Gestational SARS-CoV-2 infection is associated with placental expression of immune and trophoblast genes.

INTRODUCTION: Maternal SARS-CoV-2 infection during pregnancy is associated with adverse pregnancy outcomes and can have effects on the placenta, even in the absence of severe disease or vertical transmission to the fetus. This study aimed to evaluate histopathologic and molecular effects in the placenta after SARS-CoV-2 infection during pregnancy. METHODS: We performed a study of 45 pregnant participants from the Generation C prospective cohort study at the Mount Sinai Health System in New York City. We compared histologic features and the expression of 48 immune and trophoblast genes in placentas delivered from 15 SARS-CoV-2 IgG antibody positive and 30 IgG SARS-CoV-2 antibody negative mothers. Statistical analyses were performed using Fisher's exact tests, Spearman correlations and linear regression models. RESULTS: The median gestational age at the time of SARS-CoV-2 IgG serology test was 35 weeks. Two of the IgG positive participants also had a positive RT-PCR nasal swab at delivery. 82.2% of the infants were delivered at term (≥37 weeks), and gestational age at delivery did not differ between the SARS-CoV-2 antibody positive and negative groups. No significant differences were detected between the groups in placental histopathology features. Differential expression analyses revealed decreased expression of two trophoblast genes (PSG3 and CGB3) and increased expression of three immune genes (CXCL10, TLR3 and DDX58) in placentas delivered from SARS-CoV-2 IgG positive participants. DISCUSSION: SARS-CoV-2 infection during pregnancy is associated with gene expression changes of immune and trophoblast genes in the placenta at birth which could potentially contribute to long-term health effects in the offspring.

Histology and Transcriptome Profiles of the Mammary Gland across Critical Windows of Development in Sprague Dawley Rats.

Breast development occurs through well-defined stages representing 'windows of susceptibility' to adverse environmental exposures that potentially modify breast cancer risk. Systematic characterization of morphology and transcriptome during normal breast development lays the foundation of our understanding of cancer etiology. We examined mammary glands in female Sprague Dawley rats across six developmental stages - pre-pubertal, peri-pubertal, pubertal, lactation, adult parous and adult nulliparous. We investigated histology by Hematoxylin and Eosin and Mallory's Trichrome stain, proliferative and apoptotic rate by immunohistochemistry and whole-transcriptome by microarrays. We identified differentially expressed genes between adjacent developmental stages by linear models, underlying pathways by gene ontology analysis and gene networks and hubs active across developmental stages by coexpression network analysis. Mammary gland development was associated with large-scale changes in the transcriptome; particularly from pre-pubertal to peri-pubertal period and the lactation period were characterized by distinct patterns of gene expression with unique biological functions such as immune processes during pre-pubertal development and cholesterol biosynthesis during lactation. These changes were reflective of the shift in mammary gland histology, from a rudimentary organ during early stages to a secretory organ during lactation followed by regression with age. Hub genes within mammary gene networks included metabolic genes such as Pparg during the pre-pubertal stage and tight junction-related genes claudins and occludins in lactating mammary glands. Transcriptome profile paired with histology enhanced our understanding of mammary development, which is fundamental in understanding the etiologic mechanism of breast cancer, especially pertaining to windows of susceptibility to environmental exposures that may alter breast cancer risk.

Novel Predictors of Breast Cancer Survival Derived from miRNA Activity Analysis.

