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The high heritability of amyotrophic lateral sclerosis (ALS) contrasts with its low molecular diagnosis rate post-genetic testing, pointing to potential undiscovered genetic factors. To aid the exploration of these factors, we introduced EpiOut, an algorithm to identify chromatin accessibility outliers that are regions exhibiting divergent accessibility from the population baseline in a single or few samples. Annotation of accessible regions with histone chromatin immunoprecipitation sequencing and Hi-C indicates that outliers are concentrated in functional loci, especially among promoters interacting with active enhancers. Across different omics levels, outliers are robustly replicated, and chromatin accessibility outliers are reliable predictors of gene expression outliers and aberrant protein levels. When promoter accessibility does not align with gene expression, our results indicate that molecular aberrations are more likely to be linked to post-transcriptional regulation rather than transcriptional regulation. Our findings demonstrate that the outlier detection paradigm can uncover dysregulated regions in rare diseases. EpiOut is available at github.com/uci-cbcl/EpiOut.

Acinetobacter baumannii causes severe infections in humans, resists multiple antibiotics, and survives in stressful environmental conditions due to modulations of its complex transcriptional regulatory network (TRN). Unfortunately, our global understanding of the TRN in this emerging opportunistic pathogen is limited. Here, we apply independent component analysis, an unsupervised machine learning method, to a compendium of 139 RNA-seq data sets of three multidrug-resistant A. baumannii international clonal complex I strains (AB5075, AYE, and AB0057). This analysis allows us to define 49 independently modulated gene sets, which we call iModulons. Analysis of the identified A. baumannii iModulons reveals validating parallels to previously defined biological operons/regulons and provides a framework for defining unknown regulons. By utilizing the iModulons, we uncover potential mechanisms for a RpoS-independent general stress response, define global stress-virulence trade-offs, and identify conditions that may induce plasmid-borne multidrug resistance. The iModulons provide a model of the TRN that emphasizes the importance of transcriptional regulation of virulence phenotypes in A. baumannii. Furthermore, they suggest the possibility of future interventions to guide gene expression toward diminished pathogenic potential.IMPORTANCEThe rise in hospital outbreaks of multidrug-resistant Acinetobacter baumannii infections underscores the urgent need for alternatives to traditional broad-spectrum antibiotic therapies. The success of A. baumannii as a significant nosocomial pathogen is largely attributed to its ability to resist antibiotics and survive environmental stressors. However, there is limited literature available on the global, complex regulatory circuitry that shapes these phenotypes. Computational tools that can assist in the elucidation of A. baumanniis transcriptional regulatory network architecture can provide much-needed context for a comprehensive understanding of pathogenesis and virulence, as well as for the development of targeted therapies that modulate these pathways.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

Transcription factor programs mediating the immune response to coronavirus disease 2019 (COVID-19) are not fully understood. Capturing active transcription initiation from cis-regulatory elements such as enhancers and promoters by capped small RNA sequencing (csRNA-seq), in contrast to capturing steady-state transcripts by conventional RNA-seq, allows unbiased identification of the underlying transcription factor activity and regulatory pathways. Here, we profile transcription initiation in critically ill COVID-19 patients, identifying transcription factor motifs that correlate with clinical lung injury and disease severity. Unbiased clustering reveals distinct subsets of cis-regulatory elements that delineate the cell type, pathway-specific, and combinatorial transcription factor activity. We find evidence of critical roles of regulatory networks, showing that STAT/BCL6 and E2F/MYB regulatory programs from myeloid cell populations are activated in patients with poor disease outcomes and associated with COVID-19 susceptibility genetic variants. More broadly, we demonstrate how capturing acute, disease-mediated changes in transcription initiation can provide insight into the underlying molecular mechanisms and stratify patient disease severity.

BackgroundInfection by coronavirus SARS-CoV2 is a severe and often deadly disease that has implications for the respiratory system and multiple organs across the human body. While the effects in the lung have been extensively studied, less is known about the impact COVID-19 has across other organs.MethodsHere, we contribute a single-nuclei RNA-sequencing atlas comprising six human organs across 20 autopsies where we analyzed the transcriptional changes due to COVID-19 in multiple cell types. The integration of data from multiple organs enabled the identification of systemic transcriptional changes.ResultsComputational cross-organ analysis for endothelial cells and macrophages identified systemic transcriptional changes in these cell types in COVID-19 samples. In addition, analysis of gene modules showed enrichment of specific signaling pathways across multiple organs in COVID-19 autopsies.ConclusionsAltogether, the COVID Tissue Atlas enables the investigation of both cell type-specific and cross-organ transcriptional responses to COVID-19, providing insights into the molecular networks affected by the disease and highlighting novel potential targets for therapies and drug development.FundingThe Chan-Zuckerberg Initiative, The Chan-Zuckerberg Biohub.

