Gα13 restricts nutrient driven proliferation in mucosal germinal centers.

Germinal centers (GCs) that form in mucosal sites are exposed to gut-derived factors that have the potential to influence homeostasis independent of antigen receptor-driven selective processes. The G-protein Gα13 confines B cells to the GC and limits the development of GC-derived lymphoma. We discovered that Gα13-deficiency fuels the GC reaction via increased mTORC1 signaling and Myc protein expression specifically in the mesenteric lymph node (mLN). The competitive advantage of Gα13-deficient GC B cells (GCBs) in mLN was not dependent on T cell help or gut microbiota. Instead, Gα13-deficient GCBs were selectively dependent on dietary nutrients likely due to greater access to gut lymphatics. Specifically, we found that diet-derived glutamine supported proliferation and Myc expression in Gα13-deficient GCBs in the mLN. Thus, GC confinement limits the effects of dietary glutamine on GC dynamics in mucosal tissues. Gα13 pathway mutations coopt these processes to promote the gut tropism of aggressive lymphoma.

SUMO promotes DNA repair protein collaboration to support alternative telomere lengthening in the absence of PML.

The alternative lengthening of telomeres (ALT) pathway maintains telomere length in a significant fraction of cancers that are associated with poor clinical outcomes. A better understanding of ALT mechanisms is therefore necessary for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins contributes to the formation of ALT telomere-associated PML bodies (APBs), in which telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, it is still unknown whether-and if so, how-SUMO supports ALT beyond APB formation. Here, we show that SUMO condensates that contain DNA repair proteins enable telomere maintenance in the absence of APBs. In PML knockout ALT cell lines that lack APBs, we found that SUMOylation is required for manifesting ALT features independent of PML and APBs. Chemically induced telomere targeting of SUMO produces condensate formation and ALT features in PML-null cells. This effect requires both SUMOylation and interactions between SUMO and SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are associated with the accumulation of DNA repair proteins, including Rad52, Rad51AP1, RPA, and BLM, at telomeres. Furthermore, Rad52 can undergo phase separation, enrich SUMO at telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that SUMO condensate formation promotes collaboration among DNA repair factors to support ALT telomere maintenance without PML. Given the promising effects of SUMOylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure.

RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.

Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling.

PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling.

RAD51AP1 regulates ALT-HDR through chromatin-directed homeostasis of TERRA.

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.

Competition between PRC2.1 and 2.2 subcomplexes regulates PRC2 chromatin occupancy in human stem cells.

Polycomb repressive complex 2 (PRC2) silences expression of developmental transcription factors in pluripotent stem cells by methylating lysine 27 on histone H3. Two mutually exclusive subcomplexes, PRC2.1 and PRC2.2, are defined by the set of accessory proteins bound to the core PRC2 subunits. Here we introduce separation-of-function mutations into the SUZ12 subunit of PRC2 to drive it into a PRC2.1 or 2.2 subcomplex in human induced pluripotent stem cells (iPSCs). We find that PRC2.2 occupies polycomb target genes at low levels and that homeobox transcription factors are upregulated when this complex is exclusively present. In contrast with previous studies, we find that chromatin occupancy of PRC2 increases drastically when it is forced to form PRC2.1. Additionally, several cancer-associated mutations also coerce formation of PRC2.1. We suggest that PRC2 chromatin occupancy can be altered in the context of disease or development by tuning the ratio of PRC2.1 to PRC2.2.

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.

Regulation of histone methylation by automethylation of PRC2.

Polycomb-repressive complex 2 (PRC2) is a histone methyltransferase that is critical for regulating transcriptional repression in mammals. Its catalytic subunit, EZH2, is responsible for the trimethylation of H3K27 and also undergoes automethylation. Using mass spectrometry analysis of recombinant human PRC2, we identified three methylated lysine residues (K510, K514, and K515) on a disordered but highly conserved loop of EZH2. Methylation of these lysines increases PRC2 histone methyltransferase activity, whereas their mutation decreases activity in vitro. De novo histone methylation in an EZH2 knockout cell line is greatly impeded by mutation of the automethylation lysines. EZH2 automethylation occurs intramolecularly (in cis) by methylation of a pseudosubstrate sequence on a flexible loop. This posttranslational modification and cis regulation of PRC2 are analogous to the activation of many protein kinases by autophosphorylation. We propose that EZH2 automethylation allows PRC2 to modulate its histone methyltransferase activity by sensing histone H3 tails, SAM concentration, and perhaps other effectors.

