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The 11-subunit RNA exosome is thought to regulate the mammalian noncoding transcriptome; here, a mouse model is generated in which the essential Exosc3 subunit of the RNA exosome in B cells is conditionally deleted, revealing a link between sites of genomic RNA exosome function and AID-mediated chromosomal translocations. Noncoding RNAs pinpoint AID in B cells It is difficult to identify rare non-coding RNA (ncRNA) species because of their low abundance in cells and the fact that they are rapidly degraded, mainly through the action of the cellular non-coding RNA 3′–5′ degradation complex, RNA exosome. Uttiya Basu and colleagues have generated a mouse model in which an essential subunit (exosome component 3, Exosc3 ) of the RNA exosome can be conditionally inactivated in B cells. Exosc3-deficient B cells lack the recombination and mutagenesis activities that are necessary for generating antibodies. Many non-coding RNAs normally degraded are found in these cells, including xTSS-RNAs, a type of antisense RNA encoded at transcription start sites. Surprisingly, the locations of the xTSS-RNAs correlate with sites of translocation breakages. The model suggested is that antisense transcription of the ncRNAs recruits activation-induced cytidine deaminase (AID) and results in formation of single-strand DNA; pairing with the RNAs makes R-loops that can lead to genomic instability. The vast majority of the mammalian genome has the potential to express noncoding RNA (ncRNA). The 11-subunit RNA exosome complex is the main source of cellular 3′–5′ exoribonucleolytic activity and potentially regulates the mammalian noncoding transcriptome 1 . Here we generated a mouse model in which the essential subunit Exosc3 of the RNA exosome complex can be conditionally deleted. Exosc3 -deficient B cells lack the ability to undergo normal levels of class switch recombination and somatic hypermutation, two mutagenic DNA processes used to generate antibody diversity via the B-cell mutator protein activation-induced cytidine deaminase (AID) 2 , 3 . The transcriptome of Exosc3 -deficient B cells has revealed the presence of many novel RNA exosome substrate ncRNAs. RNA exosome substrate RNAs include xTSS-RNAs, transcription start site (TSS)-associated antisense transcripts that can exceed 500 base pairs in length and are transcribed divergently from cognate coding gene transcripts. xTSS-RNAs are most strongly expressed at genes that accumulate AID-mediated somatic mutations and/or are frequent translocation partners of DNA double-strand breaks generated at Igh in B cells 4 , 5 . Strikingly, translocations near TSSs or within gene bodies occur over regions of RNA exosome substrate ncRNA expression. These RNA exosome-regulated, antisense-transcribed regions of the B-cell genome recruit AID and accumulate single-strand DNA structures containing RNA–DNA hybrids. We propose that RNA exosome regulation of ncRNA recruits AID to single-strand DNA-forming sites of antisense and divergent transcription in the B-cell genome, thereby creating a link between ncRNA transcription and overall maintenance of B-cell genomic integrity.

Noncoding RNAs are exquisitely titrated by the cellular RNA surveillance machinery for regulating diverse biological processes. The RNA exosome, the predominant 3′ RNA exoribonuclease in mammalian cells, is composed of nine core and two catalytic subunits. Here, we developed a mouse model with a conditional allele to study the RNA exosome catalytic subunit DIS3. In DIS3-deficient B cells, integrity of the immunoglobulin heavy chain ( Igh ) locus in its topologically associating domain is affected, with accumulation of DNA-associated RNAs flanking CTCF-binding elements, decreased CTCF binding to CTCF-binding elements and disorganized cohesin localization. DIS3-deficient B cells also accumulate activation-induced cytidine deaminase–mediated asymmetric nicks, altering somatic hypermutation patterns and increasing microhomology-mediated end-joining DNA repair. Altered mutation patterns and Igh architectural defects in DIS3-deficient B cells lead to decreased class-switch recombination but increased chromosomal translocations. Our observations of DIS3-mediated architectural regulation at the Igh locus are reflected genome wide, thus providing evidence that noncoding RNA processing is an important mechanism for controlling genome organization. DIS3 deficiency alters CTCF and cohesin binding at the Igh locus in B cells. These cells show decreased class-switch recombination but increased chromosomal translocations.