Purpose: Breast cancer is among the leading causes of cancer-related death; discovery of novel prognostic markers is needed to improve outcomes. Combining systems biology and epidemiology, we investigated miRNA-associated genes and breast cancer survival in a well-characterized population-based study.Experimental Design: A recently developed algorithm, ActMiR, was used to identify key miRNA "activities" corresponding to target gene degradation, which were predictive of breast cancer mortality in published databases. We profiled miRNA-associated genes in tumors from our well-characterized population-based cohort of 606 women with first primary breast cancer. Cox proportional hazards models were used to estimate HRs and 95% confidence intervals (CI), after 15+ years of follow-up with 119 breast cancer-specific deaths.Results: miR-500a activity was identified as a key miRNA for estrogen receptor-positive breast cancer mortality using public databases. From a panel of 161 miR-500a-associated genes profiled, 73 were significantly associated with breast cancer-specific mortality (FDR < 0.05) in our population, among which two clusters were observed to have opposing directions of association. For example, high level of SUSD3 was associated with reduced breast cancer-specific mortality (HR = 0.3; 95% CI, 0.2-0.4), whereas the opposite was observed for TPX2 (HR = 2.7; 95% CI, 1.8-3.9). Most importantly, we identified set of genes for which associations with breast cancer-specific mortality were independent of known prognostic factors, including hormone receptor status and PAM50-derived risk-of-recurrence scores. These results are validated in independent datasets.Conclusions: We identified novel markers that may improve prognostic efficiency while shedding light on molecular mechanisms of breast cancer progression. Clin Cancer Res; 24(3); 581-91. ©2017 AACR.

Changes in mammary histology and transcriptome profiles by low-dose exposure to environmental phenols at critical windows of development.

Exposure to environmental chemicals has been linked to altered mammary development and cancer risk at high doses using animal models. Effects at low doses comparable to human exposure remain poorly understood, especially during critical developmental windows. We investigated the effects of two environmental phenols commonly used in personal care products - methyl paraben (MPB) and triclosan (TCS) - on the histology and transcriptome of normal mammary glands at low doses mimicking human exposure during critical windows of development. Sprague-Dawley rats were exposed during perinatal, prepubertal and pubertal windows, as well as from birth to lactation. Low-dose exposure to MPB and TCS induced measurable changes in both mammary histology (by Masson's Trichrome Stain) and transcriptome (by microarrays) in a window-specific fashion. Puberty represented a window of heightened sensitivity to MPB, with increased glandular tissue and changes of expression in 295 genes with significant enrichment in functions such as DNA replication and cell cycle regulation. Long-term exposure to TCS from birth to lactation was associated with increased adipose and reduced glandular and secretory tissue, with expression alterations in 993 genes enriched in pathways such as cholesterol synthesis and adipogenesis. Finally, enrichment analyses revealed that genes modified by MPB and TCS were over-represented in human breast cancer gene signatures, suggesting possible links with breast carcinogenesis. These findings highlight the issues of critical windows of susceptibility that may confer heightened sensitivity to environmental insults and implicate the potential health effects of these ubiquitous environmental chemicals in breast cancer.

Enlarged leukocyte referent libraries can explain additional variance in blood-based epigenome-wide association studies.

AIM: We examined whether variation in blood-based epigenome-wide association studies could be more completely explained by augmenting existing reference DNA methylation libraries. MATERIALS & METHODS: We compared existing and enhanced libraries in predicting variability in three publicly available 450K methylation datasets that collected whole-blood samples. Models were fit separately to each CpG site and used to estimate the additional variability when adjustments for cell composition were made with each library. RESULTS: Calculation of the mean difference in the CpG-specific residual sums of squares error between models for an arthritis, aging and metabolic syndrome dataset, indicated that an enhanced library explained significantly more variation across all three datasets (p < 10(-3)). CONCLUSION: Pathologically important immune cell subtypes can explain important variability in epigenome-wide association studies done in blood.

Prenatal Exposure to Organophosphorous Pesticides and Fetal Growth: Pooled Results from Four Longitudinal Birth Cohort Studies.