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi+ RRE+), 3' defective (Psi+ RRE-), and 5' defective (Psi- RRE+) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4+ T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/106 cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/106 cells; 5.6%) or intact (0.6 copies/106 cells;

Eukaryotic genomes are structurally organized via the formation of multiple loops that create gene expression regulatory units called topologically associating domains (TADs). Here we revealed the KSHV TAD structure at 500 bp resolution and constructed a 3D KSHV genomic structural model with 2 kb binning. The latent KSHV genome formed very similar genomic architectures in three different naturally infected PEL cell lines and in an experimentally infected epithelial cell line. The majority of the TAD boundaries were occupied by structural maintenance of chromosomes (SMC1) cohesin complex and CCCTC-binding factor (CTCF), and the KSHV transactivator was recruited to those sites during reactivation. Triggering KSHV gene expression decreased prewired genomic loops within the regulatory unit, while contacts extending outside of regulatory borders increased, leading to formation of a larger regulatory unit with a shift from repressive to active compartments (B to A). The 3D genomic structural model proposes that the immediate early promoter region is localized on the periphery of the 3D viral genome during latency, while highly inducible noncoding RNA regions moved toward the inner space of the structure, resembling the configuration of a "bird cage" during reactivation. The compartment-like properties of viral episomal chromatin structure and its reorganization during the transition from latency may help facilitate viral gene transcription. IMPORTANCE The 3D architecture of chromatin allows for efficient arrangement, expression, and replication of genetic material. The genomes of all organisms studied to date have been found to be organized through some form of tiered domain structures. However, the architectural framework of the genomes of large double-stranded DNA viruses such as the herpesvirus family has not been reported. Prior studies with Kaposi's sarcoma-associated herpesvirus (KSHV) have indicated that the viral chromatin shares many biological properties exhibited by the host cell genome, essentially behaving as a mini human chromosome. Thus, we hypothesized that the KSHV genome may be organized in a similar manner. In this report, we describe the domain structure of the latent and lytic KSHV genome at 500 bp resolution and present a 3D genomic structural model for KSHV under each condition. These results add new insights into the complex regulation of the viral life cycle.

Human infections with methicillin-resistant Staphylococcus aureus (MRSA) are commonly treated with vancomycin, and strains with decreased susceptibility, designated as vancomycin-intermediate S. aureus (VISA), are associated with treatment failure. Here, we profiled the phenotypic, mutational, and transcriptional landscape of 10 VISA strains adapted by laboratory evolution from one common MRSA ancestor, the USA300 strain JE2. Using functional and independent component analysis, we found that: 1) despite the common genetic background and environmental conditions, the mutational landscape diverged between evolved strains and included mutations previously associated with vancomycin resistance (in vraT, graS, vraFG, walKR, and rpoBCD) as well as novel adaptive mutations (SAUSA300_RS04225, ssaA, pitAR, and sagB); 2) the first wave of mutations affected transcriptional regulators and the second affected genes involved in membrane biosynthesis; 3) expression profiles were predominantly strain-specific except for sceD and lukG, which were the only two genes significantly differentially expressed in all clones; 4) three independent virulence systems (φSa3, SaeR, and T7SS) featured as the most transcriptionally perturbed gene sets across clones; 5) there was a striking variation in oxacillin susceptibility across the evolved lineages (from a 10-fold increase to a 63-fold decrease) that also arose in clinical MRSA isolates exposed to vancomycin and correlated with susceptibility to teichoic acid inhibitors; and 6) constitutive expression of the VraR regulon explained cross-susceptibility, while mutations in walK were associated with cross-resistance. Our results show that adaptation to vancomycin involves a surprising breadth of mutational and transcriptional pathways that affect antibiotic susceptibility and possibly the clinical outcome of infections.