Author Correction: A naturally occurring antiviral ribonucleotide encoded by the human genome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Deportations and departures: Undocumented Mexican immigrants' return migration during three presidential administrations.

This study examines changes in the sociodemographic patterns of deportation and voluntary return of undocumented immigrants from the United States to Mexico during three US presidential administrations (2001 to 2019) with different immigration policies. Most previous studies examining these migration flows for the United States as a whole have relied exclusively on counts of deportees and returnees, thereby ignoring changes over the past 20 y in the characteristics of the undocumented population itself, i.e., the population at risk of deportation or voluntary return. We estimate Poisson models based on two data sources that permit us to compare changes in the sex, age, education, and marital status distributions of both deportees and voluntary return migrants with the corresponding changes in the undocumented population during the Bush, Obama, and Trump administrations: the Migration Survey on the Borders of Mexico-North (Encuesta sobre Migración en las Fronteras de México-Norte) for counts of deportees and voluntary return migrants and the Current Population Survey's Annual Social and Economic Supplement for estimated counts of the undocumented population living in the United States. We find that whereas disparities by sociodemographic characteristics in the likelihood of deportation generally increased beginning in Obama's first term, sociodemographic disparities in the likelihood of voluntary return generally decreased over this period. Despite heightened antiimmigrant rhetoric during the Trump administration, the changes in deportation and voluntary return migration to Mexico among the undocumented during Trump's term were part of a trend that began early in the Obama administration.

PRC2 direct transfer from G-quadruplex RNA to dsDNA has implications for RNA-binding chromatin modifiers.

The chromatin-modifying enzyme, Polycomb Repressive Complex 2 (PRC2), deposits the H3K27me3 epigenetic mark to negatively regulate expression at numerous target genes, and this activity has been implicated in embryonic development, cell differentiation, and various cancers. A biological role for RNA binding in regulating PRC2 histone methyltransferase activity is generally accepted, but the nature and mechanism of this relationship remains an area of active investigation. Notably, many in vitro studies demonstrate that RNA inhibits PRC2 activity on nucleosomes through mutually antagonistic binding, while some in vivo studies indicate that PRC2's RNA-binding activity is critical for facilitating its biological function(s). Here we use biochemical, biophysical, and computational approaches to interrogate PRC2's RNA and DNA-binding kinetics. Our findings demonstrate that PRC2-polynucleotide dissociation rates are dependent on the concentration of free ligand, indicating the potential for direct transfer between nucleic acid ligands without a free-enzyme intermediate. Direct transfer explains the variation in previously reported dissociation kinetics, allows reconciliation of prior in vitro and in vivo studies, and expands the potential mechanisms of RNA-mediated PRC2 regulation. Moreover, simulations indicate that such a direct transfer mechanism could be obligatory for RNA to recruit proteins to chromatin.

Structures of Vac8-containing protein complexes reveal the underlying mechanism by which Vac8 regulates multiple cellular processes.

