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1.  p53 Contributes to Differentiating Gene Expression Following Exposure to Acetaminophen and Its Less Hepatotoxic Regioisomer Both In Vitro and In Vivo 
The goal of the present study was to compare hepatic toxicogenomic signatures across in vitro and in vivo mouse models following exposure to acetaminophen (APAP) or its relatively nontoxic regioisomer 3′-hydroxyacetanilide (AMAP). Two different Affymetrix microarray platforms and one Agilent Oligonucleotide microarray were utilized. APAP and AMAP treatments resulted in significant and large changes in gene expression that were quite disparate, and likely related to their different toxicologic profiles. Ten transcripts, all of which have been implicated in p53 signaling, were identified as differentially regulated at all time-points following APAP and AMAP treatments across multiple microarray platforms. Protein-level quantification of p53 activity aligned with results from the transcriptomic analysis, thus supporting the implicated mechanism of APAP-induced toxicity. Therefore, the results of this study provide good evidence that APAP-induced p53 phosphorylation and an altered p53-driven transcriptional response are fundamental steps in APAP-induced toxicity.
PMCID: PMC4454132  PMID: 26056430
acetaminophen; AMAP; microarray; p53; TAMH; toxicogenomics
2.  A Hypothetical Protein of Alteromonas macleodii AltDE1 (amad1_06475) Predicted to be a Cold-Shock Protein with RNA Chaperone Activity 
Alteromonas macleodii AltDE1 is a deep sea protobacteria that is distinct from the surface isolates of the same species. This study was designed to elucidate the biological function of amad1_06475, a hypothetical protein of A. macleodii AltDE1. The 70 residues protein sequence showed considerable homology with cold-shock proteins (CSPs) and RNA chaperones from different organisms. Multiple sequence alignment further supported the presence of conserved csp domain on the protein sequence. The three-dimensional structure of the protein was also determined, and verified by PROCHECK, Verify3D, and QMEAN programs. The predicted structure contained five anti-parallel β-strands and RNA-binding motifs, which are characteristic features of prokaryotic CSPs. Finally, the binding of a thymidine-rich oligonucleotide and a single uracil molecule in the active site of the protein further strengthens our prediction about the function of amad1_06475 as a CSP and thereby acting as a RNA chaperone. The binding was performed by molecular docking tools and was compared with similar binding of 3PF5 (PDB) and 2HAX (PDB), major CSPs of Bacillus subtilis and Bacillus caldolyticus, respectively.
PMCID: PMC4271719  PMID: 25574135
Cold-shock protein (CSP); RNA chaperone; cold-shock domain; RNP
3.  A Comprehensive Profile of ChIP-Seq-Based PU.1/Spi1 Target Genes in Microglia 
Microglia are resident mononuclear phagocytes that play a principal role in the maintenance of normal tissue homeostasis in the central nervous system (CNS). Microglia, rapidly activated in response to proinflammatory stimuli, are accumulated in brain lesions of neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. The E26 transformation-specific (ETS) family transcription factor PU.1/Spi1 acts as a master regulator of myeloid and lymphoid development. PU.1-deficient mice show a complete loss of microglia, indicating that PU.1 plays a pivotal role in microgliogenesis. However, the comprehensive profile of PU.1/Spi1 target genes in microglia remains unknown. By analyzing a chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) dataset numbered SRP036026 with the Strand NGS program, we identified 5,264 Spi1 target protein-coding genes in BV2 mouse microglial cells. They included Spi1, Irf8, Runx1, Csf1r, Csf1, Il34, Aif1 (Iba1), Cx3cr1, Trem2, and Tyrobp. By motif analysis, we found that the PU-box consensus sequences were accumulated in the genomic regions surrounding ChIP-Seq peaks. By using pathway analysis tools of bioinformatics, we found that ChIP-Seq-based Spi1 target genes show a significant relationship with diverse pathways essential for normal function of monocytes/macrophages, such as endocytosis, Fc receptor-mediated phagocytosis, and lysosomal degradation. These results suggest that PU.1/Spi1 plays a crucial role in regulation of the genes relevant to specialized functions of microglia. Therefore, aberrant regulation of PU.1 target genes might contribute to the development of neurodegenerative diseases with accumulation of activated microglia.