In vitro antibiotic susceptibility testing often fails to accurately predict in vivo drug efficacies, in part due to differences in the molecular composition between standardized bacteriologic media and physiological environments within the body. Here, we investigate the interrelationship between antibiotic susceptibility and medium composition in Escherichia coli K-12 MG1655 as contextualized through machine learning of transcriptomics data. Application of independent component analysis, a signal separation algorithm, shows that complex phenotypic changes induced by environmental conditions or antibiotic treatment are directly traced to the action of a few key transcriptional regulators, including RpoS, Fur, and Fnr. Integrating machine learning results with biochemical knowledge of transcription factor activation reveals medium-dependent shifts in respiration and iron availability that drive differential antibiotic susceptibility. By extension, the data generation and data analytics workflow used here can interrogate the regulatory state of a pathogen under any measured condition and can be applied to any strain or organism for which sufficient transcriptomics data are available. IMPORTANCE Antibiotic resistance is an imminent threat to global health. Patient treatment regimens are often selected based on results from standardized antibiotic susceptibility testing (AST) in the clinical microbiology lab, but these in vitro tests frequently misclassify drug effectiveness due to their poor resemblance to actual host conditions. Prior attempts to understand the combined effects of drugs and media on antibiotic efficacy have focused on physiological measurements but have not linked treatment outcomes to transcriptional responses on a systems level. Here, application of machine learning to transcriptomics data identified medium-dependent responses in key regulators of bacterial iron uptake and respiratory activity. The analytical workflow presented here is scalable to additional organisms and conditions and could be used to improve clinical AST by identifying the key regulatory factors dictating antibiotic susceptibility.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

Background & aimsPancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy that invades surrounding structures and metastasizes rapidly. Although inflammation is associated with tumor formation and progression, little is known about the mechanisms of this connection. We investigate the effects of interleukin (IL) 22 in the development of pancreatic tumors in mice.MethodsWe performed studies with Pdx1-Cre;LSL-KrasG12D;Trp53+/-;Rosa26EYFP/+ (PKCY) mice, which develop pancreatic tumors, and PKCY mice with disruption of IL22 (PKCY Il22-/-mice). Pancreata were collected at different stages of tumor development and analyzed by immunohistochemistry, immunoblotting, real-time polymerase chain reaction, and flow cytometry. Some mice were given cerulean to induce pancreatitis. Pancreatic cancer cell lines (PD2560) were orthotopically injected into C57BL/6 mice or Il22-/-mice, and tumor development was monitored. Pancreatic cells were injected into the tail veins of mice, and lung metastases were quantified. Acini were collected from C57BL/6 mice and resected human pancreata and were cultured. Cell lines and acini cultures were incubated with IL22 and pharmacologic inhibitors, and protein levels were knocked down with small hairpin RNAs. We performed immunohistochemical analyses of 26 PDACs and 5 nonneoplastic pancreas specimens.ResultsWe observed increased expression of IL22 and the IL22 receptor (IL22R) in the pancreas compared with other tissues in mice; IL22 increased with pancreatitis and tumorigenesis. Flow cytometry indicated that the IL22 was produced primarily by T-helper 22 cells. PKCY Il22-/-mice did not develop precancerous lesions or pancreatic tumors. The addition of IL22 to cultured acinar cells increased their expression of markers of ductal metaplasia; these effects of IL22 were prevented with inhibitors of Janus kinase signaling to signal transducer and activator of transcription (STAT) (ruxolitinib) or mitogen-activated protein kinase kinase (MEK) (trametinib) and with STAT3 knockdown. Pancreatic cells injected into Il22-/- mice formed smaller tumors than those injected into C57BL/6. Incubation of IL22R-expressing PDAC cells with IL22 promoted spheroid formation and invasive activity, resulting in increased expression of stem-associated transcription factors (GATA4, SOX2, SOX17, and NANOG), and increased markers of the epithelial-mesenchymal transition (CDH1, SNAI2, TWIST1, and beta catenin); ruxolitinib blocked these effects. Human PDAC tissues had higher levels of IL22, phosphorylated STAT3, and markers of the epithelial-mesenchymal transition than nonneoplastic tissues. An increased level of STAT3 in IL22R-positive cells was associated with shorter survival times of patients.ConclusionsWe found levels of IL22 to be increased during pancreatitis and pancreatic tumor development and to be required for tumor development and progression in mice. IL22 promotes acinar to ductal metaplasia, stem cell features, and increased expression of markers of the epithelial-mesenchymal transition; inhibitors of STAT3 block these effects. Increased expression of IL22 by PDACs is associated with reduced survival times.