Vac8, a yeast vacuolar protein with armadillo repeats, mediates various cellular processes by changing its binding partners; however, the mechanism by which Vac8 differentially regulates these processes remains poorly understood. Vac8 interacts with Nvj1 to form the nuclear-vacuole junction (NVJ) and with Atg13 to mediate cytoplasm-to-vacuole targeting (Cvt), a selective autophagy-like pathway that delivers cytoplasmic aminopeptidase I directly to the vacuole. In addition, Vac8 associates with Myo2, a yeast class V myosin, through its interaction with Vac17 for vacuolar inheritance from the mother cell to the emerging daughter cell during cell divisions. Here, we determined the X-ray crystal structure of the Vac8-Vac17 complex and found that its interaction interfaces are bipartite, unlike those of the Vac8-Nvj1 and Vac8-Atg13 complexes. When the key amino acids present in the interface between Vac8 and Vac17 were mutated, vacuole inheritance was severely impaired in vivo. Furthermore, binding of Vac17 to Vac8 prevented dimerization of Vac8, which is required for its interactions with Nvj1 and Atg13, by clamping the H1 helix to the ARM1 domain of Vac8 and thereby preventing exposure of the binding interface for Vac8 dimerization. Consistently, the binding affinity of Vac17-bound Vac8 for Nvj1 or Atg13 was markedly lower than that of free Vac8. Likewise, free Vac17 had no affinity for the Vac8-Nvj1 and Vac8-Atg13 complexes. These results provide insights into how vacuole inheritance and other Vac8-mediated processes, such as NVJ formation and Cvt, occur independently of one another.

A novel mechanoeffector role of fibroblast S100A4 in myofibroblast transdifferentiation and fibrosis.

Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.

The Economic Iceberg of Celiac Disease: More Than the Cost of Gluten-Free Food.

Celiac disease (CeD) is a chronic autoimmune disorder of global relevance, with the potential for acute and long-term complications. However, the economic burden of CeD is rarely considered and largely thought of as limited to the cost of gluten-free food. Fortunately, recent research has shed light on the various societal costs of CeD across the health care continuum. This article summarizes the current evidence on the economic impacts of CeD, which suggest that the societal economic burden of CeD stretches beyond the cost of gluten-free food. This review provides ample evidence of larger but hidden costs related to excess health care use for complications and comorbidities, as well as reduced productivity. Although significant advances are expected in the management of CeD, their effect on the economic burden of CeD remain uncertain. The aim of this review was to inform stakeholders across society and contribute to improved policies to support patients with CeD.

DNMT1 inhibition by pUG-fold quadruplex RNA.

Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.

Targeting of Polycomb Repressive Complex 2 to RNA by Short Repeats of Consecutive Guanines.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that trimethylates H3K27, a mark of repressed chromatin. Mammalian PRC2 binds RNA promiscuously, with thousands of target transcripts in vivo. But what does PRC2 recognize in these RNAs? Here we show that purified human PRC2 recognizes G > C,U ≫ A in single-stranded RNA and has a high affinity for folded guanine quadruplex (G4) structures but little binding to duplex RNAs. Importantly, G-tract motifs are significantly enriched among PRC2-binding transcripts in vivo. DNA sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on chromatin and H3K27me3-modified nucleosomes. Collectively, the abundance of PRC2-binding RNA motifs rationalizes the promiscuous RNA binding of PRC2, and their enrichment at Polycomb target genes provides a means for RNA-mediated regulation.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

Break Wave Lithotripsy for Urolithiasis: Results of the First-in-Human International Multi-Institutional Clinical Trial.

PURPOSE: This study reports on a prospective, multicenter, single-arm, clinical trial utilizing the SonoMotion (San Mateo, California) Break Wave lithotripsy (BWL) device to fragment urinary stones. MATERIALS AND METHODS: Patients with a urinary stone underwent a single treatment of 30 minutes and peak negative pressure of 4.5 to 8 MPa. Subjects were contacted and outcomes assessed at 7, 14, and 35 days after treatment, with clinical follow-up and CT imaging 70 ± 14 days postprocedure. The primary objectives were to assess the safety (hematomas, complications, etc) and effectiveness of BWL (any fragmentation, residual fragments ≤4 mm or ≤2 mm, and completely stone-free rate) as assessed via noncontrast CT-kidneys, ureters, and bladder. RESULTS: Forty-four patients with a ureteral (43%) or renal (57%) stone were treated across 5 centers. Stone fragmentation occurred in 88% of cases; 70% had fragments ≤ 4 and 51% ≤ 2 mm, while 49% were completely stone free on CT; no serious adverse events were reported. Eighty-six percent of patients received either no analgesic medication at all (50%) or minor analgesia (36%). After determining optimal therapy settings, 36 patients were treated and the effectiveness improved exhibiting fragmentation in 92% (33/36), residual fragments ≤ 4 mm in 75% and 58% with fragments ≤ 2 mm with 58% completely stone free. Effectiveness was less in subjects with lower pole stones with 81% fragmentation, 71% having fragments ≤ 4 mm, 29% with fragments ≤ 2 mm, and 29% completely stone free; of distal ureteral stone patients, 89% were completely stone free. CONCLUSIONS: BWL offered safe and effective noninvasive stone therapy requiring little to no anesthesia and was carried out successfully in nonoperative environments. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03811171.

