+) breast cancer tissue samples, 8 human epithelial growth factor receptor 2-positive (HER2+) breast cancer tissue samples, and 3 normal breast tissues by identification, functional annotation, and prediction of upstream regulators and cell-surface biomarkers of differentially expressed genes (DEGs). Results: We identified over 5,000 DEGs in each of ER+ and HER2+ breast cancers compared to normal tissues. Functional enrichment analysis of shared DEGs indicated significant changes in the regulation of immune ­systems in the 2 subtypes. We further identified 1,871 DEGs between the 2 subtypes and disclosed great tumor heterogeneity. We identified 533 shared upregulated genes and predicted 17 upstream transcription factors, as well as identified differentially expressed cell-surface biomarkers for distinguishing our ER+ and HER2+ breast cancers. Further analysis also highlighted the limitation of the usage of HER2 alone in breast cancer classification. Conclusion: Our findings in ER+ and HER2+ breast cancers provided novel insights into heterogeneous transcriptional activities underlying complex mechanisms of oncogenesis in breast cancers.

Adipocyte Expression of SLC19A1 Links DNA Hypermethylation to Adipose Tissue Inflammation and Insulin Resistance.

Insulin resistance (IR) is promoted by a chronic low-grade inflammation in white adipose tissue (WAT). The latter might be regulated through epigenetic mechanisms such as DNA methylation. The one carbon cycle (1CC) is a central metabolic process governing DNA methylation. To identify adipocyte-expressed 1CC genes linked to WAT inflammation, IR, and their causal role. Cohort study. Outpatient academic clinic. Obese and nonobese subjects. Gene expression and DNA methylation arrays were performed in subcutaneous WAT and isolated adipocytes. In in vitro differentiated human adipocytes, gene knockdown was achieved by small interfering RNA, and analyses included microarray, quantitative polymerase chain reaction, DNA methylation by enzyme-linked immunosorbent assay and pyrosequencing, protein secretion by enzyme-linked immunosorbent assay, targeted metabolomics, and luciferase reporter and thermal shift assays. Effects on adipocyte inflammation. In adipocytes from obese individuals, global DNA hypermethylation was associated positively with gene expression of proinflammatory pathways. Among the 1CC genes, IR in vivo and proinflammatory gene expression in WAT were most strongly and inversely associated with SLC19A1, a gene encoding a membrane folate carrier. SLC19A1 knockdown in human adipocytes perturbed intracellular 1CC metabolism, induced global DNA hypermethylation, and increased expression of proinflammatory genes. Several CpG loci linked SLC19A1 to inflammation; validation studies were focused on the chemokine C-C motif chemokine ligand 2 (CCL2) in which methylation in the promoter (cg12698626) regulated CCL2 expression and CCL2 secretion through altered transcriptional activity. Reduced SLC19A1 expression in human adipocytes induces DNA hypermethylation, resulting in increased expression of specific proinflammatory genes, including CCL2. This constitutes an epigenetic mechanism that might link dysfunctional adipocytes to WAT inflammation and IR.

Novel and functional ATG12 gene variants in sporadic Parkinson's disease

Parkinson's disease (PD) is a common and progressive neurodegenerative disease, including familial and sporadic cases. To date, genetic causes for sporadic PD, majority of PD cases, remain largely unknown. Accumulating evidence indicates that dysfunctional autophagy, a highly conserved cellular process, is involved in the PD pathogenesis. We speculated that changed expression levels of autophagy-related genes (ATG) may contribute to PD development.Previously, we have genetically analyzed ATG5 and ATG7 genes in sporadic PD patients and identified several functional DNA sequence variants (DSVs). In groups of sporadic PD patients and ethic-matched healthy controls in this study, we further genetically and functionally analyzed the promoter of ATG12, a critical gene for autophagososme formation. The results showed that three DNA sequence variants (DSVs), g.115842507G>T,g.115842394C>T and g.115841817_18del, were identified three PD patients, which significantly altered transcriptional activity of ATG12 gene promoter, probably due to abolishing or creating binding sites for transcription factors. The transcriptional activity of ATG12 gene promoter was not significantly affected by other two DSVs identified in PD patients, g.115842640A>C and g.115842242G>C, which may not alter binding sites for transcription factors. Therefore, these three functional DSVs identified in PD patient may change ATG12 protein levels, contributing to PD development as a risk factor by interfering with autophagy as well as non-autophagy functions.

Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples

BackgroundPreinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown.MethodTo address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA.ResultsExamination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs.ConclusionsGene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.