The laminar architecture of the mammalian neocortex depends on the orderly generation of distinct neuronal subtypes by apical radial glia (aRG) during embryogenesis. Here, we identify critical roles for the autism risk gene Foxp1 in maintaining aRG identity and gating the temporal competency for deep-layer neurogenesis. Early in development, aRG express high levels of Foxp1 mRNA and protein, which promote self-renewing cell divisions and deep-layer neuron production. Foxp1 levels subsequently decline during the transition to superficial-layer neurogenesis. Sustained Foxp1 expression impedes this transition, preserving a population of cells with aRG identity throughout development and extending the early neurogenic period into postnatal life. FOXP1 expression is further associated with the initial formation and expansion of basal RG (bRG) during human corticogenesis and can promote the formation of cells exhibiting characteristics of bRG when misexpressed in the mouse cortex. Together, these findings reveal broad functions for Foxp1 in cortical neurogenesis.

Candida albicans is a commensal member of the human microbiota that colonizes multiple niches in the body including the skin, oral cavity, and gastrointestinal and genitourinary tracts of healthy individuals. It is also the most common human fungal pathogen isolated from patients in clinical settings. C. albicans can cause a number of superficial and invasive infections, especially in immunocompromised individuals. The ability of C. albicans to succeed as both a commensal and a pathogen, and to thrive in a wide range of environmental niches within the host, requires sophisticated transcriptional regulatory programs that can integrate and respond to host specific environmental signals. Identifying and characterizing the transcriptional regulatory networks that control important developmental processes in C. albicans will shed new light on the strategies used by C. albicans to colonize and infect its host. Here, we discuss the transcriptional regulatory circuits controlling three major developmental processes in C. albicans: biofilm formation, the white-opaque phenotypic switch, and the commensal-pathogen transition. Each of these three circuits are tightly knit and, through our analyses, we show that they are integrated together by extensive regulatory crosstalk between the core regulators that comprise each circuit.

ObjectiveThe NCoR-1 and NCoR-2 corepressors are products of an early gene duplication near the beginning of vertebrate evolution and play both overlapping and divergent roles in development and physiology. Alternative-splicing of NCoR-1 and NCoR-2 further customizes their functions. To better understand the evolutionary basis of this phenomenon we extended our prior study of NCoR-1 and NCoR-2 alternative-splicing to an expanded series of species.ResultsAlternative-splicing of NCoR-2 was observed in all vertebrates examined whereas alternative-splicing of NCoR-1 was largely limited to placental mammals. Notably the most prominent of the NCoR-1 alternative-splicing events specific to the placental lineage, in exon 37 that plays a key role in murine metabolism, mimics in many features an analogous alternative-splicing event that appeared in NCoR-2 much earlier at the beginning of the vertebrate radiation. Detection of additional alternative-splicing events, at exons 28 in NCoR-1 or NCoR-2, was limited to the Rodentia or Primates examined, indicating both corepressor paralogs continued to acquire additional splice variations more recently and independently of one another. Our results suggest that the NCoR-1/NCoR-2 paralogs have been subject to a mix of shared and distinct selective pressures, resulting in the pattern of divergent and convergent alternative-splicing observed in extant species.

Epigenomic regulation and lineage-specific gene expression act in concert to drive cellular differentiation, but the temporal interplay between these processes is largely unknown. Using neural induction from human pluripotent stem cells (hPSCs) as a paradigm, we interrogated these dynamics by performing RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and assay for transposase accessible chromatin using sequencing (ATAC-seq) at seven time points during early neural differentiation. We found that changes in DNA accessibility precede H3K27ac, which is followed by gene expression changes. Using massively parallel reporter assays (MPRAs) to test the activity of 2,464 candidate regulatory sequences at all seven time points, we show that many of these sequences have temporal activity patterns that correlate with their respective cell-endogenous gene expression and chromatin changes. A prioritization method incorporating all genomic and MPRA data further identified key transcription factors involved in driving neural fate. These results provide a comprehensive resource of genes and regulatory elements that orchestrate neural induction and illuminate temporal frameworks during differentiation.

