Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

Multimodal Analysis of Composition and Spatial Architecture in Human Squamous Cell Carcinoma.

To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.

Combined protein and nucleic acid imaging reveals virus-dependent B cell and macrophage immunosuppression of tissue microenvironments.

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.

Robust single-cell matching and multimodal analysis using shared and distinct features.

The ability to align individual cellular information from multiple experimental sources is fundamental for a systems-level understanding of biological processes. However, currently available tools are mainly designed for single-cell transcriptomics matching and integration, and generally rely on a large number of shared features across datasets for cell matching. This approach underperforms when applied to single-cell proteomic datasets due to the limited number of parameters simultaneously accessed and lack of shared markers across these experiments. Here, we introduce a cell-matching algorithm, matching with partial overlap (MARIO) that accounts for both shared and distinct features, while consisting of vital filtering steps to avoid suboptimal matching. MARIO accurately matches and integrates data from different single-cell proteomic and multimodal methods, including spatial techniques and has cross-species capabilities. MARIO robustly matched tissue macrophages identified from COVID-19 lung autopsies via codetection by indexing imaging to macrophages recovered from COVID-19 bronchoalveolar lavage fluid by cellular indexing of transcriptomes and epitopes by sequencing, revealing unique immune responses within the lung microenvironment of patients with COVID.

Silencing of aryl hydrocarbon receptor repressor restrains Th17 cell immunity in autoimmune hepatitis.

Th17-cells play a key role in the pathogenesis of autoimmune hepatitis (AIH). Dysregulation of Th17-cells in AIH is linked to defective response to aryl-hydrocarbon-receptor (AhR) activation. AhR modulates adaptive immunity and is regulated by aryl-hydrocarbon-receptor-repressor (AHRR), which inhibits AhR transcriptional activity. In this study, we investigated whether defective Th17-cell response to AhR derives from aberrant AHRR regulation in AIH. Th17-cells, obtained from the peripheral blood of AIH patients (n = 30) and healthy controls (n = 30) were exposed to AhR endogenous ligands, and their response assessed in the absence or presence of AHRR silencing. Therapeutic effects of AHRR blockade were tested in a model of Concanavalin-A (Con-A)-induced liver injury in humanized mice. AHRR was markedly upregulated in AIH Th17-cells, following exposure to l-kynurenine, an AhR endogenous ligand. In patients, silencing of AHRR boosted Th17-cell response to l-kynurenine, as reflected by increased levels of CYP1A1, the main gene controlled by AhR; and decreased IL17A expression. Blockade of AHRR limited the differentiation of naïve CD4-cells into Th17 lymphocytes; and modulated Th17-cell metabolic profile by increasing the levels of uridine via ATP depletion or pyrimidine salvage. Treatment with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA) oligonucleotides to silence human AHRR in vivo, reduced ALT levels, attenuated lymphocyte infiltration on histology, and heightened frequencies of regulatory immune subsets in NOD/scid/gamma mice, reconstituted with human CD4 cells, and exposed to Con-A. In conclusion, blockade of AHRR in AIH restores Th17-cell response to AHR, and limits Th17-cell differentiation through generation of uridine. In vivo, silencing of AHRR attenuates liver damage in NOD/scid/gamma mice. Blockade of AHRR might therefore represent a novel therapeutic strategy to modulate effector Th17-cell immunity and restore homeostasis in AIH.

Morphologic and molecular analysis of liver injury after SARS-CoV-2 vaccination reveals distinct characteristics.

BACKGROUND & AIMS: Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH. METHODS: Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n = 6) and from patients with an initial diagnosis of AIH (n = 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing. RESULTS: Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8+ effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4+ effector T cells and CD79a+ B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH. CONCLUSIONS: Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis. IMPACT AND IMPLICATIONS: Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis.

SYCP2 Translocation-Mediated Dysregulation and Frameshift Variants Cause Human Male Infertility.