PMCID: PMC4262374  PMID: 25574134
ChIP-Seq; GenomeJack; KeyMolnet; microglia; microgliopathy; Nasu–Hakola disease; PU.1; Spi1; Strand NGS
4.  Endothelin-1 but not Endothelial Nitric Oxide Synthase Gene Polymorphism is Associated with Sickle Cell Disease in Africa 
Sickle cell disease shows marked variability in severity and pathophysiology among individuals, probably linked to differential expression of various adhesion molecules. In this study, we investigated the differential distribution, genomic diversity and haplotype frequency of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) polymorphisms, recently implicated as important in modification of disease severity. One hundred and forty five sickle cell disease patients (HbSS) and 244 adult and pediatric controls, without sickle cell disease (HbAA), were recruited from Mali. Genotypic analysis of the functionally significant eNOS variants (T786C, G894T and intron 4) and endothelin-1 (G5665T) was carried out with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Our results show that the wild type alleles are the most frequent for all eNOS variants between cases and controls. Allelic and genotypic frequencies of eNOS polymorphic groups are not significantly different between cases and controls (P > 0.05). In addition, there is no association between eNOS variants and sickle cell disease, contrary to published reports. On the other hand, we report that endothelin-1 (G5665T) mutant variant had the lowest allelic frequency, and is significantly associated with sickle cell disease in Africa (P < 0.05). Similarly, haplotype frequencies were the same between cases and controls, except for the haplotype combining all mutant variants (T, C, 4a; P = 0.01). eNOS polymorphic variants are less frequent, with no significance with sickle cell disease in Africa. On the other hand, endothelin-1 is associated with sickle cell disease, and has the capacity to redefine pathophysiology and possibly serve as modulator of disease phenotype.
PMCID: PMC4051816  PMID: 24932102
endothelial nitric oxide synthase; endothelin-1; sickle cell disease; polymorphisms; pathophysiology
5.  Species-Specific Regulation of t-PA and PAI-1 Gene Expression in Human and Rat Astrocytes 
In recent years, the role and physiological regulation of the serine protease tissue-type plasminogen activator (t-PA) and its inhibitors, including plasminogen activator inhibitor type-1 (PAI-1), in the brain have received much attention. However, as studies focusing these issues are difficult to perform in humans, a great majority of the studies conducted to date have utilized rodent in vivo and/or in vitro models. In view of the species-specific structural differences present in both the t-PA and the PAI-1 promoters, we have compared the response of these genes in astrocytes of rat and human origin. We reveal marked quantitative and qualitative species-specific differences in gene induction following treatment with various physiological and pathological stimuli. Thus, our findings are of importance for the interpretation of previous and future results related to t-PA and PAI-1 expression.
PMCID: PMC4024051  PMID: 24855337
astrocytes; tissue-type plasminogen activator; plasminogen activator inhibitor type-1; gene expression; species-specific
6.  Overfeeding Dairy Cattle During Late-Pregnancy Alters Hepatic PPARα-Regulated Pathways Including Hepatokines: Impact on Metabolism and Peripheral Insulin Sensitivity 
Hepatic metabolic gene networks were studied in dairy cattle fed control (CON, 1.34 Mcal/kg) or higher energy (overfed (OVE), 1.62 Mcal/kg) diets during the last 45 days of pregnancy. A total of 57 target genes encompassing PPARα-targets/co-regulators, hepatokines, growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, lipogenesis, and lipoprotein metabolism were evaluated on −14, 7, 14, and 30 days around parturition. OVE versus CON cows were in more negative energy balance (NEB) postpartum and had greater serum non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), and liver triacylglycerol (TAG) concentrations. Milk synthesis rate did not differ. Liver from OVE cows responded to postpartal NEB by up-regulating expression of PPARα-targets in the fatty acid oxidation and ketogenesis pathways, along with gluconeogenic genes. Hepatokines (fibroblast growth factor 21 (FGF21), angiopoietin-like 4 (ANGPTL4)) and apolipoprotein A-V (APOA5) were up-regulated postpartum to a greater extent in OVE than CON. OVE led to greater blood insulin prepartum, lower NEFA:insulin, and greater lipogenic gene expression suggesting insulin sensitivity was not impaired. A lack of change in APOB, MTTP, and PNPLA3 coupled with upregulation of PLIN2 postpartum in cows fed OVE contributed to TAG accumulation. Postpartal responses in NEFA and FGF21 with OVE support a role of this hepatokine in diminishing adipose insulin sensitivity.
PMCID: PMC3981572  PMID: 24737933
nutrition; nuclear receptor; obesity; lactation
7.  Oxygen and Cell Fate Decisions 
Molecular oxygen has been known to play a critical role in a wide range of biological processes including glycolysis, mitochondrial respiration, angiogenesis, pulmonary functions, and cardiovascular activities. An emerging theme has developed in recent years that oxygen has significant impact on embryonic development, maintenance of stem cells, and cellular differentiation or cell fate decisions. Among the notable observations, early embryonic development takes place in a hypoxic microenvironment. Hematopoietic stem cells appear to be located in hypoxic regions within the bone marrow. Majority of the current observations have shown that hypoxia seems to prevent cellular differentiation and to maintain pluripotency of stem/progenitor cells. Genetic studies have demonstrated a critical role of hypoxia-inducible factors 1α and 2α in embryonic development. These intriguing observations demonstrate an important role of molecular oxygen in such fundamental biological processes as stem cell maintenance and regulation of cell fate decisions. Herein, we describe some of the latest advances in the biology of molecular oxygen and provide our perspectives on the potential impact of these interesting findings.