Heart failure is associated with hypertrophying of cardiomyocytes and changes in transcriptional activity. Studies from rapidly dividing cells in culture have suggested that transcription may be compartmentalized into factories within the nucleus, but this phenomenon has not been tested in vivo and the role of nuclear architecture in cardiac gene regulation is unknown. While alterations to transcription have been linked to disease, little is known about the regulation of the spatial organization of transcription and its properties in the pathological setting. In the present study, we investigate the structural features of endogenous transcription factories in the heart and determine the principles connecting chromatin structure to transcriptional regulation in vivo. Super-resolution imaging of endogenous RNA polymerase II clusters in neonatal and adult cardiomyocytes revealed distinct properties of transcription factories in response to pathological stress: neonatal nuclei demonstrated changes in number of clusters, with parallel increases in nuclear area, while the adult nuclei underwent changes in size and intensity of RNA polymerase II foci. Fluorescence in situ hybridization-based labeling of genes revealed locus-specific relationships between expression change and anatomical localization-with respect to nuclear periphery and heterochromatin regions, both sites associated with gene silencing-in the nuclei of cardiomyocytes in hearts (but not liver hepatocytes) of mice subjected to pathologic stimuli that induce heart failure. These findings demonstrate a role for chromatin organization and rearrangement of nuclear architecture for cell type-specific transcription in vivo during disease. RNA polymerase II ChIP and chromatin conformation capture studies in the same model system demonstrate formation and reorganization of distinct nuclear compartments regulating gene expression. These findings reveal locus-specific compartmentalization of stress-activated, housekeeping and silenced genes in the anatomical context of the endogenous nucleus, revealing basic principles of global chromatin structure and nuclear architecture in the regulation of gene expression in healthy and diseased conditions.

Developmental Pluripotency-Associated-4 (DPPA4) is one of the few core pluripotency genes lacking clearly defined molecular and cellular functions. Here, we used a proteomics screening approach of human embryonic stem cell (hESC) nuclear extract to determine DPPA4 molecular functions through identification of novel cofactors. Unexpectedly, the signaling molecule ERBB3-binding protein 1 (EBP1) was the strongest candidate binding partner for DPPA4 in hESC. EBP1 is a growth factor signaling mediator present in two isoforms, p48 and p42. The two isoforms generally have opposing functions, however their roles in pluripotent cells have not been established. We found that DPPA4 preferentially binds p48 in pluripotent and NTERA-2 cells, but this interaction is largely absent in non-pluripotent cells and is reduced with differentiation. The DPPA4-EBP1 interaction is mediated at least in part in DPPA4 by the highly conserved SAF-A/B, Acinus and PIAS (SAP) domain. Functionally, we found that DPPA4 transcriptional repressive function in reporter assays is significantly increased by specific p48 knockdown, an effect that was abolished with an interaction-deficient DPPA4 ΔSAP mutant. Thus, DPPA4 and EBP1 may cooperate in transcriptional functions through their physical association in a pluripotent cell specific context. Our study identifies EBP1 as a novel pluripotency cofactor and provides insight into potential mechanisms used by DPPA4 in regulating pluripotency through its association with EBP1. Stem Cells 2018;36:671-682.

The activity-regulated cytoskeletal (Arc) gene is implicated in numerous synaptic plasticity paradigms, including long-term potentiation and depression and homeostatic plasticity, and is critical for consolidating memory. How Arc facilitates these forms of plasticity is not fully understood. Unlike other neuronal immediate-early genes, Arc encodes a protein that shuttles between the somatodendritic and nuclear compartments to regulate synaptic plasticity. Little attention has been paid to Arc's role in the nucleus. Here, we highlight the regulatory elements and signaling cascades required to induce Arc transcription and discuss the significance of Arc nuclear localization for synaptic plasticity and scaling. We integrate these findings into the context of cognitive function and disease and propose a model in which Arc mediates an effect on memory as a "chaser" of synaptic activity through homeostatic scaling.