BACKGROUND: Organophosphorous (OP) pesticides are associated with reduced fetal growth in animals, but human studies are inconsistent. OBJECTIVES: We pooled data from four cohorts to examine associations of prenatal OP exposure with birth weight (n = 1,169), length (n = 1,152), and head circumference (n = 1,143). METHODS: Data were from the CHAMACOS, HOME, Columbia, and Mount Sinai birth cohorts. Concentrations of three diethyl phosphate (ΣDEP) and three dimethyl phosphate (ΣDMP) metabolites of OP pesticides [summed to six dialkyl phosphates (ΣDAPs)] were measured in maternal urine. Linear regression and mixed-effects models were used to examine associations with birth outcomes. RESULTS: We found no significant associations of ΣDEP, ΣDMP, or ΣDAPs with birth weight, length, or head circumference overall. However, among non-Hispanic black women, increasing urinary ΣDAP and ΣDMP concentrations were associated with decreased birth length (ß = -0.4 cm; 95% CI: -0.9, 0.0 and ß = -0.4 cm; 95% CI: -0.8, 0.0, respectively, for each 10-fold increase in metabolite concentration). Among infants with the PON1192RR genotype, ΣDAP and ΣDMP were negatively associated with length (ß = -0.4 cm; 95% CI: -0.9, 0.0 and ß = -0.5 cm; 95% CI: -0.9, -0.1). CONCLUSIONS: This study confirms previously reported associations of prenatal OP exposure among black women with decreased infant size at birth, but finds no evidence of smaller birth weight, length, or head circumference among whites or Hispanics. Contrary to our hypothesis, we found stronger inverse associations of DAPs and birth outcome in infants with the less susceptible PON1192RR genotype. The large pooled data set facilitated exploration of interactions by race/ethnicity and PON1 genotype, but was limited by differences in study populations. CITATION: Harley KG, Engel SM, Vedar MG, Eskenazi B, Whyatt RM, Lanphear BP, Bradman A, Rauh VA, Yolton K, Hornung RW, Wetmur JG, Chen J, Holland NT, Barr DB, Perera FP, Wolff MS. 2016. Prenatal exposure to organophosphorous pesticides and fetal growth: pooled results from four longitudinal birth cohort studies. Environ Health Perspect 124:1084-1092; http://dx.doi.org/10.1289/ehp.1409362.

Prenatal Organophosphorus Pesticide Exposure and Child Neurodevelopment at 24 Months: An Analysis of Four Birth Cohorts.

BACKGROUND: Organophosphorus pesticides (OPs) are used in agriculture worldwide. Residential use was common in the United States before 2001. OBJECTIVES: We conducted a pooled analysis of four birth cohorts (children's centers; n = 936) to evaluate associations of prenatal exposure to OPs with child development at 24 months. METHODS: Using general linear models, we computed site-specific and pooled estimates of the association of total dialkyl (ΣDAP), diethyl (ΣDEP), and dimethylphosphate (ΣDMP) metabolite concentrations in maternal prenatal urine with mental and psychomotor development indices (MDI/PDI) and evaluated heterogeneity by children's center, race/ethnicity, and PON1 genotype. RESULTS: There was significant heterogeneity in the center-specific estimates of association for ΣDAP and ΣDMP and the MDI (p = 0.09, and p = 0.05, respectively), as well as heterogeneity in the race/ethnicity-specific estimates for ΣDAP (p = 0.06) and ΣDMP (p = 0.02) and the MDI. Strong MDI associations in the CHAMACOS population per 10-fold increase in ΣDAP (ß = -4.17; 95% CI: -7.00, -1.33) and ΣDMP (ß = -3.64; 95% CI: -5.97, -1.32) were influential, as were associations among Hispanics (ß per 10-fold increase in ΣDAP = -2.91; 95% CI: -4.71, -1.12). We generally found stronger negative associations of ΣDAP and ΣDEP with the 24-month MDI for carriers of the 192Q PON1 allele, particularly among blacks and Hispanics. CONCLUSIONS: Data pooling was complicated by center-related differences in subject characteristics, eligibility, and changes in regulations governing residential use of OPs during the study periods. Pooled summary estimates of prenatal exposure to OPs and neurodevelopment should be interpreted with caution because of significant heterogeneity in associations by center, race/ethnicity, and PON1 genotype. Subgroups with unique exposure profiles or susceptibilities may be at higher risk for adverse neurodevelopment following prenatal exposure. CITATION: Engel SM, Bradman A, Wolff MS, Rauh VA, Harley KG, Yang JH, Hoepner LA, Barr DB, Yolton K, Vedar MG, Xu Y, Hornung RW, Wetmur JG, Chen J, Holland NT, Perera FP, Whyatt RM, Lanphear BP, Eskenazi B. 2016. Prenatal organophosphorus pesticide exposure and child neurodevelopment at 24 months: an analysis of four birth cohorts. Environ Health Perspect 124:822-830; http://dx.doi.org/10.1289/ehp.1409474.

