Double Knockout Mice Research Articles

Rapid and Accurate Remethylation of Dnmt3a Deficient Hematopoietic Cells with Restoration of DNMT3A Activity

Heterozygous loss-of-function mutations in the DNMT3A gene are the most common cause of clonal hematopoiesis, and among the most common initiating events for Acute Myeloid Leukemia (AML). A reduction of DNMT3A activity causes a canonical, focal hypomethylation phenotype in hematopoietic cells, which is associated with immortalization of hematopoietic stem cells, and blockage of myeloid differentiation. The methylation defect can be partially reversed with restoration of physiologic levels of DNMT3A expression over a period of several months (Ketkar et al., PNAS, 2020; PMID 35313694).To identify a more efficient remethylation strategy, we first characterized the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic cell deficiency of Dnmt3a, Dnmt3b (or both enzymes), or expressing the dominant negative Dnmt3aR878H mutation (equivalent to R882H in humans; the most common mutation found in AML patients). Using these different mouse models as substrates, we then defined the patterns and completeness of DNA remethylation after “adding back” supraphysiologic levels of wild type DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive “chaperone” that augments the activity of DNMT3A and DNMT3B in early embryogenesis), with MSCV-based retroviruses transduced into primary mouse hematopoietic stem/progenitor cell s. Overexpression of WT DNMT3A for 2 weeks in vitro (~3 fold) can accurately reverse the differentially methylated regions (DMRs, which are >99% hypomethylated) of Dnmt3a deficient hematopoietic cells, or cells expressing the R878H mutation. The DMRs of Dnmt3b deficient mouse bone marrow cells can be corrected by overexpression of DNMT3A, DNMT3B1, or DNMT3B3; however, DNMT3B3 failed to remethylate the DMRs in Dnmt3a/Dnmt3b double knockout mouse bone marrow cells. Since DNMT3B3 is an inactive enzyme that does not cause remethylation in the absence of DNMT3A, these data suggest that DNMT3B3 functions in a DNMT3A-dependent manner in hematopoietic cells. Importantly, both DNMT3B3 and DNMT3L have been shown to facilitate DNA methylation by acting as a chaperone for DNMT3A (Duymich et al., Nat. Communication, 2016; PMID 27121154). In vitro, DNMT3L copurified with DNMT3A leads to an increase in DNMT3A methyltransferase activity that is ~5 times greater than DNMT3B3 copurified with DNMT3A. Remarkably, overexpression of DNMT3L (which is not expressed in adult hematopoietic cells, or AML cells) can completely correct the hypomethylation phenotype of Dnmt3aR878H/+ bone marrow cells within 2 weeks of in vitro overexpression, or 4 weeks of in vivo overexpression ( Figure 1A and 1B), probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele in these heterozygous mutant cells. Two weeks of overexpression of DNMT3A or DNMT3L in Dnmt3aR878H/+ bone marrow cells (cultured in vitro with IL-3, SCF, FLT3L, and TPO to maintain HSPC populations) can induce a differentiation response, increasing the fraction of mature myeloid cells in the cultures by ~4-fold. This finding was recapitulated in vivo where one month of overexpression of DNMT3A or DNMT3L in transplanted Dnmt3aR878H/+ mouse bone marrow cells increased the fraction of mature myeloid cells by 2-fold by DNMT3L and 4-fold by DNMT3A. Together,these data show that the focal, canonical DNA hypomethylation phenotype of Dnmt3a R878H/+ hematopoietic cells can be accurately and efficiently corrected by overexpressing WT DNMT3A or DNMT3L for several weeks. We have also shown that the restoration of methylation in Dnmt3a R878H/+ mutant cells alters the developmental fate of progenitors, increasing the proportion of mature myeloid cells. These data suggest that DNMT3L expression may represent a novel approach for restoring DNMT3A activity in AMLs initiated by DNMT3A mutations, and could possibly alter AML cell fate. Additional studies are underway to determine whether the correction of DNMT3A activity in Dnmt3aR878H/+ -initiated AML cells with retroviral addback of DNMT3A or DNMT3L will cause a differentiation response that alters AML cell fate.

