Non-coding Transcription Instructs Chromatin Folding and Compartmentalization to Dictate Enhancer-Promoter Communication and T Cell Fate.

It is now established that Bcl11b specifies T cell fate. Here, we show that in developing T cells, the Bcl11b enhancer repositioned from the lamina to the nuclear interior. Our search for factors that relocalized the Bcl11b enhancer identified a non-coding RNA named ThymoD (thymocyte differentiation factor). ThymoD-deficient mice displayed a block at the onset of T cell development and developed lymphoid malignancies. We found that ThymoD transcription promoted demethylation at CTCF bound sites and activated cohesin-dependent looping to reposition the Bcl11b enhancer from the lamina to the nuclear interior and to juxtapose the Bcl11b enhancer and promoter into a single-loop domain. These large-scale changes in nuclear architecture were associated with the deposition of activating epigenetic marks across the loop domain, plausibly facilitating phase separation. These data indicate how, during developmental progression and tumor suppression, non-coding transcription orchestrates chromatin folding and compartmentalization to direct with high precision enhancer-promoter communication.

Comprehensive characterization of neutrophil genome topology.

Neutrophils are responsible for the first line of defense against invading pathogens. Their nuclei are uniquely structured as multiple lobes that establish a highly constrained nuclear environment. Here we found that neutrophil differentiation was not associated with large-scale changes in the number and sizes of topologically associating domains (TADs). However, neutrophil genomes were enriched for long-range genomic interactions that spanned multiple TADs. Population-based simulation of spherical and toroid genomes revealed declining radii of gyration for neutrophil chromosomes. We found that neutrophil genomes were highly enriched for heterochromatic genomic interactions across vast genomic distances, a process named supercontraction. Supercontraction involved genomic regions located in the heterochromatic compartment in both progenitors and neutrophils or genomic regions that switched from the euchromatic to the heterochromatic compartment during neutrophil differentiation. Supercontraction was accompanied by the repositioning of centromeres, pericentromeres, and long interspersed nuclear elements (LINEs) to the neutrophil nuclear lamina. We found that Lamin B receptor expression was required to attach centromeric and pericentromeric repeats but not LINE-1 elements to the lamina. Differentiating neutrophils also repositioned ribosomal DNA and mininucleoli to the lamina-a process that was closely associated with sharply reduced ribosomal RNA expression. We propose that large-scale chromatin reorganization involving supercontraction and recruitment of heterochromatin and nucleoli to the nuclear lamina facilitates the folding of the neutrophil genome into a confined geometry imposed by a multilobed nuclear architecture.

Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate.

The genome is folded into domains located in compartments that are either transcriptionally inert or transcriptionally permissive. Here we used genome-wide strategies to characterize domains during B cell development. Structured interaction matrix analysis showed that occupancy by the architectural protein CTCF was associated mainly with intradomain interactions, whereas sites bound by the histone acetyltransferase p300 or the transcription factors E2A or PU.1 were associated with intra- and interdomain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitor cells, the transcriptionally inactive locus encoding early B cell factor (Ebf1) was sequestered at the nuclear lamina, which thereby preserved their multipotency. After development into the pro-B cell stage, Ebf1 and other genes switched compartments to establish new intra- and interdomain interactions associated with a B lineage-specific transcription signature.

The E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hif1a and c-myc/p19Arf pathways to suppress innate variant TFH cell development, thymocyte expansion, and lymphomagenesis.

It is now well established that the E and Id protein axis regulates multiple steps in lymphocyte development. However, it remains unknown how E and Id proteins mechanistically enforce and maintain the naïve T-cell fate. Here we show that Id2 and Id3 suppressed the development and expansion of innate variant follicular helper T (TFH) cells. Innate variant TFH cells required major histocompatibility complex (MHC) class I-like signaling and were associated with germinal center B cells. We found that Id2 and Id3 induced Foxo1 and Foxp1 expression to antagonize the activation of a TFH transcription signature. We show that Id2 and Id3 acted upstream of the Hif1a/Foxo/AKT/mTORC1 pathway as well as the c-myc/p19Arf module to control cellular expansion. We found that mice depleted for Id2 and Id3 expression developed colitis and αß T-cell lymphomas. Lymphomas depleted for Id2 and Id3 expression displayed elevated levels of c-myc, whereas p19Arf abundance declined. Transcription signatures of Id2- and Id3-depleted lymphomas revealed similarities to genetic deficiencies associated with Burkitt lymphoma. We propose that, in response to antigen receptor and/or cytokine signaling, the E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hif1a and c-myc/p19Arf pathways to control cellular expansion and homeostatic proliferation.

AID targeting: old mysteries and new challenges.