Human Dendritic Cell Response Signatures Distinguish 1918, Pandemic, and Seasonal H1N1 Influenza Viruses.

UNLABELLED: Influenza viruses continue to present global threats to human health. Antigenic drift and shift, genetic reassortment, and cross-species transmission generate new strains with differences in epidemiology and clinical severity. We compared the temporal transcriptional responses of human dendritic cells (DC) to infection with two pandemic (A/Brevig Mission/1/1918, A/California/4/2009) and two seasonal (A/New Caledonia/20/1999, A/Texas/36/1991) H1N1 influenza viruses. Strain-specific response differences included stronger activation of NF-κB following infection with A/New Caledonia/20/1999 and a unique cluster of genes expressed following infection with A/Brevig Mission/1/1918. A common antiviral program showing strain-specific timing was identified in the early DC response and found to correspond with reported transcript changes in blood during symptomatic human influenza virus infection. Comparison of the global responses to the seasonal and pandemic strains showed that a dramatic divergence occurred after 4 h, with only the seasonal strains inducing widespread mRNA loss. IMPORTANCE: Continuously evolving influenza viruses present a global threat to human health; however, these host responses display strain-dependent differences that are incompletely understood. Thus, we conducted a detailed comparative study assessing the immune responses of human DC to infection with two pandemic and two seasonal H1N1 influenza strains. We identified in the immune response to viral infection both common and strain-specific features. Among the stain-specific elements were a time shift of the interferon-stimulated gene response, selective induction of NF-κB signaling by one of the seasonal strains, and massive RNA degradation as early as 4 h postinfection by the seasonal, but not the pandemic, viruses. These findings illuminate new aspects of the distinct differences in the immune responses to pandemic and seasonal influenza viruses.

Mouse dendritic cell (DC) influenza virus infectivity is much lower than that for human DCs and is hemagglutinin subtype dependent.

We show that influenza A H1N1 virus infection leads to very low infectivity in mouse dendritic cells (DCs) in vitro compared with that in human DCs. This holds when H3 or H5 replaces H1 in recombinant viruses. Viruslike particles confirm the difference between mouse and human, suggesting that reduced virus entry contributes to lower mouse DC infectivity. Low infectivity of mouse DCs should be considered when they are used to study responses of DCs that are actually infected.

DNA methylation in peripheral blood measured by LUMA is associated with breast cancer in a population-based study.

Our purpose was to identify epigenetic markers of breast cancer risk, which can be reliably measured in peripheral blood and are amenable for large population screening. We used 2 independent assays, luminometric methylation assay (LUMA) and long interspersed elements-1 (LINE-1) to measure "global methylation content" in peripheral blood DNA from a well-characterized population-based case-control study. We examined associations between methylation levels and breast cancer risk among 1055 cases and 1101 controls and potential influences of 1-carbon metabolism on global methylation. Compared with women in the lowest quintile of LUMA methylation, those in the highest quintile had a 2.41-fold increased risk of breast cancer (95% confidence interval: 1.83-3.16; P, trend<0.0001). The association did not vary by other key tumor characteristics and lifestyle risk factors. Consistent with LUMA findings, genome-wide methylation profiling of a subset of samples revealed greater promoter hypermethylation in breast cancer case participants (P=0.04); higher LUMA was associated with higher promoter methylation in the controls (P=0.05). LUMA levels were also associated with functional sodium nitroprusside in key 1-carbon metabolizing genes, MTHFR C677T (P=0.001) and MTRR A66G (P=0.018). LINE-1 methylation was associated with neither breast cancer risk nor 1-carbon metabolism. Our results show that global promoter hypermethylation measured in peripheral blood was associated with breast cancer risk.

