Post-transcriptional splicing can occur in a slow-moving zone around the gene.

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

CTCF/cohesin organize the ground state of chromatin-nuclear speckle association.

The interchromatin space in the cell nucleus contains various membrane-less nuclear bodies. Recent findings indicate that nuclear speckles, comprising a distinct nuclear body, exhibit interactions with certain chromatin regions in a ground state. Key questions are how this ground state of chromatin-nuclear speckle association is established and what are the gene regulatory roles of this layer of nuclear organization. We report here that chromatin structural factors CTCF and cohesin are required for full ground state association between DNA and nuclear speckles. Disruption of ground state DNA-speckle contacts via either CTCF depletion or cohesin depletion had minor effects on basal level expression of speckle-associated genes, however we show strong negative effects on stimulus-dependent induction of speckle-associated genes. We identified a putative speckle targeting motif (STM) within cohesin subunit RAD21 and demonstrated that the STM is required for chromatin-nuclear speckle association. In contrast to reduction of CTCF or RAD21, depletion of the cohesin releasing factor WAPL stabilized cohesin on chromatin and DNA-speckle contacts, resulting in enhanced inducibility of speckle-associated genes. In addition, we observed disruption of chromatin-nuclear speckle association in patient derived cells with Cornelia de Lange syndrome (CdLS), a congenital neurodevelopmental diagnosis involving defective cohesin pathways, thus revealing nuclear speckles as an avenue for therapeutic inquiry. In summary, our findings reveal a mechanism to establish the ground organizational state of chromatin-speckle association, to promote gene inducibility, and with relevance to human disease.

Nuclear speckles regulate HIF-2α programs and correlate with patient survival in kidney cancer.

Nuclear speckles are membrane-less bodies within the cell nucleus enriched in RNA biogenesis, processing, and export factors. In this study we investigated speckle phenotype variation in human cancer, finding a reproducible speckle signature, based on RNA expression of speckle-resident proteins, across >20 cancer types. Of these, clear cell renal cell carcinoma (ccRCC) exhibited a clear correlation between the presence of this speckle expression signature, imaging-based speckle phenotype, and clinical outcomes. ccRCC is typified by hyperactivation of the HIF-2α transcription factor, and we demonstrate here that HIF-2α drives physical association of a select subset of its target genes with nuclear speckles. Disruption of HIF-2α-driven speckle association via deletion of its speckle targeting motifs (STMs)-defined in this study-led to defective induction of speckle-associating HIF-2α target genes without impacting non-speckle-associating HIF-2α target genes. We further identify the RNA export complex, TREX, as being specifically altered in speckle signature, and knockdown of key TREX component, ALYREF, also compromises speckle-associated gene expression. By integrating tissue culture functional studies with tumor genomic and imaging analysis, we show that HIF-2α gene regulatory programs are impacted by specific manipulation of speckle phenotype and by abrogation of speckle targeting abilities of HIF-2α. These findings suggest that, in ccRCC, a key biological function of nuclear speckles is to modulate expression of a specific subset of HIF-2α-regulated target genes that, in turn, influence patient outcomes. We also identify STMs in other transcription factors, suggesting that DNA-speckle targeting may be a general mechanism of gene regulation.

TOX2 coordinates with TET2 to positively regulate central memory differentiation in human CAR T cells.

Chimeric antigen receptor (CAR) T cell therapy is used in treating human hematological malignancies, but its efficacy is limited by T cell exhaustion (TEX). TEX arises at the expense of central memory T cells (TCM), which exhibit robust antitumor efficacy. Reduction of the TET2 gene led to increased TCM differentiation in a patient with leukemia who experienced a complete remission. We show that loss of TET2 led to increased chromatin accessibility at exhaustion regulators TOX and TOX2, plus increased expression of TOX2. Knockdown of TOX increased the percentage of TCM. However, unexpectedly, knockdown of TOX2 decreased TCM percentage and reduced proliferation. Consistently, a TCM gene signature was reduced in the TOX2 knockdown, and TOX2 bound to promoters of numerous TCM genes. Our results thus suggest a role for human TOX2, in contrast to exhaustion regulator TOX, as a potentiator of central memory differentiation of CAR T cells, with plausible utility in CAR T cell cancer therapy via modulated TOX2 expression.

Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

An NK-like CAR T cell transition in CAR T cell dysfunction.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.

p53 mediates target gene association with nuclear speckles for amplified RNA expression.

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.

Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis.

Metastasis is the leading cause of cancer-related deaths, and greater knowledge of the metastatic microenvironment is necessary to effectively target this process. Microenvironmental changes occur at distant sites prior to clinically detectable metastatic disease; however, the key niche regulatory signals during metastatic progression remain poorly characterized. Here, we identify a core immune suppression gene signature in pre-metastatic niche formation that is expressed predominantly by myeloid cells. We target this immune suppression program by utilizing genetically engineered myeloid cells (GEMys) to deliver IL-12 to modulate the metastatic microenvironment. Our data demonstrate that IL12-GEMy treatment reverses immune suppression in the pre-metastatic niche by activating antigen presentation and T cell activation, resulting in reduced metastatic and primary tumor burden and improved survival of tumor-bearing mice. We demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.

Dendritic Cell Paucity Leads to Dysfunctional Immune Surveillance in Pancreatic Cancer.

Here, we utilized spontaneous models of pancreatic and lung cancer to examine how neoantigenicity shapes tumor immunity and progression. As expected, neoantigen expression during lung adenocarcinoma development leads to T cell-mediated immunity and disease restraint. By contrast, neoantigen expression in pancreatic ductal adenocarcinoma (PDAC) results in exacerbation of a fibro-inflammatory microenvironment that drives disease progression and metastasis. Pathogenic TH17 responses are responsible for this neoantigen-induced tumor progression in PDAC. Underlying these divergent T cell responses in pancreas and lung cancer are differences in infiltrating conventional dendritic cells (cDCs). Overcoming cDC deficiency in early-stage PDAC leads to disease restraint, while restoration of cDC function in advanced PDAC restores tumor-restraining immunity and enhances responsiveness to radiation therapy.

TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion.

Exhausted CD8+ T (Tex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (Teff) or memory (Tmem) CD8+ T cells. Tex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, Tex cells are a distinct immune subset, with a unique chromatin landscape compared with Teff and Tmem cells. However, the mechanisms that govern the transcriptional and epigenetic development of Tex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of Tex cells in mice. TOX is largely dispensable for the formation of Teff and Tmem cells, but it is critical for exhaustion: in the absence of TOX, Tex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in Tex cells. Robust expression of TOX therefore results in commitment to Tex cells by translating persistent stimulation into a distinct Tex cell transcriptional and epigenetic developmental program.

p63 establishes epithelial enhancers at critical craniofacial development genes.

The transcription factor p63 is a key mediator of epidermal development. Point mutations in p63 in patients lead to developmental defects, including orofacial clefting. To date, knowledge on how pivotal the role of p63 is in human craniofacial development is limited. Using an inducible transdifferentiation model, combined with epigenomic sequencing and multicohort meta-analysis of genome-wide association studies data, we show that p63 establishes enhancers at craniofacial development genes to modulate their transcription. Disease-specific substitution mutation in the DNA binding domain or sterile alpha motif protein interaction domain of p63, respectively, eliminates or reduces establishment of these enhancers. We show that enhancers established by p63 are highly enriched for single-nucleotide polymorphisms associated with nonsyndromic cleft lip ± cleft palate (CL/P). These orthogonal approaches indicate a strong molecular link between p63 enhancer function and CL/P, illuminating molecular mechanisms underlying this developmental defect and revealing vital regulatory elements and new candidate causative genes.

Comparison of genotoxic versus nongenotoxic stabilization of p53 provides insight into parallel stress-responsive transcriptional networks.