Activation-induced cytidine deaminase (AID) mediates cytosine deamination and underlies two central processes in antibody diversification: somatic hypermutation and class-switch recombination. AID deamination is not exclusive to immunoglobulin loci; it can instigate DNA lesions in non-immunoglobulin genes and thus stringent checks are in place to constrain and restrict its activity. Recent findings have provided new insights into the mechanisms that target AID activity to specific genomic regions, revealing an involvement for noncoding RNAs associated with polymerase pausing and with enhancer transcription as well as genomic architecture. We review these findings and integrate them into a model for multilevel regulation of AID expression and targeting in immunoglobulin and non-immunoglobulin loci. Within this framework we discuss gaps in understanding, and outline important areas of further research.

JAML immunotherapy targets recently activated tumor-infiltrating CD8+ T cells.

Junctional adhesion molecule-like protein (JAML) serves as a co-stimulatory molecule in γδ T cells. While it has recently been described as a cancer immunotherapy target in mice, its potential to cause toxicity, specific mode of action with regard to its cellular targets, and whether it can be targeted in humans remain unknown. Here, we show that JAML is induced by T cell receptor engagement, reveal that this induction is linked to cis-regulatory interactions between the CD3D and JAML gene loci. When compared with other immunotherapy targets plagued by low target specificity and end-organ toxicity, we find JAML to be mostly restricted to and highly expressed by tissue-resident memory CD8+ T cells in multiple cancer types. By delineating the key cellular targets and functional consequences of agonistic anti-JAML therapy in a murine melanoma model, we show its specific mode of action and the reason for its synergistic effects with anti-PD-1.

Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

Single-cell eQTL analysis of activated T cell subsets reveals activation and cell type-dependent effects of disease-risk variants.

The impact of genetic variants on cells challenged in biologically relevant contexts has not been fully explored. Here, we activated CD4+ T cells from 89 healthy donors and performed a single-cell RNA sequencing assay with >1 million cells to examine cell type-specific and activation-dependent effects of genetic variants. Single-cell expression quantitative trait loci (sc-eQTL) analysis of 19 distinct CD4+ T cell subsets showed that the expression of over 4000 genes is significantly associated with common genetic polymorphisms and that most of these genes show their most prominent effects in specific cell types. These genes included many that encode for molecules important for activation, differentiation, and effector functions of T cells. We also found new gene associations for disease-risk variants identified from genome-wide association studies and highlighted the cell types in which their effects are most prominent. We found that biological sex has a major influence on activation-dependent gene expression in CD4+ T cell subsets. Sex-biased transcripts were significantly enriched in several pathways that are essential for the initiation and execution of effector functions by CD4+ T cells like TCR signaling, cytokines, cytokine receptors, costimulatory, apoptosis, and cell-cell adhesion pathways. Overall, this DICE (Database of Immune Cell Expression, eQTLs, and Epigenomics) subproject highlights the power of sc-eQTL studies for simultaneously exploring the activation and cell type-dependent effects of common genetic variants on gene expression (https://dice-database.org).

The ORF3 protein of hepatitis E virus delays degradation of activated growth factor receptors by interacting with CIN85 and blocking formation of the Cbl-CIN85 complex.

Hepatitis E virus (HEV) causes an acute self-limiting disease that is endemic in developing countries. Previous studies suggested that the ORF3 protein (pORF3) of HEV is required for infection in vivo and is likely to modulate the host response. Our previous work showed that pORF3 localizes to early and recycling endosomes and causes a delay in the postinternalization trafficking of epidermal growth factor receptor (EGFR) to late endosomes/lysosomes. Here we report that pORF3 also delays the trafficking and degradation of activated hepatocyte growth factor receptor (c-Met) and delineate the mechanistic details of these effects. A mutant ORF3 protein, which does not localize to endosomes, also showed similar effects on growth factor receptor trafficking, making this effect independent of the endosomal localization of pORF3. The ORF3 protein was found to interact with CIN85, a multidomain adaptor protein implicated in the Cbl-mediated downregulation of receptor tyrosine kinases. This interaction competed with the formation of the growth factor receptor-Cbl-CIN85 complex, resulting in the reduced ubiquitination of CIN85 and trafficking of the growth factor receptor complex toward late endosomes/lysosomes. We propose that through its effects on growth factor receptor trafficking, pORF3 prolongs endomembrane growth factor signaling and promotes cell survival to contribute positively to viral replication and pathogenesis.

COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types.

Common genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in TH2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.

Promoter-interacting expression quantitative trait loci are enriched for functional genetic variants.