The influence of one-carbon metabolism on gene promoter methylation in a population-based breast cancer study.

Abnormal methylation in gene promoters is a hallmark of the cancer genome; however, factors that may influence promoter methylation have not been well elucidated. As the one-carbon metabolism pathway provides the universal methyl donor for methylation reactions, perturbation of this pathway might influence DNA methylation and, ultimately, affect gene functions. Utilizing approximately 800 breast cancer tumor tissues from a large population-based study, we investigated the relationships between dietary and genetic factors involved in the one-carbon metabolism pathway and promoter methylation of a panel of 13 breast cancer-related genes. We found that CCND2, HIN1 and CHD1 were the most "dietary sensitive" genes, as methylation of their promoters was associated with intakes of at least two out of the eight dietary methyl factors examined. On the other hand, some micronutrients (i.e., B 2 and B 6) were more "epigenetically active" as their intake levels correlated with promoter methylation status in 3 out of the 13 breast cancer genes evaluated. Both positive (hypermethylation) and inverse (hypomethylation) associations with high micronutrient intake were observed. Unlike what we saw for dietary factors, we did not observe any clear patterns between one-carbon genetic polymorphisms and the promoter methylation status of the genes examined. Our results provide preliminary evidence that one-carbon metabolism may have the capacity to influence the breast cancer epigenome. Given that epigenetic alterations are thought to occur early in cancer development and are potentially reversible, dietary modifications may offer promising venues for cancer intervention and prevention.

Differential methylation of imprinted genes in growth-restricted placentas.

A complex network of epigenetic factors participates in regulating the monoallelic expression of a small subset of genes (~1%) in the human genome. This phenomenon goes under the definition of genomic imprinting, a parent-of-origin effect that, when altered during early embryogenesis, may influence fetal development into adulthood. Pertubations in genomic imprinting have been associated with placental and fetal growth restrictions. We analyzed the differential DNA methylation of all known imprinted genes on 10 appropriate-for-gestational-age, clinically normal, placentas and 7 severe intrauterine growth-restricted placentas. Samples were pooled according to the diagnosis and analyzed by methylated DNA immunoprecipitation (MeDIP) on a tiling microarray platform. The distribution of the differentially methylated regions (DMRs) identified in growth-restricted placentas showed a slight tendency toward hypermethylation. Imprinted genes not expressed in placenta showed a unique DMR profile with the fewest hyper- and hypomethylated DMRs. Promoter and CpG island DMRs were sporadic and randomly distributed. The vast majority of DMR identified (~99%) were mapped in introns, showing no common sequence features. Also, by using the more advanced array data mining softwares, no significant patterns emerged. In contrast, differential methylation showed a highly significant correlation with gene length. Overall these data suggest that differential methylation changes in growth-restricted placentas occur throughout the genomic regions, encompassing genes actively expressed in the placenta. These findings warrant caution in interpreting the significance of genes carrying clustered DMRs because the distribution of DMRs in a gene may be attributed as a function of its length rather than as a specific biological role.

Prenatal exposure to organophosphates, paraoxonase 1, and cognitive development in childhood.