PMCID: PMC2733087  PMID: 19606268
adipogenesis; chondrogenesis; differentiation; hypoxia; myogenesis; oxygen; placenta; preadipocytes; progenitor cells; stem cells; trophoblasts
8.  DNA Conformational Transitions Induced by Supercoiling Control Transcription in Chromatin 
Regulation of transcription in eukaryotes is considered in the light of recent findings demonstrating the presence of negative and positive superhelical tension in chromatin. This tension induces conformational transitions in DNA duplex. Particularly, the transition into A-form renders DNA accessible and waylaying for initiation of transcription producing RNA molecules long known to belong to the A-conformation. Competition between conformational transitions in various DNA sequences for the energy of elastic spring opens a possibility for understanding of fine tuning of transcription at a distance.
PMCID: PMC3956857  PMID: 24653646
DNA conformation; nucleosomes; transcription; RNA-polymerase; topoisomerase II
9.  Tissue-Specific Gene Expression and Regulation in Liver and Muscle Following Chronic Corticosteroid Administration 
Although corticosteroids (CSs) affect gene expression in multiple tissues, the array of genes that are regulated by these catabolic steroids is diverse, highly tissue specific, and depends on their functions in the tissue. Liver has many important functions in performing and regulating diverse metabolic processes. Muscle, in addition to its mechanical role, is critical in maintaining systemic energy homeostasis and accounts for about 80% of insulin-directed glucose disposal. Consequently, a better understanding of CS pharmacogenomic effects in these tissues would provide valuable information regarding the tissue-specificity of transcriptional dynamics, and would provide insights into the underlying molecular mechanisms of action for both beneficial and detrimental effects.
We performed an integrated analysis of transcriptional data from liver and muscle in response to methylprednisolone (MPL) infusion, which included clustering and functional annotation of clustered gene groups, promoter extraction and putative transcription factor (TF) identification, and finally, regulatory closeness (RC) identification.
This analysis allowed the identification of critical transcriptional responses and CS-responsive functions in liver and muscle during chronic MPL administration, the prediction of putative transcriptional regulators relevant to transcriptional responses of CS-affected genes which are also potential secondary bio-signals altering expression levels of target-genes, and the exploration of the tissue-specificity and biological significance of gene expression patterns, CS-responsive functions, and transcriptional regulation.
The analysis provided an integrated description of the genomic and functional effects of chronic MPL infusion in liver and muscle.
PMCID: PMC3956809  PMID: 24653645
liver; muscle; glucocorticoids; corticosteroids; gene expression; gene regulation; promoter analysis
10.  Tetracycline Derivative Minocycline Inhibits Autophagy and Inflammation in Concanavalin-A-Activated Human Hepatoma Cells 
Inhibition of soluble matrix metalloproteinase (MMP) activity is among the non-antibiotic cellular effects exerted by the anti-inflammatory tetracycline derivative minocycline. The impact of minocycline on the signal transduction functions of membrane-bound MMPs is however unknown. We assessed minocycline in a concanavalin-A (ConA)-activated human HepG2 hepatoma cell model, a condition known to increase the expression of membrane type-1 MMP (MT-MMP) and to trigger inflammatory and autophagy processes. We found that minocycline inhibited ConA-induced formation of autophagic acidic vacuoles, green fluorescent microtubule-associated protein 1 light chain 3 (GFP-LC3) puncta formation, gene and protein expression of autophagy biomarker BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), invasion biomarker MT1-MMP, and inflammation biomarker cyclooxygenase (COX)-2. Gene silencing of MT1-MMP abrogated ConA-induced formation of autophagic acidic vacuoles and ConA-induced expressions of BNIP3 and COX-2. Minocycline was also shown to inhibit ConA-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation as well as gene expression of NANOS1, a biomarker believed to colocalize with MT1-MMP and the specific silencing of which further inhibited ConA-induced STAT3 phosphorylation. Collectively, our data demonstrate that part of minocycline’s effects on autophagy could be exerted through the inhibition of MT1-MMP signaling functions, which contribute to the autophagy and inflammatory phenotype of ConA-activated HepG2 cells.