The tumor suppressor protein p53 is activated in response to diverse intrinsic and extrinsic cellular stresses and controls a broad cell-protective gene network. Whether p53:DNA binding and subsequent transcriptional activation differs downstream of these diverse intrinsic and extrinsic activators is controversial. Using primary human fibroblasts, we assessed the genome-wide profile of p53 binding, chromatin structure, and transcriptional dynamics after either genotoxic or nongenotoxic activation of p53. Activation of p53 by treatment with either etoposide or the small-molecule MDM2 inhibitor nutlin 3A yields strikingly similar genome-wide binding of p53 and concomitant changes to local chromatin modifications and structure. DNA damage, but not p53 activation per se, leads to increased expression of genes in an inflammatory cytokine pathway. The NF-κB pathway inhibitor Bay 11-7082 abrogates etoposide-mediated activation of the inflammation gene signature but does not affect expression of canonical p53 target genes. Our data demonstrate that differential activation of p53 within the same cell type leads to highly similar genome-wide binding, chromatin dynamics, and gene expression dynamics and that DNA damage-mediated signaling through NF-κB likely controls the observed pro-inflammatory cytokine gene expression pattern.

Growth phase-specific changes in the composition of E. coli transcription complexes.

In E. coli, a single oligomeric enzyme transcribes the genomic DNA, while multiple auxiliary proteins and regulatory RNA interact with the core RNA polymerase (RP) during different stages of the transcription cycle to influence its function. In this work, using fast protein isolation techniques combined with mass spectrometry (MS) and immuno-analyses, we studied growth phase-specific changes in the composition of E. coli transcription complexes. We show that RP isolated from actively growing cells is represented by prevalent double copy assemblies and single copy RP-RNA and RP-RNA-RapA complexes. We demonstrate that RpoD/σ70 obtained in fast purification protocols carries tightly associated RNA and show evidence pointing to a role of sigma-associated RNA in the formation of native RP-(RNA)-RpoD/σ70 (holoenzyme) complexes. We report that enzymes linked functionally to the metabolism of lipopolysaccharides co-purify with RP-RNA complexes and describe two classes of RP-associated molecules (phospholipids and putative phospholipid-rNT species). We hypothesize that these modifications could enable anchoring of RP-RNA and RNA in cell membranes. We also report that proteins loosely associated with ribosomes and degradosomes (S1, Hfq) co-purify with RP-RNA complexes isolated from actively growing cells - a result consistent with their proposed roles as adaptor-proteins. In contrast, GroEL, SecB, and SecA co-purified with RP obtained from cells harvested in early stationary phase. Our results demonstrate that fast, affinity chromatography-based isolation of large multi-protein assemblies in combination with MS can be used as a tool for analysis of their composition and the profiling of small protein-associated molecules (SPAM).

The Role of BPIFA1 in Upper Airway Microbial Infections and Correlated Diseases.

The mucosa is part of the first line of immune defense against pathogen exposure in humans and prevents viral and bacterial infection of the soft palate, lungs, uvula, and nasal cavity that comprise the ear-nose-throat (ENT) region. Bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1) is a secretory protein found in human upper aerodigestive tract mucosa. This innate material is secreted in mucosal fluid or found in submucosal tissue in the human soft palate, lung, uvula, and nasal cavity. BPIFA1 is a critical component of the innate immune response that prevents upper airway diseases. This review will provide a brief introduction of the roles of BPIFA1 in the upper airway (with a focus on the nasal cavity, sinus, and middle ear), specifically its history, identification, distribution in various human tissues, function, and diagnostic value in various upper airway infectious diseases.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28.

KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity.

TRIM28 Controls Genomic Imprinting through Distinct Mechanisms during and after Early Genome-wide Reprogramming.

Genomic imprinting depends on the establishment and maintenance of DNA methylation at imprinting control regions. However, the mechanisms by which these heritable marks influence allele-specific expression are not fully understood. By analyzing maternal, zygotic, maternal-zygotic, and conditional Trim28 mutants, we found that the transcription factor TRIM28 controls genomic imprinting through distinct mechanisms at different developmental stages. During early genome-wide reprogramming, both maternal and zygotic TRIM28 are required for the maintenance of methylation at germline imprints. However, in conditional Trim28 mutants, Gtl2-imprinted gene expression was lost despite normal methylation levels at the germline IG-DMR. These results provide evidence that TRIM28 controls imprinting after early embryonic reprogramming through a mechanism other than the maintenance of germline imprints. Additionally, our finding that secondary imprints were hypomethylated in TRIM28 mutants uncovers a requirement of TRIM28 after genome-wide reprogramming for interpreting germline imprints and regulating DNA methylation at imprinted gene promoters.