BACKGROUND: Prenatal exposure to organophosphate pesticides has been shown to negatively affect child neurobehavioral development. Paraoxonase 1 (PON1) is a key enzyme in the metabolism of organophosphates. OBJECTIVE: We examined the relationship between biomarkers of organophosphate exposure, PON1, and cognitive development at ages 12 and 24 months and 6-9 years. METHODS: The Mount Sinai Children's Environmental Health Study enrolled a multiethnic prenatal population in New York City between 1998 and 2002 (n = 404). Third-trimester maternal urine samples were collected and analyzed for organophosphate metabolites (n = 360). Prenatal maternal blood was analyzed for PON1 activity and genotype. Children returned for neurodevelopment assessments ages 12 months (n = 200), 24 months (n = 276), and 6-9 (n = 169) years of age. RESULTS: Prenatal total dialkylphosphate metabolite level was associated with a decrement in mental development at 12 months among blacks and Hispanics. These associations appeared to be enhanced among children of mothers who carried the PON1 Q192R QR/RR genotype. In later childhood, increasing prenatal total dialkyl- and dimethylphosphate metabolites were associated with decrements in perceptual reasoning in the maternal PON1 Q192R QQ genotype, which imparts slow catalytic activity for chlorpyrifos oxon, with a monotonic trend consistent with greater decrements with increasing prenatal exposure. CONCLUSION: Our findings suggest that prenatal exposure to organophosphates is negatively associated with cognitive development, particularly perceptual reasoning, with evidence of effects beginning at 12 months and continuing through early childhood. PON1 may be an important susceptibility factor for these deleterious effects.

Role of cell-to-cell variability in activating a positive feedback antiviral response in human dendritic cells.

In the first few hours following Newcastle disease viral infection of human monocyte-derived dendritic cells, the induction of IFNB1 is extremely low and the secreted type I interferon response is below the limits of ELISA assay. However, many interferon-induced genes are activated at this time, for example DDX58 (RIGI), which in response to viral RNA induces IFNB1. We investigated whether the early induction of IFNBI in only a small percentage of infected cells leads to low level IFN secretion that then induces IFN-responsive genes in all cells. We developed an agent-based mathematical model to explore the IFNBI and DDX58 temporal dynamics. Simulations showed that a small number of early responder cells provide a mechanism for efficient and controlled activation of the DDX58-IFNBI positive feedback loop. The model predicted distributions of single cell responses that were confirmed by single cell mRNA measurements. The results suggest that large cell-to-cell variation plays an important role in the early innate immune response, and that the variability is essential for the efficient activation of the IFNB1 based feedback loop.

Linking emulsion PCR haplotype analysis.

The experimental measurement of haplotype requires the determination of two or more genotypes on the same DNA molecule. Because such measurements are much more complicated than measurements of genotypes, haplotypes are typically inferred using population data for linkage disequilibrium between the markers of interest. We have developed a method for molecular haplotyping, linking emulsion PCR (LE-PCR), and have demonstrated that the method is sufficiently robust to determine haplotypes for multiple markers in a population setting. LE-PCR uses emulsion PCR to isolate single template molecules for simultaneous PCR of widely spaced markers and uses linking PCR to fuse these amplicons into one short amplicon, which maintains the phase of the markers. LE-PCR is illustrated for polymorphisms in human paraoxonase 1 (PON1) that have been shown to affect transcriptional activity and substrate specificity in the detoxification of organophosphates.

A common polymorphism in the caspase recruitment domain of RIG-I modifies the innate immune response of human dendritic cells.

Infection of human dendritic cells (DCs) by negative-strand RNA viruses, such as Newcastle disease virus, leads to the induction of the IFNbeta gene, IFNB1, through the activation of the RNA helicase RIG-I, which is encoded by DDX58. Expression levels of IFNB1 and DDX58 in infected DCs showed positive correlations at the population and the single-cell levels. DDX58 has a common and potentially functional single nucleotide polymorphism, rs10813831 (A/G), encoding an Arg7Cys amino acid change in the RIG-I protein caspase recruitment domain (CARD). Quantitative RT-PCR analysis on Newcastle disease virus-infected primary DCs from 130 individuals revealed a significant association of the Arg7Cys single nucleotide polymorphism with increased IFNB1 and DDX58 transcription. Allelic imbalance analysis ruled out allele-specific DDX58 message levels and suggested that the observed association between Arg7Cys and IFNB1 and DDX58 transcription originated from a functional change in RIG-I due to the amino acid substitution in the CARD. DDX58 transfection experiments in 293T cells confirmed a biological functional difference between RIG-I 7Cys and the more common RIG-I 7Arg. Taken together, these data indicate that the innate immune response to viral infection of human cells is modified by a functional polymorphism in the RIG-I CARD.