Role of Nhp6 and Hmo1 in SWI/SNF occupancy and nucleosome landscape at gene regulatory regions

Diverse chromatin modifiers are involved in regulation of gene expression at the level of transcriptional regulation. Among these modifiers are ATP-dependent chromatin remodelers, where the SWI/SNF complex is the founding member. It has been observed that High Mobility Group (HMG) proteins can influence the activity of a number of these chromatin remodelers. In this context, we have previously demonstrated that the yeast HMG proteins Nhp6 and Hmo1 can stimulate SWI/SNF activity. Here, we studied the genome-wide binding patterns of Nhp6, Hmo1 and the SWI/SNF complex, finding that most of gene promoters presenting high occupancy of this complex also display high enrichment of these HMG proteins. Using deletion mutant strains we demonstrate that binding of SWI/SNF is significantly reduced at numerous genomic locations by deletion of NHP6 and/or deletion of HMO1. Moreover, alterations in the nucleosome landscape take place at gene promoters undergoing reduced SWI/SNF binding. Additional analyses show that these effects also correlate with alterations in transcriptional activity. Our results suggest that, besides the ability to stimulate SWI/SNF activity, these HMG proteins are able to assist the loading of this complex onto gene regulatory regions.

Clinical response to non-surgical periodontal treatment in patients with interleukin-6 and interleukin-10 polymorphisms.

BackgroundGenetic polymorphisms are commonly associated with altered transcriptional activity and possibly make individuals more susceptible to periodontal disease development, increased disease severity and poor treatment outcome. The study aimed to determine the effect of Interleukin-6 (IL-6) -572 G/C (rs1800796) and IL-10 -592 C/A (rs1800872) polymorphisms on the outcomes of non-surgical periodontal therapy in a Caucasian population.Material and MethodsSixty-eight patients with chronic periodontal disease were grouped according to their genotype: IL-6, IL-10, IL-6 and IL-10 susceptible (SCP) and non-susceptible (NSCP). All individuals were clinically evaluated at the first visit, and blood sample were collected from patients after checking the inclusion and exclusion criteria of the study. All patients received non-surgical periodontal therapy from a single-blinded periodontist. Clinical periodontal measurements were repeated 45 days after therapy.ResultsThis population mean aged 47.63 years included 52.2% females and 58.2% non-smokers. Following DNA separation and genotyping, 65.7% of patients were homozygous carriers of the IL-6 - 572G; 49.3% were carriers of the IL-10 -592A- allele (AA and CA genotypes); and 35.8% carried SCP genotypes for both polymorphisms. The clinical parameters after therapy were not associated with the genotype status. The multiple logistic regression analysis did not show any statistically significant association between the genotypes and the variables tested.Conclusions Within the limitations of this longitudinal study, it can be suggested that IL-6 -572 G/C and IL-10 -592 C/A polymorphisms as well as their combination do not influence the outcome of nonsurgical periodontal therapy in Caucasian patients diagnosed with chronic periodontal disease. Key words:Gene polymorphism, genetics, interleukins, periodontal disease, treatment outcome.

Altered nuclear dynamics in MDX myofibers.

Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to progressive muscle degeneration and weakness. Although the genetic basis is known, the pathophysiology of dystrophic skeletal muscle remains unclear. We examined nuclear movement in wild-type (WT) and muscular dystrophy mouse model for DMD (MDX) (dystrophin-null) mouse myofibers. We also examined expression of proteins in the linkers of nucleoskeleton and cytoskeleton (LINC) complex, as well as nuclear transcriptional activity via histone H3 acetylation and polyadenylate-binding nuclear protein-1. Because movement of nuclei is not only LINC dependent but also microtubule dependent, we analyzed microtubule density and organization in WT and MDX myofibers, including the application of a unique 3D tool to assess microtubule core structure. Nuclei in MDX myofibers were more mobile than in WT myofibers for both distance traveled and velocity. MDX muscle shows reduced expression and labeling intensity of nesprin-1, a LINC protein that attaches the nucleus to the microtubule and actin cytoskeleton. MDX nuclei also showed altered transcriptional activity. Previous studies established that microtubule structure at the cortex is disrupted in MDX myofibers; our analyses extend these findings by showing that microtubule structure in the core is also disrupted. In addition, we studied malformed MDX myofibers to better understand the role of altered myofiber morphology vs. microtubule architecture in the underlying susceptibility to injury seen in dystrophic muscles. We incorporated morphological and microtubule architectural concepts into a simplified finite element mathematical model of myofiber mechanics, which suggests a greater contribution of myofiber morphology than microtubule structure to muscle biomechanical performance.NEW & NOTEWORTHY Microtubules provide the means for nuclear movement but show altered organization in the muscular dystrophy mouse model (MDX) (dystrophin-null) muscle. Here, MDX myofibers show increased nuclear movement, altered transcriptional activity, and altered linkers of nucleoskeleton and cytoskeleton complex expression compared with healthy myofibers. Microtubule architecture was incorporated in finite element modeling of passive stretch, revealing a role of fiber malformation, commonly found in MDX muscle. The results suggest that alterations in microtubule architecture in MDX muscle affect nuclear movement, which is essential for muscle function.