Autotaxin (ATX, Enpp2) is a secreted lysophospholipase D catalysing the production of lysophosphatidic acid, a pleiotropic growth factor-like lysophospholipid. Increased ATX expression has been detected in a number of chronic inflammatory diseases and different types of cancer, while genetic interventions have proven a role for ATX in disease pathogenesis. Therefore, ATX has emerged as a potential drug target and a large number of ATX inhibitors have been developed exhibiting promising therapeutic potential. However, the embryonic lethality of ATX null mice and the ubiquitous expression of ATX and LPA receptors in adult life question the suitability of ATX as a drug target. Here we show that inducible, ubiquitous genetic deletion of ATX in adult mice, as well as long-term potent pharmacologic inhibition, are well tolerated, alleviating potential toxicity concerns of ATX therapeutic targeting.

The angiopoietin (ANGPT)–TIE2/TEK signaling pathway is essential for blood and lymphatic vascular homeostasis. ANGPT1 is a potent TIE2 activator, whereas ANGPT2 functions as a context-dependent agonist/antagonist. In disease, ANGPT2-mediated inhibition of TIE2 in blood vessels is linked to vascular leak, inflammation, and metastasis. Using conditional knockout studies in mice, we show TIE2 is predominantly activated by ANGPT1 in the cardiovascular system and by ANGPT2 in the lymphatic vasculature. Mechanisms underlying opposing actions of ANGPT2 in blood vs. lymphatic endothelium are poorly understood. Here we show the endothelial-specific phosphatase VEPTP (vascular endothelial protein tyrosine phosphatase) determines TIE2 response to ANGPT2. VEPTP is absent from lymphatic endothelium in mouse in vivo, permitting ANGPT2/TIE2-mediated lymphangiogenesis. Inhibition of VEPTP converts ANGPT2 into a potent TIE2 activator in blood endothelium. Our data support a model whereby VEPTP functions as a rheostat to modulate ANGPT2 ligand effect on TIE2.

Fibrodysplasia ossificans progressiva (FOP) is a rare disease characterized by heterotopic ossification (HO) in connective tissues and painful flare-ups. In the phase 2 LUMINA-1 trial, adult patients with FOP were randomized to garetosmab, an activin A-blocking antibody ( n  = 20) or placebo ( n  = 24) in period 1 (28 weeks), followed by an open-label period 2 (28 weeks; n  = 43). The primary end points were safety and for period 1, the activity and size of HO lesions. All patients experienced at least one treatment-emergent adverse event during period 1, notably epistaxis, madarosis and skin abscesses. Five deaths (5 of 44; 11.4%) occurred in the open-label period and, while considered unlikely to be related, causality cannot be ruled out. The primary efficacy end point in period 1 (total lesion activity by PET–CT) was not met ( P  = 0.0741). As the development of new HO lesions was suppressed in period 1, the primary efficacy end point in period 2 was prospectively changed to the number of new HO lesions versus period 1. No placebo patients crossing over to garetosmab developed new HO lesions (0% in period 2 versus 40.9% in period 1; P  = 0.0027). Further investigation of garetosmab in FOP is ongoing. ClinicalTrials.gov identifier NCT03188666 . In the LUMINA-1 trial for fibrodysplasia ossificans progressiva, garetosmab, an activin A monoclonal antibody, did not lead to significant changes in heterotopic ossification lesion activity in pre-existing lesions in period 1. Garetosmab prevented the formation of new lesions in both periods 1 and 2.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by activin A, an effect countered by inhibition of anti-activin A via monoclonal antibody treatment. Hence, we surmised that anti-ACVR1 antibodies that block activation of ACVR1 by ligands should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated anti-ACVR1 monoclonal antibodies that block ACVR1's activation by its ligands. Surprisingly, in vivo, these anti-ACVR1 antibodies stimulated HO and activated signaling of FOP-mutant ACVR1. This property was restricted to FOP-mutant ACVR1 and resulted from anti-ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild-type ACVR1 was inhibited by anti-ACVR1 antibodies. These results uncover an additional property of FOP-mutant ACVR1 and indicate that anti-ACVR1 antibodies should not be considered as therapeutics for FOP.