Unexplained infertility affects 2%-3% of reproductive-aged couples. One approach to identifying genes involved in infertility is to study subjects with this clinical phenotype and a de novo balanced chromosomal aberration (BCA). While BCAs may reduce fertility by production of unbalanced gametes, a chromosomal rearrangement may also disrupt or dysregulate genes important in fertility. One such subject, DGAP230, has severe oligozoospermia and 46,XY,t(20;22)(q13.3;q11.2). We identified exclusive overexpression of SYCP2 from the der(20) allele that is hypothesized to result from enhancer adoption. Modeling the dysregulation in budding yeast resulted in disrupted structural integrity of the synaptonemal complex, a common cause of defective spermatogenesis in mammals. Exome sequencing of infertile males revealed three heterozygous SYCP2 frameshift variants in additional subjects with cryptozoospermia and azoospermia. In sum, this investigation illustrates the power of precision cytogenetics for annotation of the infertile genome, suggests that these mechanisms should be considered as an alternative etiology to that of segregation of unbalanced gametes in infertile men harboring a BCA, and provides evidence of SYCP2-mediated male infertility in humans.

Inflammatory molecular endotypes of nasal polyps derived from White and Japanese populations.

BACKGROUND: Emerging evidence suggests that chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous disease with disparate inflammatory characteristics between different racial groups and geographies. Currently, little is known about possible underlying distinguishing factors between these inflammatory differences. OBJECTIVE: Our aim was to interrogate differences in CRSwNP disease between White/non-Asian patients and Japanese patients by using whole transcriptome and single-cell RNA gene expression profiling of nasal polyps (NPs). METHODS: We performed whole transcriptome RNA sequencing with endotype stratification of NPs from 8 White patients (residing in the United States) and 9 Japanese patients (residing in Japan). Reproducibility was confirmed by quantitative PCR in an independent validation set of 46 White and 31 Japanese patients. Single-cell RNA sequencing (scRNAseq) was used to stratify key cell types for contributory transcriptional signatures. RESULTS: Unsupervised clustering analysis identified 2 major endotypes that were present within both cohorts of patients with NPs and had previously been reported at the cytokine level: (1) type 2 endotype and (2) non-type 2 endotype. Importantly, there was a statistically significant difference in the proportion of these endotypes between these geographically distinct subgroups with NPs (P = .03). Droplet-based single-cell RNA sequencing further identified prominent type 2 inflammatory transcript expression: C-C motif chemokine ligand 13 (CCL13) and CCL18 in M2 macrophages, as well as cystatin SN (CST1) and CCL26 in basal, suprabasal, and secretory epithelial cells. CONCLUSION: NPs from both racial groups harbor the same 2 major endotypes, which we have determined to be present in differing ratios between each cohort with CRSwNP disease. Distinct inflammatory and epithelial cells contribute to the type 2 inflammatory profiles observed.

Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival.

Placental Pathology Findings during and after SARS-CoV-2 Infection: Features of Villitis and Malperfusion.

Since the outbreak of coronavirus disease 2019 (COVID-19), there has been a debate whether pregnant women are at a specific risk for COVID-19 and whether it might be vertically transmittable through the placenta. We present a series of five placentas of SARS coronavirus 2 (SARS-CoV-2)-positive women who had been diagnosed with mild symptoms of COVID-19 or had been asymptomatic before birth. We provide a detailed histopathologic description of morphological changes accompanied by an analysis of presence of SARS-CoV-2 in the placental tissue. All placentas were term deliveries (40th and 41st gestational weeks). One SARS-CoV-2-positive patient presented with cough and dyspnoea. This placenta showed prominent lymphohistiocytic villitis and intervillositis and signs of maternal and foetal malperfusion. Viral RNA was present in both placenta tissue and the umbilical cord and could be visualized by in situ hybridization in the decidua. SARS-CoV-2 tests were negative at the time of delivery of 3/5 women, and their placentas did not show increased inflammatory infiltrates. Signs of maternal and/or foetal malperfusion were present in 100% and 40% of cases, respectively. There was no transplacental transmission to the infants. In our cohort, we can document different time points regarding SARS-CoV-2 infection. In acute COVID-19, prominent lymphohistiocytic villitis may occur and might potentially be attributable to SARS-CoV-2 infection of the placenta. Furthermore, there are histopathological signs of maternal and foetal malperfusion, which might have a relationship to an altered coagulative or microangiopathic state induced by SARS-CoV-2, yet this cannot be proven considering a plethora of confounding factors.

Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma.

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

RNA Sequencing Analyses of Gene Expression during Epstein-Barr Virus Infection of Primary B Lymphocytes.

Epstein-Barr virus (EBV) infection of human primary resting B lymphocytes (RBLs) leads to the establishment of lymphoblastoid cell lines (LCLs) that can grow indefinitely in vitro EBV transforms RBLs through the expression of viral latency genes, and these genes alter host transcription programs. To globally measure the transcriptome changes during EBV transformation, primary human resting B lymphocytes (RBLs) were infected with B95.8 EBV for 0, 2, 4, 7, 14, 21, and 28 days, and poly(A) plus RNAs were analyzed by transcriptome sequencing (RNA-seq). Analyses of variance (ANOVAs) found 3,669 protein-coding genes that were differentially expressed (false-discovery rate [FDR] < 0.01). Ninety-four percent of LCL genes that are essential for LCL growth and survival were differentially expressed. Pathway analyses identified a significant enrichment of pathways involved in cell proliferation, DNA repair, metabolism, and antiviral responses. RNA-seq also identified long noncoding RNAs (lncRNAs) differentially expressed during EBV infection. Clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) and CRISPR activation (CRISPRa) found that CYTOR and NORAD lncRNAs were important for LCL growth. During EBV infection, type III EBV latency genes were expressed rapidly after infection. Immediately after LCL establishment, EBV lytic genes were also expressed in LCLs, and ∼4% of the LCLs express gp350. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) and POLR2A chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET) data linked EBV enhancers to 90% of EBV-regulated genes. Many genes were linked to enhancers occupied by multiple EBNAs or NF-κB subunits. Incorporating these assays, we generated a comprehensive EBV regulome in LCLs.IMPORTANCE Epstein-Barr virus (EBV) immortalization of resting B lymphocytes (RBLs) is a useful model system to study EBV oncogenesis. By incorporating transcriptome sequencing (RNA-seq), chromatin immune precipitation followed by deep sequencing (ChIP-seq), chromatin interaction analysis followed by paired-end tag sequencing (ChIA-PET), and genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen, we identified key pathways that EBV usurps to enable B cell growth and transformation. Multiple layers of regulation could be achieved by cooperations between multiple EBV transcription factors binding to the same enhancers. EBV manipulated the expression of most cell genes essential for lymphoblastoid cell line (LCL) growth and survival. In addition to proteins, long noncoding RNAs (lncRNAs) regulated by EBV also contributed to LCL growth and survival. The data presented in this paper not only allowed us to further define the molecular pathogenesis of EBV but also serve as a useful resource to the EBV research community.

TAF Family Proteins and MEF2C Are Essential for Epstein-Barr Virus Super-Enhancer Activity.