Arsenic trioxide induces cell cycle arrest and alters DNA methylation patterns of cell cycle regulatory genes in colorectal cancer cells

AimsCell cycle dysregulation is important in tumorigenesis. Transcriptional silencing of cell cycle regulatory genes, due to DNA methylation, is a common epigenetic event in malignancies. As2O3 has been shown to induce cell cycle arrest and also to be a potential hypomethylating agent. Our study aimed to investigate DNA methylation patterns of cell cycle regulatory genes promoters, the effects of Arsenic trioxide (As2O3) on the methylated genes and cell cycle distribution in colorectal cancer (CRC) cell lines. Main methodsThe methylation-specific PCR (MSP) and/or restriction enzyme-based methods were used to study the promoter methylation patterns of 24 cell cycle regulatory genes in CRC cell lines. Gene expression level and cell cycle distribution were determined by Real-time PCR and flow cytometric analyses, respectively. Key findingsOur methylation analysis indicated that only promoters of RBL1 (p107), CHFR and p16 genes were aberrantly methylated in three cell lines. As2O3 significantly decreased DNA methylation in promoter regions of these genes and restored their expression. We found that As2O3 significantly reduced the expression of DNA methyltransferase 1 (DNMT1) and increased arsenic methyltransferase (AS3MT). Furthermore, As2O3 altered transcriptional activity of several unmethylated cell cycle regulatory genes including cyclin B1, E1, D1, GADD45A and p21. Cell cycle flow cytometry analysis showed As2O3 induced G2/M arrest in all three cell lines. SignificanceThese data suggest that demethylation and alteration in the expression level of the cell cycle-related genes may be possible mechanisms in As2O3-induced cell cycle arrest in colorectal cancer cells.

Alanine Expansions Associated with Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-mediated Dimerization and Nuclear Import

Heterozygous mutations of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to congenital central hypoventilation syndrome (CCHS), a neurodevelopmental disorder characterized by a failure in the autonomic control of breathing. Polyalanine expansions in the 20-residues region of the C terminus of PHOX2B are the major mutations responsible for CCHS. Elongation of the alanine stretch in PHOX2B leads to a protein with altered DNA binding, transcriptional activity, and nuclear localization and the possible formation of cytoplasmic aggregates; furthermore, the findings of various studies support the idea that CCHS is not due to a pure loss of function mechanism but also involves a dominant negative effect and/or toxic gain of function for PHOX2B mutations. Because PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant negative effects of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A using a co-immunoprecipitation assay and the mammalian two-hybrid system. Our findings show that PHOX2B forms homodimers and heterodimerizes weakly with mutated proteins, exclude the direct involvement of the polyalanine tract in dimer formation, and indicate that mutated proteins retain partial ability to form heterodimers with PHOX2A. Moreover, in this study, we investigated the effects of the longest polyalanine expansions on the homeodomain-mediated nuclear import, and our data clearly show that the expanded C terminus interferes with this process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity.

Pro-inflammatory cytokines and their epistatic interactions in genetic susceptibility to schizophrenia.

BackgroundIn schizophrenia, genetic background may provide a substrate for intrinsic maldevelopment of the brain through environmental influences, by recruiting neurotrophic factors and cytokines, to trigger the changes that lead to impaired neuronal functions. Cytokines being the key regulators of immune/inflammatory reactions are also known to influence the dopaminergic, noradrenergic, and serotonergic neurotransmission. Therefore, functional polymorphisms in cytokine genes may result in imbalances in the pro- and anti-inflammatory cytokine production.MethodsWe screened polymorphisms in pro- and anti-inflammatory cytokine genes using a case-control association study in a South Indian population. The role of allele, genotype, haplotype, and diplotypes of these cytokine genes and their epistatic interactions were assessed in contributing to the risk of developing schizophrenia. Meta-analysis for the reported associations was also monitored for global significance.ResultsThe pro-inflammatory cytokine gene polymorphisms in IL1Ars1800587, IL6rs1800796, TNFArs361525, and IFNGrs2069718 were associated with schizophrenia. The study also provides significant evidence for strong epistatic interactions among pro-inflammatory cytokine genes IL6 and IFNG in the development of schizophrenia. In silico analysis suggested that associated risk variants were indicative of altered transcriptional activity with higher production of IL1α, IL-6, TNF-α, and IFN-ɤ cytokines. Meta-analysis indicated heterogeneity among study population while IL1Ars1800587 was found to be globally significant.ConclusionsIt is important to identify the nature of inflammatory response that can be amplified by the environment, to influence either Th1 response or Th2 response. The associated functional variants in the study are involved with increased expression resulting in higher production of the pro-inflammatory cytokines IL-1α, IL-6, TNF-α, and IFN-γ. The interaction of immunological stressors with these high producer alleles of pro-inflammatory cytokines may suggest that even a lower threshold may be sufficient to induce a resultant chronic effect on the psycho-social and environmental stressors that may result in the development and pathogenesis of schizophrenia. Understanding environmental factors that influence the expression of these pro-inflammatory cytokine genes or their interaction can possibly help in dissecting the phenotypic variation and therapeutic response to antipsychotics in schizophrenia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0569-8) contains supplementary material, which is available to authorized users.