PMCID: PMC3948715  PMID: 24634581
hepatocellular carcinoma cells; concanavalin-A; autophagy; inflammation; minocycline; antibiotic; cancer
11.  Discovery of Emphysema Relevant Molecular Networks from an A/J Mouse Inhalation Study Using Reverse Engineering and Forward Simulation (REFS™) 
Chronic obstructive pulmonary disease (COPD) is a respiratory disorder caused by extended exposure of the airways to noxious stimuli, principally cigarette smoke (CS). The mechanisms through which COPD develops are not fully understood, though it is believed that the disease process includes a genetic component, as not all smokers develop COPD. To investigate the mechanisms that lead to the development of COPD/emphysema, we measured whole genome gene expression and several COPD-relevant biological endpoints in mouse lung tissue after exposure to two CS doses for various lengths of time. A novel and powerful method, Reverse Engineering and Forward Simulation (REFS™), was employed to identify key molecular drivers by integrating the gene expression data and four measured COPD-relevant endpoints (matrix metalloproteinase (MMP) activity, MMP-9 levels, tissue inhibitor of metalloproteinase-1 levels and lung weight). An ensemble of molecular networks was generated using REFS™, and simulations showed that it could successfully recover the measured experimental data for gene expression and COPD-relevant endpoints. The ensemble of networks was then employed to simulate thousands of in silico gene knockdown experiments. Thirty-three molecular key drivers for the above four COPD-relevant endpoints were therefore identified, with the majority shown to be enriched in inflammation and COPD.
PMCID: PMC3937248  PMID: 24596455
Bayesian network; chronic obstructive pulmonary disease (COPD); reverse engineering and forward simulation (REFS™)
12.  Gene Expression and Gene Ontology Enrichment Analysis for H3K4me3 and H3K4me1 in Mouse Liver and Mouse Embryonic Stem Cell Using ChIP-Seq and RNA-Seq 
Recent study has identified the cis-regulatory elements in the mouse genome as well as their genomic localizations. Recent discoveries have shown the enrichment of H3 lysine 4 trimethylation (H3K4me3) binding as an active promoter and the presence of H3 lysine 4 monomethylation (H3K4me1) outside promoter regions as a mark for an enhancer. In this work, we further identified highly expressed genes by H3K4me3 mark or by both H3K4me3 and H3K4me1 marks in mouse liver using ChIP-Seq and RNA-Seq. We found that in mice, the liver carries embryonic stem cell-related functions while the embryonic stem cell also carries liver-related functions. We also identified novel genes in RNA-Seq experiments for mouse liver and for mouse embryonic stem cells. These genes are not currently in the Ensemble gene database at NCBI.
PMCID: PMC3921077  PMID: 24526835
gene expression; gene ontology; ChIP-Seq; RNA-Seq; H3K4me3; H3K4me1
13.  Central Role of the PPARγ Gene Network in Coordinating Beef Cattle Intramuscular Adipogenesis in Response to Weaning Age and Nutrition 
Adipogenic/lipogenic transcriptional networks regulating intramuscular fat deposition (IMF) in response to weaning age and dietary starch level were studied. The longissimus muscle (LM) of beef steers on an early weaning (141 days age) plus high-starch diet (EWS) or a normal weaning (NW, 222 days age) plus starch creep-feed diet (CFS) was biopsied at 0 (EW), 25, 50, 96 (NW), 167, and 222 (pre-slaughter) days. Expression patterns of 35 target genes were studied. From NW through slaughter, all steers received the same high-starch diet. In EWS steers the expression of PPARG, other adipogenic (CEBPA, ZFP423) and lipogenic (THRSP, SREBF1, INSIG1) activators, and several enzymes (FASN, SCD, ELOVL6, PCK1, DGAT2) that participate in the process of IMF increased gradually to a peak between 96 and 167 days on treatment. Steers in NW did not achieve similar expression levels even by 222 days on treatment, suggesting a blunted response even when fed a high-starch diet after weaning. High-starch feeding at an early age (EWS) triggers precocious and sustained adipogenesis, resulting in greater marbling.
PMCID: PMC3894150  PMID: 24516329
adipogenesis; nutrition; transcriptomics; marbling; weaning
14.  Innate Immunity Interactome Dynamics 
Innate immune response involves protein–protein interactions, deoxyribonucleic acid (DNA)–protein interactions and signaling cascades. So far, thousands of protein–protein interactions have been curated as a static interaction map. However, protein–protein interactions involved in innate immune response are dynamic. We recorded the dynamics in the interactome during innate immune response by combining gene expression data of lipopolysaccharide (LPS)-stimulated dendritic cells with protein–protein interactions data. We identified the differences in interactome during innate immune response by constructing differential networks and identifying protein modules, which were up-/down-regulated at each stage during the innate immune response. For each protein complex, we identified enriched biological processes and pathways. In addition, we identified core interactions that are conserved throughout the innate immune response and their enriched gene ontology terms and pathways. We defined two novel measures to assess the differences between network maps at different time points. We found that the protein interaction network at 1 hour after LPS stimulation has the highest interactions protein ratio, which indicates a role for proteins with large number of interactions in innate immune response. A pairwise differential matrix allows for the global visualization of the differences between different networks. We investigated the toll-like receptor subnetwork and found that S100A8 is down-regulated in dendritic cells after LPS stimulation. Identified protein complexes have a crucial role not only in innate immunity, but also in circadian rhythms, pathways involved in cancer, and p53 pathways. The study confirmed previous work that reported a strong correlation between cancer and immunity.