Super-enhancers (SEs) are clusters of enhancers marked by extraordinarily high and broad chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) signals for H3K27ac or other transcription factors (TFs). SEs play pivotal roles in development and oncogenesis. Epstein-Barr virus (EBV) super-enhancers (ESEs) are co-occupied by all essential EBV oncogenes and EBV-activated NF-κB subunits. Perturbation of ESEs stops lymphoblastoid cell line (LCL) growth. To further characterize ESEs and identify proteins critical for ESE function, MYC ESEs were cloned upstream of a green fluorescent protein (GFP) reporter. Reporters driven by MYC ESEs 525 kb and 428 kb upstream of MYC (525ESE and 428ESE) had very high activities in LCLs but not in EBV-negative BJAB cells. EBNA2 activated MYC ESE-driven luciferase reporters. CRISPRi targeting 525ESE significantly decreased MYC expression. Genome-wide CRISPR screens identified factors essential for ESE activity. TBP-associated factor (TAF) family proteins, including TAF8, TAF11, and TAF3, were essential for the activity of the integrated 525ESE-driven reporter in LCLs. TAF8 and TAF11 knockout significantly decreased 525ESE activity and MYC transcription. MEF2C was also identified to be essential for 525ESE activity. Depletion of MEF2C decreased 525ESE reporter activity, MYC expression, and LCL growth. MEF2C cDNA resistant to CRIPSR cutting rescued MEF2C knockout and restored 525ESE reporter activity and MYC expression. MEF2C depletion decreased IRF4, EBNA2, and SPI1 binding to 525ESE in LCLs. MEF2C depletion also affected the expression of other ESE target genes, including the ETS1 and BCL2 genes. These data indicated that in addition to EBNA2, TAF family members and MEF2C are essential for ESE activity, MYC expression, and LCL growth.IMPORTANCE SEs play critical roles in cancer development. Since SEs assemble much bigger protein complexes on enhancers than typical enhancers (TEs), they are more sensitive than TEs to perturbations. Understanding the protein composition of SEs that are linked to key oncogenes may identify novel therapeutic targets. A genome-wide CRISPR screen specifically identified proteins essential for MYC ESE activity but not simian virus 40 (SV40) enhancer. These proteins not only were essential for the reporter activity but also were also important for MYC expression and LCL growth. Targeting these proteins may lead to new therapies for EBV-associated cancers.

Epstein-Barr Virus LMP1-Mediated Oncogenicity.

Epstein-Barr virus latent membrane protein 1 (LMP1) is expressed in multiple human malignancies, including nasopharyngeal carcinoma and Hodgkin and immunosuppression-associated lymphomas. LMP1 mimics CD40 signaling to activate multiple growth and survival pathways, in particular, NF-κB. LMP1 has critical roles in Epstein-Barr virus (EBV)-driven B-cell transformation, and its expression causes fatal lymphoproliferative disease in immunosuppressed mice. Here, we review recent developments in studies of LMP1 signaling, LMP1-induced host dependency factors, mouse models of LMP1 lymphomagenesis, and anti-LMP1 immunotherapy approaches.

Epstein-Barr virus nuclear antigen 3A partially coincides with EBNA3C genome-wide and is tethered to DNA through BATF complexes.

Epstein-Barr Virus (EBV) conversion of B-lymphocytes to Lymphoblastoid Cell Lines (LCLs) requires four EBV nuclear antigen (EBNA) oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and EBNALP associate with EBV and cell enhancers, up-regulate the EBNA promoter, MYC, and EBV Latent infection Membrane Proteins (LMPs), which up-regulate BCL2 to protect EBV-infected B-cells from MYC proliferation-induced cell death. LCL proliferation induces p16(INK4A) and p14(ARF)-mediated cell senescence. EBNA3A and EBNA3C jointly suppress p16(INK4A) and p14(ARF), enabling continuous cell proliferation. Analyses of the EBNA3A human genome-wide ChIP-seq landscape revealed 37% of 10,000 EBNA3A sites to be at strong enhancers; 28% to be at weak enhancers; 4.4% to be at active promoters; and 6.9% to be at weak and poised promoters. EBNA3A colocalized with BATF-IRF4, ETS-IRF4, RUNX3, and other B-cell Transcription Factors (TFs). EBNA3A sites clustered into seven unique groups, with differing B-cell TFs and epigenetic marks. EBNA3A coincidence with BATF-IRF4 or RUNX3 was associated with stronger EBNA3A ChIP-Seq signals. EBNA3A was at MYC, CDKN2A/B, CCND2, CXCL9/10, and BCL2, together with RUNX3, BATF, IRF4, and SPI1. ChIP-re-ChIP revealed complexes of EBNA3A on DNA with BATF. These data strongly support a model in which EBNA3A is tethered to DNA through a BATF-containing protein complexes to enable continuous cell proliferation.