Change in Volumes and Location of Preterm White Matter Injury over a Period of 15 Years.

OBJECTIVE: To evaluate whether white matter injury (WMI) volumes and spatial distribution, which are important predictors of neurodevelopmental outcomes in preterm infants, have changed over a period of 15 years. STUDY DESIGN: Five hundred and twenty-eight infants born <32 weeks' gestational age from 2 sequential prospective cohorts (cohort 1: 2006 through 2012; cohort 2: 2014 through 2019) underwent early-life (median 32.7 weeks postmenstrual age) and/or term-equivalent-age MRI (median 40.7 weeks postmenstrual age). WMI were manually segmented for quantification of volumes. There were 152 infants with WMI with 74 infants in cohort 1 and 78 in cohort 2. Multivariable linear regression models examined change in WMI volume across cohorts while adjusting for clinical confounders. Lesion maps assessed change in WMI location across cohorts. RESULTS: There was a decrease in WMI volume in cohort 2 compared with cohort 1 (ß = -0.6, 95% CI [-0.8, -0.3], P < .001) with a shift from more central to posterior location of WMI. There was a decrease in clinical illness severity of infants across cohorts. CONCLUSIONS: We found a decrease in WMI volume and shift to more posterior location in very preterm infants over a period of 15 years. This may potentially reflect more advanced maturation of white matter at the time of injury which may be related to changes in clinical practice over time.

Incidence of checkpoint inhibitor-associated inflammatory arthritis, immunomodulation and mortality in cancer patients on immunotherapy: a retrospective cohort study.

OBJECTIVES: Immune checkpoint inhibitor (ICI) associated inflammatory arthritis (ICI-IA) occurs in 4-6% of ICI-treated patients based on one observational study. We identified cases of ICI-IA using administrative claims to study its incidence and characteristics at the population level. METHODS: We used the Medicare 5% sample to identify patients initiating ICIs. Cancer patients were identified by having ≥ 2 ICD-9/10-CM diagnosis codes from an oncologist for lung cancer, melanoma, or renal/urothelial cancer. ICI-IA was defined as having two Medicare claims ≥ 30 days apart with combinations of ICD-9/10-CM diagnosis codes that favored specificity. ICI-IA was identified in patients with a musculoskeletal diagnosis after ICI initiation, who had i.) no inflammatory arthritis or inflammatory rheumatic disease before ICI initiation ever, and ii) no musculoskeletal complaint in the one year prior to ICI. We examined DMARD utilization and visits to rheumatology in patients with ICI-IA. Landmark analysis and a time varying Cox proportional hazards model for overall survival was constructed. RESULTS: The incidence of ICI-IA was 7.2 (6.1-8.4) per 100 patient years. Patients with ICI-IA were mean (SD) age 73.5(7.0) years, 48% women, 91% white. Median(IQR) time from ICI initiation to first ICI-IA diagnosis was 124(56, 252) days. Only 24(16%) received care from a rheumatologist, and 24(16%) were prescribed a DMARD (46% by a rheumatologist). The HR for mortality in patients with ICI-IA was 0.86 (95% CI 0.59-1.26, p= 0.45). CONCLUSIONS: The incidence of ICI-IA identified in claims data is similar to that reported in observational studies, however, few patients are treated with a DMARD or see a rheumatologist. There was no difference in overall survival between ICI-treated patients with and without ICI-IA.