Expression quantitative trait loci (eQTLs) studies provide associations of genetic variants with gene expression but fall short of pinpointing functionally important eQTLs. Here, using H3K27ac HiChIP assays, we mapped eQTLs overlapping active cis-regulatory elements that interact with their target gene promoters (promoter-interacting eQTLs, pieQTLs) in five common immune cell types (Database of Immune Cell Expression, Expression quantitative trait loci and Epigenomics (DICE) cis-interactome project). This approach allowed us to identify functionally important eQTLs and show mechanisms that explain their cell-type restriction. We also devised an approach to eQTL discovery that relies on HiChIP-based promoter interaction maps as a structural framework for deciding which SNPs to test for association with gene expression, and observe ultra-long-distance pieQTLs (>1 megabase away), including several disease-risk variants. We validated the functional role of pieQTLs using reporter assays, CRISPRi, dCas9-tiling guides and Cas9-mediated base-pair editing. In this article we present a method for functional eQTL discovery and provide insights into relevance of noncoding variants for cell-specific gene regulation and for disease association beyond conventional eQTL mapping.

Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection.

The hepatitis E virus (HEV), a nonenveloped RNA virus, is the causative agent of hepatitis E. The mode by which HEV attaches to and enters into target cells for productive infection remains unidentified. Open reading frame 2 (ORF2) of HEV encodes its major capsid protein, pORF2, which is likely to have the determinants for virus attachment and entry. Using an approximately 56-kDa recombinant pORF2 that can self-assemble as virus-like particles, we demonstrated that cell surface heparan sulfate proteoglycans (HSPGs), specifically syndecans, play a crucial role in the binding of pORF2 to Huh-7 liver cells. Removal of cell surface heparan sulfate by enzymatic (heparinase) or chemical (sodium chlorate) treatment of cells or competition with heparin, heparan sulfate, and their oversulfated derivatives caused a marked reduction in pORF2 binding to the cells. Syndecan-1 is the most abundant proteoglycan present on these cells and, hence, plays a key role in pORF2 binding. Specificity is likely to be dictated by well-defined sulfation patterns on syndecans. We show that pORF2 binds syndecans predominantly via 6-O sulfation, indicating that binding is not entirely due to random electrostatic interactions. Using an in vitro infection system, we also showed a marked reduction in HEV infection of heparinase-treated cells. Our results indicate that, analogous to some enveloped viruses, a nonenveloped virus like HEV may have also evolved to use HSPGs as cellular attachment receptors.

The hepatitis E virus ORF3 protein stabilizes HIF-1alpha and enhances HIF-1-mediated transcriptional activity through p300/CBP.

The hepatitis E virus (HEV) causes hepatitis E and is an important human pathogen. We have previously shown that the HEV open reading frame 3 (ORF3) protein promotes survival of the host cell. Here we report finding increased expression of glycolytic pathway enzymes in ORF3-expressing cells. Promoter analysis of these genes revealed the ubiquitous presence of hypoxia inducible factor (HIF) responsive element (HRE). Dominant-negative and siRNA studies showed increased expression of glycolytic pathway genes by the ORF3 to be mediated by the HIF-1 transcription factor. Our results showed that HIF-1alpha, a highly unstable subunit of the HIF-1, was stabilized in ORF3-expressing cells. This was through phosphatidylinositol-3-kinase (PI3K) mediated activation of Akt/protein kinase B. Enhanced binding to the consensus HRE and increased transactivation activity of HIF-1 were also observed in ORF3-expressing cells. The HIF complex recruits the transcriptional adapter/histone acetyltransferase protein p300/CBP to target gene promoters and p300/CBP phosphorylation is required for this interaction. We show that ORF3-mediated extracellularly regulated kinase (Erk) activation was responsible for the observed increase in phosphorylation and transactivation activity of p300/CBP. Our results reveal a two-pronged strategy through which the ORF3 protein might modulate the energy homeostasis in HEV infected cells and thus contribute to pathogenesis.

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies.

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (>100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

COVID-19 genetic risk variants are associated with expression of multiple genes in diverse immune cell types.

Common genetic polymorphisms associated with severity of COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Here, we assessed the effects of 679 COVID-19-risk variants on gene expression in a wide-range of immune cell types. Severe COVID-19-risk variants were significantly associated with the expression of 11 protein-coding genes, and overlapped with either target gene promoter or cis -regulatory regions that interact with target promoters in the cell types where their effects are most prominent. For example, we identified that the association between variants in the 3p21.31 risk locus and the expression of CCR2 in classical monocytes is likely mediated through an active cis-regulatory region that interacted with CCR2 promoter specifically in monocytes. The expression of several other genes showed prominent genotype-dependent effects in non-classical monocytes, NK cells, B cells, or specific T cell subtypes, highlighting the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations.

Plasma Cell Fate Is Orchestrated by Elaborate Changes in Genome Compartmentalization and Inter-chromosomal Hubs.