Epstein-Barr virus nuclear antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A.

Epstein-Barr virus nuclear antigen 3C (EBNA3C) repression of CDKN2A p14(ARF) and p16(INK4A) is essential for immortal human B-lymphoblastoid cell line (LCL) growth. EBNA3C ChIP-sequencing identified >13,000 EBNA3C sites in LCL DNA. Most EBNA3C sites were associated with active transcription; 64% were strong H3K4me1- and H3K27ac-marked enhancers and 16% were active promoters marked by H3K4me3 and H3K9ac. Using ENCODE LCL transcription factor ChIP-sequencing data, EBNA3C sites coincided (±250 bp) with RUNX3 (64%), BATF (55%), ATF2 (51%), IRF4 (41%), MEF2A (35%), PAX5 (34%), SPI1 (29%), BCL11a (28%), SP1 (26%), TCF12 (23%), NF-κB (23%), POU2F2 (23%), and RBPJ (16%). EBNA3C sites separated into five distinct clusters: (i) Sin3A, (ii) EBNA2/RBPJ, (iii) SPI1, and (iv) strong or (v) weak BATF/IRF4. EBNA3C signals were positively affected by RUNX3, BATF/IRF4 (AICE) and SPI1/IRF4 (EICE) cooccupancy. Gene set enrichment analyses correlated EBNA3C/Sin3A promoter sites with transcription down-regulation (P < 1.6 × 10(-4)). EBNA3C signals were strongest at BATF/IRF4 and SPI1/IRF4 composite sites. EBNA3C bound strongly to the p14(ARF) promoter through SPI1/IRF4/BATF/RUNX3, establishing RBPJ-, Sin3A-, and REST-mediated repression. EBNA3C immune precipitated with Sin3A and conditional EBNA3C inactivation significantly decreased Sin3A binding at the p14(ARF) promoter (P < 0.05). These data support a model in which EBNA3C binds strongly to BATF/IRF4/SPI1/RUNX3 sites to enhance transcription and recruits RBPJ/Sin3A- and REST/NRSF-repressive complexes to repress p14(ARF) and p16(INK4A) expression.

Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2.

Epstein-Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (E2) are coexpressed in EBV-infected B lymphocytes and are critical for lymphoblastoid cell line outgrowth. LP removes NCOR and RBPJ repressive complexes from promoters, enhancers, and matrix-associated deacetylase bodies, whereas E2 activates transcription from distal enhancers. LP ChIP-seq analyses identified 19,224 LP sites of which ~50% were ± 2 kb of a transcriptional start site. LP sites were enriched for B-cell transcription factors (TFs), YY1, SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, SMC3, NFκB, TBLR, and EBF. E2 sites were also highly enriched for LP-associated cell TFs and were more highly occupied by RBPJ and EBF. LP sites were highly marked by H3K4me3, H3K27ac, H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcription. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 sites were more similar to LP than to E2 sites in associated cell TFs, RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Az occupancy, and were more highly transcribed than LP or E2 sites. Gene affected by CTCF and LP cooccupancy were more highly expressed than genes affected by CTCF alone. LP was at myc enhancers and promoters and of MYC regulated ccnd2, 23 med complex components, and MYC regulated cell survival genes, igf2r and bcl2. These data implicate LP and associated TFs and DNA looping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatin-remodeling complexes in repressor depletion and gene activation necessary for lymphoblastoid cell line growth and survival.

Structural analysis and dimerization profile of the SCAN domain of the pluripotency factor Zfp206.

Zfp206 (also named as Zscan10) belongs to the subfamily of C(2)H(2) zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 Å crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C(2)H(2) transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development.