Mmp2 Deficiency Leads to Defective Parturition and High Dystocia Rates in Mice.

Parturition is the final and essential step for mammalian reproduction. While the uterus is quiescent during pregnancy, fundamental changes arise in the myometrial contractility, inducing fetal expulsion. Extracellular matrix (ECM) remodeling is fundamental for these events. The gelatinases subgroup of matrix metalloproteinases (MMPs), MMP2 and MMP9, participate in uterine ECM remodeling throughout pregnancy and parturition. However, their loss-of-function effect is unknown. Here, we determined the result of eliminating Mmp2 and/or Mmp9 on parturition in vivo, using single- and double-knockout (dKO) mice. The dystocia rates were measured in each genotype, and uterine tissue was collected from nulliparous synchronized females at the ages of 2, 4, 9 and 12 months. Very high percentages of dystocia (40-55%) were found in the Mmp2-/- and dKO females, contrary to the Mmp9-/- and wild-type females. The histological analysis of the uterus and cervix revealed that Mmp2-/- tissues undergo marked structural alterations, including highly enlarged myometrial, endometrial and luminal cavity. Increased collagen deposition was also demonstrated, suggesting a mechanism of extensive fibrosis in the Mmp2-/- myometrium, which may result in dystocia. Overall, this study describes a new role for MMP2 in myometrium remodeling during mammalian parturition process, highlighting a novel cause for dystocia due to a loss in MMP2 activity in the uterine tissue.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

Endothelial nitric oxide synthase (eNOS) S1176 phosphorylation status governs atherosclerotic lesion formation.

We have previously demonstrated the in vivo importance of the Akt-eNOS substrate-kinase relationship, as defective postnatal angiogenesis characteristic of global Akt1-null mice is rescued when bred to 'gain-of-function' eNOS S1176D mutant mice. While multiple studies support the vascular protective role of endothelial NO generation, the causal role of Akt1-dependent eNOS S1176 phosphorylation during atherosclerotic plaque formation is not yet clear. We herein bred congenic 'loss-of-function' eNOS S1176A and 'gain-of-function' eNOS S1176D mutant mice to the exacerbated atherogenic Akt1-/-; ApoE-/- double knockout mice to definitively test the importance of Akt-mediated eNOS S1176 phosphorylation during atherogenesis. We find that a single amino acid substitution at the eNOS S1176 phosphorylation site yields divergent effects on atherosclerotic plaque formation, as an eNOS phospho-mimic aspartate (D) substitution at S1176 leads to favorable lipid profiles and decreased indices of atherosclerosis, even when on a proatherogenic Akt1 global deletion background. Conversely, mice harboring an unphosphorylatable mutation to alanine (S1176A) result in increased plasma lipids, increased lesion formation and cellular apoptosis, phenocopying the physiological consequence of eNOS deletion and/or impaired enzyme function. Furthermore, gene expression analyses of whole aortas indicate a combinatorial detriment from NO deficiency and Western Diet challenge, as 'loss-of-function' eNOS S1176A mice on a Western Diet present a unique expression pattern indicative of augmented T-cell activity when compared to eNOS S1176D mice. By using genetic epistasis approaches, we conclusively demonstrate that Akt-mediated eNOS S1176 phosphorylation and subsequent eNOS activation remains to be the most physiologically relevant method of NO production to promote athero-protective effects.

Ablation of Long Noncoding RNA Hoxb3os Exacerbates Cystogenesis in Mouse Polycystic Kidney Disease.