PMCID: PMC3885269  PMID: 24453478
innate immunity; protein-protein interactions; gene expression; differential networks; interactome dynamics
15.  Pathway Analysis of ChIP-Seq-Based NRF1 Target Genes Suggests a Logical Hypothesis of their Involvement in the Pathogenesis of Neurodegenerative Diseases 
Nuclear respiratory factor 1 (NRF1) serves as a transcription factor that activates the expression of a wide range of nuclear genes essential for mitochondrial biogenesis and function, including mitochondrial respiratory complex subunits, heme biosynthetic enzymes, and regulatory factors involved in the replication and transcription of mitochondrial DNA. Increasing evidence indicates that mitochondrial function is severely compromised in the brains of aging-related neurodegenerative diseases. To identify the comprehensive set of human NRF1 target genes potentially relevant to the pathogenesis of neurodegenerative diseases, we analyzed the NRF1 chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) dataset retrieved from the Encyclopedia of DNA Elements (ENCODE) project. Overall, we identified 2,470 highly stringent ChIP-Seq peaks on protein-coding genes in SK-N-SH human neuroblastoma cells. They were accumulated in the proximal promoter regions with an existence of the NRF1-binding consensus sequence. The set of ChIP-Seq-based NRF1 target genes included known NRF1 targets such as EIF2S1, EIF2S2, CYCS, FMR1, FXR2, E2F6, CD47, and TOMM34. By pathway analysis, the molecules located in the core pathways related to mitochondrial respiratory function were determined to be highly enriched in NRF1 target genes. Furthermore, we found that NRF1 target genes play a pivotal role in regulation of extra-mitochondrial biological processes, including RNA metabolism, splicing, cell cycle, DNA damage repair, protein translation initiation, and ubiquitin-mediated protein degradation. We identified a panel of neurodegenerative disease-related genes, such as PARK2 (Parkin), PARK6 (Pink1), PARK7 (DJ-1), and PAELR (GPR37) for Parkinson’s disease, as well as PSENEN (Pen2) and MAPT (tau) for Alzheimer’s disease, as previously unrecognized NRF1 targets. These results suggest a logical hypothesis that aberrant regulation of NRF1 and its targets might contribute to the pathogenesis of human neurodegenerative diseases via perturbation of diverse mitochondrial and extra-mitochondrial functions.
PMCID: PMC3825669  PMID: 24250222
Alzheimer’s disease; binding sites; ChIP-Seq; GenomeJack; NRF1; Parkinson’s disease
16.  CEBPG Exhibits Allele-Specific Expression in Human Bronchial Epithelial Cells 
Inter-individual variation in CCAAT/enhancer binding protein gamma (CEBPG) transcript expression in normal human bronchial epithelial cells (NBEC) is associated with predisposition to lung cancer. We hypothesize that this inter-individual variation is in part explained by cis-acting genetic variation in CEBPG. To test this hypothesis we measured transcript expression derived from each parental copy of CEBPG (ie, allele-specific expression; ASE). There was a significant 2.9-fold higher cell cycle-specific variation in ASE of CEBPG rs2772 A compared to C allele (P < 0.001). In 20% of NBEC samples, CEBPG rs2772 A allele was expressed on average 2.10 fold greater than rs2772 C allele. These data support the hypothesis that genetic variation in linkage disequilibrium with rs2772 influences regulation of CEBPG transcript expression through a trans-effect downstream of RNA polymerase II transcription and confirm that cis-acting genetic variation contributes to inter-individual variation in CEBPG transcript expression in NBEC, which is associated with variation in lung cancer risk.