ObjectivesThe expression and activity of the breast cancer resistance protein (ABCG2) contributes toward the pharmacokinetics of endogenous and xenobiotic substrates. The effect of genetic variation on the activity of cis-regulatory elements and nuclear response elements in the ABCG2 locus and their contribution toward ABCG2 expression have not been investigated systematically. In this study, the effect of genetic variation on the in vitro and in vivo enhancer activity of six previously identified liver enhancers in the ABCG2 locus was examined.MethodsReference and variant liver enhancers were tested for their ability to alter luciferase activity in vitro in HepG2 and HEK293T cell lines and in vivo using a hydrodynamic tail vein assay. Positive in vivo single-nucleotide polymorphisms (SNPs) were tested for association with gene expression and for altered protein binding in electrophoretic mobility shift assays.ResultsMultiple SNPs were found to alter enhancer activity in vitro. Four of these variants (rs9999111, rs12508471, ABCG2RE1*2, and rs149713212) decreased and one (rs2725263) increased enhancer activity in vivo. In addition, rs9999111 and rs12508471 were associated with ABCG2 expression in lymphoblastoid cell lines, lymphocytes, and T cells, and showed increased HepG2 nuclear protein binding.ConclusionThis study identifies SNPs within regulatory regions of the ABCG2 locus that alter enhancer activity in vitro and in vivo. Several of these SNPs correlate with tissue-specific ABCG2 expression and alter DNA/protein binding. These SNPs could contribute toward reported tissue-specific variability in ABCG2 expression and may influence the correlation between ABCG2 expression and disease risk or the pharmacokinetics and pharmacodynamics of breast cancer resistance protein substrates.

Environmental insults are often detected by multiple sensors that activate diverse signaling pathways and transcriptional regulators, leading to a tailored transcriptional output. To understand how a tailored response is coordinated, we examined the inflammatory response elicited in mouse macrophages by ionizing radiation (IR). RNA-sequencing studies revealed that most radiation-induced genes were strongly dependent on only one of a small number of sensors and signaling pathways, notably the DNA damage-induced kinase ATM, which regulated many IR-response genes, including interferon response genes, via an atypical IRF1-dependent, STING-independent mechanism. Moreover, small, defined sets of genes activated by p53 and NRF2 accounted for the selective response to radiation in comparison to a microbial inducer of inflammation. Our findings reveal that genes comprising an environmental response are activated by defined sensing mechanisms with a high degree of selectivity, and they identify distinct components of the radiation response that might be susceptible to therapeutic perturbation.

Campylobacter jejuni promotes commensalism in the intestinal tracts of avian hosts and diarrheal disease in humans, yet components of intestinal environments recognized as spatial cues specific for different intestinal regions by the bacterium to initiate interactions in either host are mostly unknown. By analyzing a C. jejuni acetogenesis mutant defective in converting acetyl coenzyme A (Ac-CoA) to acetate and commensal colonization of young chicks, we discovered evidence for in vivo microbiota-derived short-chain fatty acids (SCFAs) and organic acids as cues recognized by C. jejuni that modulate expression of determinants required for commensalism. We identified a set of C. jejuni genes encoding catabolic enzymes and transport systems for amino acids required for in vivo growth whose expression was modulated by SCFAs. Transcription of these genes was reduced in the acetogenesis mutant but was restored upon supplementation with physiological concentrations of the SCFAs acetate and butyrate present in the lower intestinal tracts of avian and human hosts. Conversely, the organic acid lactate, which is abundant in the upper intestinal tract where C. jejuni colonizes less efficiently, reduced expression of these genes. We propose that microbiota-generated SCFAs and lactate are cues for C. jejuni to discriminate between different intestinal regions. Spatial gradients of these metabolites likely allow C. jejuni to locate preferred niches in the lower intestinal tract and induce expression of factors required for intestinal growth and commensal colonization. Our findings provide insights into the types of cues C. jejuni monitors in the avian host for commensalism and likely in humans to promote diarrheal disease.IMPORTANCECampylobacter jejuni is a commensal of the intestinal tracts of avian species and other animals and a leading cause of diarrheal disease in humans. The types of cues sensed by C. jejuni to influence responses to promote commensalism or infection are largely lacking. By analyzing a C. jejuni acetogenesis mutant, we discovered a set of genes whose expression is modulated by lactate and short-chain fatty acids produced by the microbiota in the intestinal tract. These genes include those encoding catabolic enzymes and transport systems for amino acids that are required by C. jejuni for in vivo growth and intestinal colonization. We propose that gradients of these microbiota-generated metabolites are cues for spatial discrimination between areas of the intestines so that the bacterium can locate niches in the lower intestinal tract for optimal growth for commensalism in avian species and possibly infection of human hosts leading to diarrheal disease.

The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E2 program appears to depend on GR SUMOylation, which leads to stable trans-recruitment of the GR-N-CoR/SMRT-HDAC3 corepressor complex on these enhancers. Together, these results uncover a mechanism by which competitive recruitment of DNA-binding nuclear receptors/transcription factors in trans to hot spot enhancers serves as an effective biological strategy for trans-repression, with clear implications for breast cancer and other diseases.