Wnt3a is a major regulator of bone metabolism however, very few of its target genes are known in bone. Wnt3a preferentially signals through transmembrane receptors Frizzled and co-receptors Lrp5/6 to activate the canonical signaling pathway. Previous studies have shown that the canonical Wnt co-receptors Lrp5 and Lrp6 also play an essential role in normal postnatal bone homeostasis, yet, very little is known about specific contributions by these co-receptors in Wnt3a-dependent signaling. We used high-throughput sequencing technology to identify target genes regulated by Wnt3a in osteoblasts and to elucidate the role of Lrp5 and Lrp6 in mediating Wnt3a signaling. Our study identified 782 genes regulated by Wnt3a in primary calvarial osteoblasts. Wnt3a up-regulated the expression of several key regulators of osteoblast proliferation/ early stages of differentiation while inhibiting genes expressed in later stages of osteoblastogenesis. We also found that Lrp6 is the key mediator of Wnt3a signaling in osteoblasts and Lrp5 played a less significant role in mediating Wnt3a signaling.

The Wnt antagonist Sost has emerged as a key regulator of bone homeostasis through the modulation of Lrp4/5/6 Wnt coreceptors. In humans, lack of Sclerostin causes sclerosteosis and van Buchem (VB) disease, two generalized skeletal hyperostosis disorders that result from hyperactive Wnt signaling. Unlike sclerosteosis, VB patients lack SOST coding mutations but carry a homozygous 52 kb noncoding deletion that is essential for the transcriptional activation of SOST in bone. We recently identified a putative bone enhancer, ECR5 , in the VB deletion region, and showed that the transcriptional activity of ECR5 is controlled by Mef2C transcription factor in vitro. Here we report that mice lacking ECR5 or Mef2C through Col1-Cre osteoblast/osteocyte-specific ablation result in high bone mass (HBM) due to elevated bone formation rates. We conclude that the absence of the Sost -specific long-range regulatory element ECR5 causes VB disease in rodents, and that Mef2C is the main transcription factor responsible for ECR5 -dependent Sost transcriptional activation in the adult skeleton.

Activin A functions in BMP signaling in two ways: it either engages ACVR1B to activate Smad2/3 signaling or binds ACVR1 to form a non-signaling complex (NSC). Although the former property has been studied extensively, the roles of the NSC remain unexplored. The genetic disorder fibrodysplasia ossificans progressiva (FOP) provides a unique window into ACVR1/Activin A signaling because in that disease Activin can either signal through FOP-mutant ACVR1 or form NSCs with wild-type ACVR1. To explore the role of the NSC, we generated ‘agonist-only’ Activin A muteins that activate ACVR1B but cannot form the NSC with ACVR1. Using one of these muteins, we demonstrate that failure to form the NSC in FOP results in more severe disease pathology. These results provide the first evidence for a biological role for the NSC in vivo and pave the way for further exploration of the NSC’s physiological role in corresponding knock-in mice.

Anterior cruciate ligament (ACL) injuries often result in post-traumatic osteoarthritis (PTOA). To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced transcriptional changes in knee joints of three mouse strains with varying susceptibility to OA: STR/ort (highly susceptible), C57BL/6J (moderately susceptible) and super-healer MRL/MpJ (not susceptible). Right knee joints of the mice were injured using a non-invasive tibial compression injury model and global gene expression was quantified before and at 1-day, 1-week, and 2-weeks post-injury using RNA-seq. Following injury, injured and uninjured joints of STR/ort and injured C57BL/6J joints displayed significant cartilage degeneration while MRL/MpJ had little cartilage damage. Gene expression analysis suggested that prolonged inflammation and elevated catabolic activity in STR/ort injured joints, compared to the other two strains may be responsible for the severe PTOA phenotype observed in this strain. MRL/MpJ had the lowest expression values for several inflammatory cytokines and catabolic enzymes activated in response to ACL injury. Furthermore, we identified several genes highly expressed in MRL/MpJ compared to the other two strains including B4galnt2 and Tpsab1 which may contribute to enhanced healing in the MRL/MpJ. Overall, this study has increased our knowledge of early molecular changes associated with PTOA development.