PMCID: PMC3712557  PMID: 23888109
allele-specific expression; CEBPG; normal bronchial epithelial cells; lung cancer; cystic fibrosis; emphysema; cell-cycle; proliferation; airway epithelium
17.  Dietary Lipid During Late-Pregnancy and Early-Lactation to Manipulate Metabolic and Inflammatory Gene Network Expression in Dairy Cattle Liver with a Focus on PPARs 
Polyunsaturated (PUFA) long-chain fatty acids (LCFAs) are more potent in eliciting molecular and tissue functional changes in monogastrics than saturated LCFA. From −21 through 10 days relative to parturition dairy cows were fed no supplemental LCFA (control), saturated LCFA (SFAT; mainly 16:0 and 18:0), or fish oil (FISH; high-PUFA). Twenty-seven genes were measured via quantitative RT-PCR in liver tissue on day −14 and day 10. Expression of nuclear receptor co-activators (CARM1, MED1), LCFA metabolism (ACSL1, SCD, ACOX1), and inflammation (IL6, TBK1, IKBKE) genes was lower with SFAT than control on day −14. Expression of SCD, however, was markedly lower with FISH than control or SFAT on both −14 and 10 days. FISH led to further decreases in expression on day 10 of LCFA metabolism (CD36, PLIN2, ACSL1, ACOX1), intracellular energy (UCP2, STK11, PRKAA1), de novo cholesterol synthesis (SREBF2), inflammation (IL6, TBK1, IKBKE), and nuclear receptor signaling genes (PPARD, MED1, NRIP1). No change in expression was observed for PPARA and RXRA. The increase of DGAT2, PLIN2, ACSL1, and ACOX1 on day 10 versus −14 in cows fed SFAT suggested upregulation of both beta-oxidation and lipid droplet (LD) formation. However, liver triacylglycerol concentration was similar among treatments. The hepatokine FGF21 and the gluconeogenic genes PC and PCK1 increased markedly on day 10 versus −14 only in controls. At the levels supplemented, the change in the profile of metabolic genes after parturition in cows fed saturated fat suggested a greater capacity for uptake of fatty acids and intracellular handling without excessive storage of LD.
PMCID: PMC3699062  PMID: 23825924
dairy cows; fat supplementation; hepatic gene network
18.  Investigation of dmyc Promoter and Regulatory Regions 
Products of the myc gene family integrate extracellular signals by modulating a wide range of their targets involved in cellular biogenesis and metabolism; the purpose of this integration is to regulate cell death, proliferation, and differentiation. However, understanding the regulation of myc at the transcription level remains a challenge. We performed rapid amplification of dmyc cDNA ends (5′ RACE) and mapped the transcription start site at P1 promoter, 18 base pairs upstream of the start of the known EST GM01143 and within the 5′ UTR. Our data show that the first TATA box, previously computationally predicted, is utilized to generate dmyc full length mRNA. The largest transcript contains all three exons, generated after the removal of the introns by constitutively regulated splicing events. Further investigation of Downstream Promoter Element (DPE) was achieved by studying lacZ reporter activity; investigation revealed that this element and its upstream cluster of binding sites are required for the dmyc intron 2 activity. These findings may provide valuable tools for further analysis of dmyc cis-elements.
PMCID: PMC3663572  PMID: 23761963
dmyc; Drosophila; 5′ RACE; RNA splicing; TATA-box; Downstream Promoter Element (DPE)
19.  Yin Yang 1 and Adipogenic Gene Network Expression in Longissimus Muscle of Beef Cattle in Response to Nutritional Management 
Among 36 differentially-expressed genes during growth in longissimus muscle (LM) of Angus steers, Yin Yang 1 (YY1) had the most relationships with other genes including some associated with adipocyte differentiation. The objective of this study was to examine the effect of nutritional management on mRNA expression of YY1 along with its targets genes PPARG, GTF2B, KAT2B, IGFBP5 and STAT5B. Longissimus from Angus and Angus × Simmental steers (7 total/treatment) on early weaning plus high-starch (EWS), normal weaning plus starch creep feeding (NWS), or normal weaning without starch creep feeding (NWN) was biopsied at 0, 96, and 240 days on treatments. Results suggest that YY1 does not exert control of adipogenesis in LM, and its expression is not sensitive to weaning age. Among the YY1-related genes, EWS led to greater IGFBP5 during growing and finishing phases. Pro-adipogenic transcriptional regulation was detected in EWS due to greater PPARG and VDR at 96 and 240 d vs. 0 d. GTF2B and KAT2B expression was lower in response to NWS and EWS than NWN, and was most pronounced at 240 d. The increase in PPARG and GTF2B expression between 96 and 240 d underscored the existence of a molecular programming mechanism that was sensitive to age and dietary starch. Such response partly explains the greater carcass fat deposition observed in response to NWS.