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology.

MYC is a key driver of cellular transformation and is deregulated in most human cancers. Studies of MYC and its interactors have provided mechanistic insight into its role as a regulator of gene transcription. MYC has been previously linked to chromatin regulation through its interaction with INI1 (SMARCB1/hSNF5/BAF47), a core member of the SWI/SNF chromatin remodeling complex. INI1 is a potent tumor suppressor that is inactivated in several types of cancers, most prominently as the hallmark alteration in pediatric malignant rhabdoid tumors. However, the molecular and functional interaction of MYC and INI1 remains unclear. Here, we characterize the MYC-INI1 interaction in mammalian cells, mapping their minimal binding domains to functionally significant regions of MYC (leucine zipper) and INI1 (repeat motifs), and demonstrating that the interaction does not interfere with MYC-MAX interaction. Protein-protein interaction network analysis expands the MYC-INI1 interaction to the SWI/SNF complex and a larger network of chromatin regulatory complexes. Genome-wide analysis reveals that the DNA-binding regions and target genes of INI1 significantly overlap with those of MYC. In an INI1-deficient rhabdoid tumor system, we observe that with re-expression of INI1, MYC and INI1 bind to common target genes and have opposing effects on gene expression. Functionally, INI1 re-expression suppresses cell proliferation and MYC-potentiated transformation. Our findings thus establish the antagonistic roles of the INI1 and MYC transcriptional regulators in mediating cellular and oncogenic functions.

A major virulence attribute of Candida albicans is its ability to form biofilms, densely packed communities of cells adhered to a surface. These biofilms are intrinsically resistant to conventional antifungal therapeutics, the host immune system, and other environmental factors, making biofilm-associated infections a significant clinical challenge. Here, we review current knowledge on the development, regulation, and molecular mechanisms of C. albicans biofilms.

Nucleotide changes in gene regulatory elements can have a major effect on interindividual differences in drug response. For example, by reviewing all published pharmacogenomic genome-wide association studies, we show here that 96.4% of the associated single nucleotide polymorphisms reside in noncoding regions. We discuss how sequencing technologies are improving our ability to identify drug response-associated regulatory elements genome-wide and to annotate nucleotide variants within them. We highlight specific examples of how nucleotide changes in these elements can affect drug response and illustrate the techniques used to find them and functionally characterize them. Finally, we also discuss challenges in the field of drug-responsive regulatory elements that need to be considered in order to translate these findings into the clinic.

In humans, microbial cells (including bacteria, archaea, and fungi) greatly outnumber host cells. Candida albicans is the most prevalent fungal species of the human microbiota; this species asymptomatically colonizes many areas of the body, particularly the gastrointestinal and genitourinary tracts of healthy individuals. Alterations in host immunity, stress, resident microbiota, and other factors can lead to C. albicans overgrowth, causing a wide range of infections, from superficial mucosal to hematogenously disseminated candidiasis. To date, most studies of C. albicans have been carried out in suspension cultures; however, the medical impact of C. albicans (like that of many other microorganisms) depends on its ability to thrive as a biofilm, a closely packed community of cells. Biofilms are notorious for forming on implanted medical devices, including catheters, pacemakers, dentures, and prosthetic joints, which provide a surface and sanctuary for biofilm growth. C. albicans biofilms are intrinsically resistant to conventional antifungal therapeutics, the host immune system, and other environmental perturbations, making biofilm-based infections a significant clinical challenge. Here, we review our current knowledge of biofilms formed by C. albicans and closely related fungal species.

Enhancers control the timing, location and expression levels of their target genes. Nucleotide variation in enhancers has been shown to lead to numerous phenotypes, including human disease. While putative enhancer sequences and nucleotide variation within them can now be detected in a rapid manner using various genomic technologies, the understanding of the functional consequences of these variants still remains largely unknown. Massively parallel reporter assays (MPRAs) can overcome this hurdle by providing the ability to test thousands of sequences and nucleotide variants within them for enhancer activity en masse. Here, we describe this technology and specifically focus on how it is being used to obtain an increased understanding of enhancer regulatory code and grammar.