The transition from the follicular B to the plasma cell stage is associated with large-scale changes in cell morphology. Here, we examine whether plasma cell development is also associated with changes in nuclear architecture. We find that the onset of plasma cell development is concomitant with a decline in remote genomic interactions; a gain in euchromatic character at loci encoding for factors that specify plasma cell fate, including Prdm1 and Atf4; and establishment of de novo inter-chromosomal hubs. We find that, in developing plasma cells and concurrent with transcriptional silencing, the Ebf1 locus repositions from an euchromatic to peri-centromeric heterochromatic environment. Finally, we find that inter-chromosomal hubs are enriched for the deposition of either H3K27Ac or H3K27me3. These data indicate that plasma cell fate is orchestrated by elaborate changes in genome topology and that epigenetic marks, linked with super-enhancers or transcriptionally repressed regions, are enriched at inter-chromosomal hubs.

The hepatitis E virus ORF3 protein modulates epidermal growth factor receptor trafficking, STAT3 translocation, and the acute-phase response.

The hepatitis E virus (HEV) causes acute viral hepatitis, but its characterization is hampered by the lack of an efficient in vitro infection system that can be used to study the effects of HEV proteins on cellular processes. Previous studies suggest that the viral ORF3 protein (pORF3) is essential for infection in vivo and is likely to modulate the host response. Here, we report that pORF3 localizes to early and recycling endosomes and causes a delay in the postinternalization trafficking of epidermal growth factor receptor (EGFR) to late endosomes/lysosomes. The cytoplasmic phosphorylated signal transducer and activator of transcription 3 (pSTAT3) proteins require growth factor receptor endocytosis for their translocation from the cytoplasm to nucleus. Consequently, lower levels of pSTAT3 were found in the nuclei of ORF3-expressing Huh7 human hepatoma cells stimulated with EGF. This results in downregulation of the acute-phase response, a major determinant of inflammation in the host. We propose that through its effects on EGFR trafficking, pORF3 prolongs endomembrane growth factor signaling and promotes cell survival. The effects on STAT3 translocation would result in a reduced inflammatory response. Both of these events are likely to contribute positively to viral replication.

Identification of significant chromatin contacts from HiChIP data by FitHiChIP.

HiChIP/PLAC-seq is increasingly becoming popular for profiling 3D chromatin contacts among regulatory elements and for annotating functions of genetic variants. Here we describe FitHiChIP, a computational method for loop calling from HiChIP/PLAC-seq data, which jointly models the non-uniform coverage and genomic distance scaling of contact counts to compute statistical significance estimates. We also develop a technique to filter putative bystander loops that can be explained by stronger adjacent loops. Compared to existing methods, FitHiChIP performs better in recovering contacts reported by Hi-C, promoter capture Hi-C and ChIA-PET experiments and in capturing previously validated promoter-enhancer interactions. FitHiChIP loop calls are reproducible among replicates and are consistent across different experimental settings. Our work also provides a framework for differential HiChIP analysis with an option to utilize ChIP-seq data for further characterizing differential loops. Even though designed for HiChIP, FitHiChIP is also applicable to other conformation capture assays.

Molecular biology and pathogenesis of hepatitis E virus.

The hepatitis E virus (HEV) is a small RNA virus and the etiological agent for hepatitis E, a form of acute viral hepatitis. The virus has a feco-oral transmission cycle and is transmitted through environmental contamination, mainly through drinking water. Recent studies on the isolation of HEV-like viruses from animal species also suggest zoonotic transfer of the virus. The absence of small animal models of infection and efficient cell culture systems has precluded virological studies on the replication cycle and pathogenesis of HEV. A vaccine against HEV has undergone successful clinical testing and diagnostic tests are available. This review describes HEV epidemiology, clinical presentation, pathogenesis, molecular virology and the host response to HEV infection. The focus is on published literature in the past decade.

The UPR Activator ATF6 Responds to Proteotoxic and Lipotoxic Stress by Distinct Mechanisms.

The unfolded protein response (UPR) is induced by proteotoxic stress of the endoplasmic reticulum (ER). Here we report that ATF6, a major mammalian UPR sensor, is also activated by specific sphingolipids, dihydrosphingosine (DHS) and dihydroceramide (DHC). Single mutations in a previously undefined transmembrane domain motif that we identify in ATF6 incapacitate DHS/DHC activation while still allowing proteotoxic stress activation via the luminal domain. ATF6 thus possesses two activation mechanisms: DHS/DHC activation and proteotoxic stress activation. Reporters constructed to monitor each mechanism show that phenobarbital-induced ER membrane expansion depends on transmembrane domain-induced ATF6. DHS/DHC addition preferentially induces transcription of ATF6 target lipid biosynthetic and metabolic genes over target ER chaperone genes. Importantly, ATF6 containing a luminal achromatopsia eye disease mutation, unresponsive to proteotoxic stress, can be activated by fenretinide, a drug that upregulates DHC, suggesting a potential therapy for this and other ATF6-related diseases including heart disease and stroke.