PMCID: PMC3653888  PMID: 23700364
early weaning; gene expression; adipogenesis; nutrition; Yin Yang 1; steers
20.  A Comprehensive Profile of ChIP-Seq-Based STAT1 Target Genes Suggests the Complexity of STAT1-Mediated Gene Regulatory Mechanisms 
Interferon-gamma (IFNγ) plays a key role in macrophage activation, T helper and regulatory cell differentiation, defense against intracellular pathogens, tissue remodeling, and tumor surveillance. The diverse biological functions of IFNγ are mediated by direct activation of signal transducer and activator of transcription 1 (STAT1) as well as numerous downstream effector genes. Because a perturbation in STAT1 target gene networks is closely associated with development of autoimmune diseases and cancers, it is important to characterize the global picture of these networks. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) provides a highly efficient method for genome-wide profiling of DNA-binding proteins. We analyzed the STAT1 ChIP-Seq dataset of IFNγ-stimulated HeLa S3 cells derived from the ENCODE project, along with transcriptome analysis on microarray. We identified 1,441 stringent ChIP-Seq peaks of protein-coding genes. They were located in the promoter (21.5%) and more often in intronic regions (72.2%) with an existence of IFNγ-activated site (GAS) elements. Among the 1,441 STAT1 target genes, 212 genes are known IFN-regulated genes (IRGs) and 194 genes (13.5%) are actually upregulated in response to IFNγ by transcriptome analysis. The panel of upregulated genes constituted IFN-signaling molecular networks pivotal for host defense against infections, where interferon-regulatory factor (IRF) and STAT transcription factors serve as a hub on which biologically important molecular connections concentrate. The genes with the peak location in intronic regions showed significantly lower expression levels in response to IFNγ. These results indicate that the binding of STAT1 to GAS is not sufficient to fully activate target genes, suggesting the high complexity of STAT1-mediated gene regulatory mechanisms.
PMCID: PMC3623615  PMID: 23645984
binding sites; ChIP-seq; GenomeJack; interferon-gamma; STAT1
21.  Butyrate Induced IGF2 Activation Correlated with Distinct Chromatin Signatures Due to Histone Modification 
Histone modification has emerged as a very important mechanism regulating the transcriptional status of the genome. Insulin-like growth factor 2 (IGF2) is a peptide hormone controlling various cellular processes, including proliferation and apoptosis. H19 gene is closely linked to IGF2 gene, and IGF2 and H19 are reciprocally regulated imprinted genes. The epigenetic signature of H19 promoter (hypermethylation) on the paternal allele plays a vital role in allowing the expression of the paternal allele of IGF2.46 Our previous studies demonstrate that butyrate regulates the expression of IGF2 as well as genes encoding IGF Binding proteins. To obtain further understanding of histone modification and its regulatory potentials in controlling IGF2/H19 gene expression, we investigated the histone modification status of some key histones associated with the expression of IGF2/H19 genes in bovine cells using RNA-seq in combination with Chip-seq technology. A high-resolution map of the major chromatin modification at the IGF2/H19 locus induced by butyrate was constructed to illustrate the fundamental association of the chromatin modification landscape that may play a role in the activation of the IGF2 gene. High-definition epigenomic landscape mapping revealed that IGF2 and H19 have distinct chromatin modification patterns at their coding and promoter regions, such as TSSs and TTSs. Moreover, the correlation between the differentially methylated regions (DMRs) of IGF2/H19 locus and histone modification (acetylation and methylation) indicated that epigenetic signatures/markers of DNA methylation, histone methylation and histone acetylation were differentially distributed on the expressed IGF2 and silenced H19 genes. Our evidence also suggests that butyrate-induced regional changes of histone acetylation statusin the upstream regulation domain of H19 may be related to the reduced expression of H19 and strong activation of IGF2. Our results provided insights into the mechanism of butyrate-induced loss of imprinting (LOI) of IGF2 and regulation of gene expression by histone modification.
PMCID: PMC3623616  PMID: 23645985
bovine; butyrate; ChIP-seq; chromatin; histone modification; IGF2
22.  Global Consensus Theorem and Self-Organized Criticality: Unifying Principles for Understanding Self-Organization, Swarm Intelligence and Mechanisms of Carcinogenesis 
Complex biological systems manifest a large variety of emergent phenomena among which prominent roles belong to self-organization and swarm intelligence. Generally, each level in a biological hierarchy possesses its own systemic properties and requires its own way of observation, conceptualization, and modeling. In this work, an attempt is made to outline general guiding principles in exploration of a wide range of seemingly dissimilar phenomena observed in large communities of individuals devoid of any personal intelligence and interacting with each other through simple stimulus-response rules. Mathematically, these guiding principles are well captured by the Global Consensus Theorem (GCT) equally applicable to neural networks and to Lotka-Volterra population dynamics. Universality of the mechanistic principles outlined by GCT allows for a unified approach to such diverse systems as biological networks, communities of social insects, robotic communities, microbial communities, communities of somatic cells, social networks and many other systems. Another cluster of universal laws governing the self-organization in large communities of locally interacting individuals is built around the principle of self-organized criticality (SOC). The GCT and SOC, separately or in combination, provide a conceptual basis for understanding the phenomena of self-organization occurring in large communities without involvement of a supervisory authority, without system-wide informational infrastructure, and without mapping of general plan of action onto cognitive/behavioral faculties of its individual members. Cancer onset and proliferation serves as an important example of application of these conceptual approaches. In this paper, the point of view is put forward that apparently irreconcilable contradictions between two opposing theories of carcinogenesis, that is, the Somatic Mutation Theory and the Tissue Organization Field Theory, may be resolved using the systemic approaches provided by GST and SOC.