Genomic loss of imprinting in first-trimester human placenta.

OBJECTIVE: The purpose of this study was to investigate imprinting patterns in first-trimester human placentas. STUDY DESIGN: Using samples of 17 first-trimester and 14 term placentas from uncomplicated pregnancies, we assessed loss of imprinting (LOI) at the RNA level in a panel of 14 genes that are known to be imprinted in the placenta with the use of a quantitative allele-specific reverse transcriptase polymerase chain reaction analysis of those genes that contained readout single nucleotide polymorphisms in their transcripts. RESULTS: There is significant LOI (ie, biallelic expression) in all 14 genes in first-trimester placentas. LOI was more variable and generally at lower levels at term. Although there is little difference in gene expression, the level of LOI is higher in the first-trimester placentas, compared with term placentas. CONCLUSION: Genomic imprinting appears to be a dynamic maturational process across gestation in human placenta. In contrast with prevailing theories, epigenetic imprints may continue to evolve past 12 weeks of gestation.

Coregulation mapping based on individual phenotypic variation in response to virus infection.

BACKGROUND: Gene coregulation across a population is an important aspect of the considerable variability of the human immune response to virus infection. Methodology to investigate it must rely on a number of ingredients ranging from gene clustering to transcription factor enrichment analysis. RESULTS: We have developed a methodology to investigate the gene to gene correlations for the expression of 34 genes linked to the immune response of Newcastle Disease Virus (NDV) infected conventional dendritic cells (DCs) from 145 human donors. The levels of gene expression showed a large variation across individuals. We generated a map of gene co-expression using pairwise correlation and multidimensional scaling (MDS). The analysis of these data showed that among the 13 genes left after filtering for statistically significant variations, two clusters are formed. We investigated to what extent the observed correlation patterns can be explained by the sharing of transcription factors (TFs) controlling these genes. Our analysis showed that there was a significant positive correlation between MDS distances and TF sharing across all pairs of genes. We applied enrichment analysis to the TFs having binding sites in the promoter regions of those genes. This analysis, after Gene Ontology filtering, indicated the existence of two clusters of genes (CCL5, IFNA1, IFNA2, IFNB1) and (IKBKE, IL6, IRF7, MX1) that were transcriptionally co-regulated. In order to facilitate the use of our methodology by other researchers, we have also developed an interactive coregulation explorer web-based tool called CorEx. It permits the study of MDS and hierarchical clustering of data combined with TF enrichment analysis. We also offer web services that provide programmatic access to MDS, hierarchical clustering and TF enrichment analysis. CONCLUSIONS: MDS mapping based on correlation in conjunction with TF enrichment analysis represents a useful computational method to generate predictions underlying gene coregulation across a population.

Antiviral response dictated by choreographed cascade of transcription factors.

The dendritic cell (DC) is a master regulator of immune responses. Pathogenic viruses subvert normal immune function in DCs through the expression of immune antagonists. Understanding how these antagonists interact with the host immune system requires knowledge of the underlying genetic regulatory network that operates during an uninhibited antiviral response. To isolate and identify this network, we studied DCs infected with Newcastle disease virus, which is able to stimulate innate immunity and DC maturation through activation of RIG-I signaling, but lacks the ability to evade the human IFN response. To analyze this experimental model, we developed a new approach integrating genome-wide expression kinetics and time-dependent promoter analysis. We found that the genetic program underlying the antiviral cell-state transition during the first 18 h postinfection could be explained by a single convergent regulatory network. Gene expression changes were driven by a stepwise multifactor cascading control mechanism, where the specific transcription factors controlling expression changed over time. Within this network, most individual genes were regulated by multiple factors, indicating robustness against virus-encoded immune evasion genes. In addition to effectively recapitulating current biological knowledge, we predicted, and validated experimentally, antiviral roles for several novel transcription factors. More generally, our results show how a genetic program can be temporally controlled through a single regulatory network to achieve the large-scale genetic reprogramming characteristic of cell-state transitions.