Scl/Tal1 confers hemogenic competence and prevents ectopic cardiomyogenesis in embryonic endothelium by unknown mechanisms. We discovered that Scl binds to hematopoietic and cardiac enhancers that become epigenetically primed in multipotent cardiovascular mesoderm, to regulate the divergence of hematopoietic and cardiac lineages. Scl does not act as a pioneer factor but rather exploits a pre-established epigenetic landscape. As the blood lineage emerges, Scl binding and active epigenetic modifications are sustained in hematopoietic enhancers, whereas cardiac enhancers are decommissioned by removal of active epigenetic marks. Our data suggest that, rather than recruiting corepressors to enhancers, Scl prevents ectopic cardiogenesis by occupying enhancers that cardiac factors, such as Gata4 and Hand1, use for gene activation. Although hematopoietic Gata factors bind with Scl to both activated and repressed genes, they are dispensable for cardiac repression, but necessary for activating genes that enable hematopoietic stem/progenitor cell development. These results suggest that a unique subset of enhancers in lineage-specific genes that are accessible for regulators of opposing fates during the time of the fate decision provide a platform where the divergence of mutually exclusive fates is orchestrated.

Herpud1 is an ER-localized protein that contributes to endoplasmic reticulum (ER) homeostasis by participating in the ER-associated protein degradation pathway. The Nrf1 transcription factor is important in cellular stress pathways. We show that loss of Nrf1 function results in decreased Herpud1 expression in cells and liver tissues. Expression of Herpud1 increases in response to ER stress, but not in Nrf1 knockout cells. Transactivation studies show that Nrf1 acts through antioxidant response elements located in the Herpud1 promoter, and chromatin immunoprecipitation demonstrates that Herpud1 is a direct Nrf1 target gene. These results indicate that Nrf1 is a transcriptional activator of Herpud1 expression during ER stress, and they suggest Nrf1 is a key player in the regulation of the ER stress response in cells.

The chemokine RANTES is produced by a variety of tissues, including cells of the monocyte/macrophage lineage. RANTES expression is rapidly and transiently up-regulated in primary monocytes and the monocytic cell line Mono Mac 6 in response to stimulation by the bacterial product lipopolysaccharide (LPS). Transient transfection of Mono Mac 6 cells with RANTES reporter-promoter deletion constructs, in conjunction with DNase I footprinting and heterologous reporter gene assays, allowed identification of an LPS-responsive region within the RANTES promoter. Electrophoretic mobility shift assays (EMSA), methylation interference and EMSA supershift experiments were used to characterize sequences and transcription factors responsible for this LPS inducibility. The region, termed RANTES site G [R(G)], contains consensus sites for Ets and CRE/AP-1-like elements. Site-directed mutagenesis of the Ets site resulted in a loss of only 15 % of promoter activity, while mutation of the CRE/AP-1 site led to a loss of 40 % of LPS-induced promoter activity. The Ets site constitutively binds the Ets family member PU.1. LPS stimulation leads to an induction of ATF-3 and JunD factor binding to the CRE/AP-1 site. Thus, LPS induction of RANTES transcription is mediated, in part, through the activation and selective binding of ATF and Jun nuclear factors to the R(G) promoter module.

The chemokine RANTES is produced by a variety of tissues, including cells of the monocyte/macrophage lineage. RANTES expression is rapidly and transiently up-regulated in primary monocytes and the monocytic cell line Mono Mac 6 in response to stimulation by the bacterial product lipopolysaccharide (LPS). Transient transfection of Mono Mac 6 cells with RANTES reporter-promoter deletion constructs, in conjunction with DNase I footprinting and heterologous reporter gene assays, allowed identification of an LPS-responsive region within the RANTES promoter. Electrophoretic mobility shift assays (EMSA), methylation interference and EMSA supershift experiments were used to characterize sequences and transcription factors responsible for this LPS inducibility. The region, termed RANTES site G [R(G)], contains consensus sites for Ets and CRE/AP-1-like elements. Site-directed mutagenesis of the Ets site resulted in a loss of only 15 % of promoter activity, while mutation of the CRE/AP-1 site led to a loss of 40 % of LPS-induced promoter activity. The Ets site constitutively binds the Ets family member PU.1. LPS stimulation leads to an induction of ATF-3 and JunD factor binding to the CRE/AP-1 site. Thus, LPS induction of RANTES transcription is mediated, in part, through the activation and selective binding of ATF and Jun nuclear factors to the R(G) promoter module.