PMCID: PMC3583443  PMID: 23471309
nonlinear dynamics; global consensus theorem; swarm intelligence; self-organized criticality; Lotka-Volterra population dynamics; neural networks; biomolecular networks; carcinogenesis; darwinian evolution
23.  Implications of Systemic Dysfunction for the Etiology of Malignancy 
The current approach to treatment in oncology is to replace the generally cytotoxic chemotherapies with pharmaceutical treatment which inactivates specific molecular targets associated with cancer development and progression. The goal is to limit cellular damage to pathways perceived to be directly responsible for the malignancy. Its underlying assumptions are twofold: (1) that individual pathways are the cause of malignancy; and (2) that the treatment objective should be destruction—either of the tumor or the dysfunctional pathway. However, the extent to which data actually support these assumptions has not been directly addressed. Accumulating evidence suggests that systemic dysfunction precedes the disruption of specific genetic/molecular pathways in most adult cancers and that targeted treatments such as kinase inhibitors may successfully treat one pathway while generating unintended changes to other, non-targeted pathways. This article discusses (1) the systemic basis of malignancy; (2) better profiling of pre-cancerous biomarkers associated with elevated risk so that preventive lifestyle modifications can be instituted early to revert high-risk epigenetic changes before tumors develop; (3) a treatment emphasis in early stage tumors that would target the restoration of systemic balance by strengthening the body’s innate defense mechanisms; and (4) establishing better quantitative models of systems to capture adequate complexity for predictability at all stages of tumor progression.
PMCID: PMC3572920  PMID: 23440603
targeted therapy; complex systems; quantitative modeling; tumor microenvironment
24.  Extensive Ethnogenomic Diversity of Endothelial Nitric Oxide Synthase (eNOS) Polymorphisms 
Nitric oxide (NO) is highly reactive, produced in endothelial cells by endothelial NO synthase (eNOS) and has been implicated in sickle cell pathophysiology. We evaluated the distribution of functionally significant eNOS variants (the T786C variant in the promoter region, the Glu298Asp variant in exon 7, and the variable number of tandem repeats (VNTR) in intron 4) in Africans, African Americans and Caucasians. The C-786 variant was more common in Caucasians than in Africans and African Americans. Consistent with other findings, the Asp-298 variant had the highest frequency in Caucasians followed by African Americans, but was completely absent in Africans. The very rare intron 4 allele, eNOS 4c, was found in some Africans and African Americans, but not in Caucasians. eNOS 4d allele was present in 2 Africans. These findings suggest a consistent and widespread genomic diversity in the distribution of eNOS variants in Africans, comparative to African Americans and Caucasians.
PMCID: PMC3562081  PMID: 23400313
polymorphisms; ethnicity; endothelial nitric oxide synthase; haplotypes
25.  Effects of High Fat Feeding on Liver Gene Expression in Diabetic Goto-Kakizaki Rats 
Effects of high fat diet (HFD) on obesity and, subsequently, on diabetes are highly variable and modulated by genetics in both humans and rodents. In this report, we characterized the response of Goto-Kakizaki (GK) rats, a spontaneous polygenic model for lean diabetes and healthy Wistar-Kyoto (WKY) controls, to high fat feeding from weaning to 20 weeks of age. Animals fed either normal diet or HFD were sacrificed at 4, 8, 12, 16 and 20 weeks of age and a wide array of physiological measurements were made along with gene expression profiling using Affymetrix gene array chips. Mining of the microarray data identified differentially regulated genes (involved in inflammation, metabolism, transcription regulation, and signaling) in diabetic animals, as well as the response of both strains to HFD. Functional annotation suggested that HFD increased inflammatory differences between the two strains. Chronic inflammation driven by heightened innate immune response was identified to be present in GK animals regardless of diet. In addition, compensatory mechanisms by which WKY animals on HFD resisted the development of diabetes were identified, thus illustrating the complexity of diabetes disease progression.
PMCID: PMC3516129  PMID: 23236253
diabetes; high fat diet; gene expression; microarray

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