Long noncoding RNAs (lncRNAs) are a class of nonprotein coding RNAs with pivotal functions in development and disease. They have emerged as an exciting new drug target category for many common conditions. However, the role of lncRNAs in autosomal dominant polycystic kidney disease (ADPKD) has been understudied. This study provides evidence implicating a lncRNA in the pathogenesis of ADPKD. We report that Hoxb3os is downregulated in ADPKD and regulates mammalian target of rapamycin (mTOR)/Akt pathway in the in vivo mouse kidney. Ablating the expression of Hoxb3os in mouse polycystic kidney disease (PKD) activated mTOR complex 2 (mTORC2) signaling and exacerbated the cystic phenotype. The results from our study provide genetic proof of concept for future studies that focus on targeting lncRNAs as a treatment option in PKD. ADPKD is a monogenic disorder characterized by the formation of kidney cysts and is primarily caused by mutations in two genes, PKD1 and PKD2 . In this study, we investigated the role of lncRNA Hoxb3os in ADPKD by ablating its expression in the mouse. Hoxb3os -null mice were viable and had grossly normal kidney morphology but displayed activation of mTOR/Akt signaling and subsequent increase in kidney cell proliferation. To determine the role of Hoxb3os in cystogenesis, we crossed the Hoxb3os -null mouse to two orthologous Pkd1 mouse models: Pkhd1/Cre; Pkd1F/F (rapid cyst progression) and Pkd1RC/RC (slow cyst progression). Ablation of Hoxb3os exacerbated cyst growth in both models. To gain insight into the mechanism whereby Hoxb3os inhibition promotes cystogenesis, we performed western blot analysis of mTOR/Akt pathway between Pkd1 single-knockout and Pkd1 - Hoxb3os double-knockout (DKO) mice. Compared with single-knockout, DKO mice presented with enhanced levels of total and phosphorylated Rictor. This was accompanied by increased phosphorylation of Akt at Ser 473 , a known mTORC2 effector site. Physiologically, kidneys from DKO mice displayed between 50% and 60% increase in cell proliferation and cyst number. The results from this study indicate that ablation of Hoxb3os in mouse PKD exacerbates cystogenesis and dysregulates mTORC2.

The katanin A-subunits KATNA1 and KATNAL1 act co-operatively in mammalian meiosis and spermiogenesis to achieve male fertility.

Katanins, a class of microtubule-severing enzymes, are potent M-phase regulators in oocytes and somatic cells. How the complex and evolutionarily crucial, male mammalian meiotic spindle is sculpted remains unknown. Here, using multiple single and double gene knockout mice, we reveal that the canonical katanin A-subunit KATNA1 and its close paralogue KATNAL1 together execute multiple aspects of meiosis. We show KATNA1 and KATNAL1 collectively regulate the male meiotic spindle, cytokinesis and midbody abscission, in addition to diverse spermatid remodelling events, including Golgi organisation, and acrosome and manchette formation. We also define KATNAL1-specific roles in sperm flagellum development, manchette regulation and sperm-epithelial disengagement. Finally, using proteomic approaches, we define the KATNA1, KATNAL1 and KATNB1 mammalian testis interactome, which includes a network of cytoskeletal and vesicle trafficking proteins. Collectively, we reveal that the presence of multiple katanin A-subunit paralogs in mammalian spermatogenesis allows for 'customised cutting' via neofunctionalisation and protective buffering via gene redundancy.

Cardiomyocyte-specific disruption of the circadian BMAL1–REV-ERBα/β regulatory network impacts distinct miRNA species in the murine heart

Circadian disruption increases cardiovascular disease (CVD) risk, through poorly understood mechanisms. Given that small RNA species are critical modulators of cardiac physiology/pathology, we sought to determine the extent to which cardiomyocyte circadian clock (CCC) disruption impacts cardiac small RNA species. Accordingly, we collected hearts from cardiomyocyte-specific Bmal1 knockout (CBK; a model of CCC disruption) and littermate control (CON) mice at multiple times of the day, followed by small RNA-seq. The data reveal 47 differentially expressed miRNAs species in CBK hearts. Subsequent bioinformatic analyses predict that differentially expressed miRNA species in CBK hearts influence processes such as circadian rhythmicity, cellular signaling, and metabolism. Of the induced miRNAs in CBK hearts, 7 are predicted to be targeted by the transcriptional repressors REV-ERBα/β (integral circadian clock components that are directly regulated by BMAL1). Similar to CBK hearts, cardiomyocyte-specific Rev-erbα/β double knockout (CM-RevDKO) mouse hearts exhibit increased let-7c-1-3p, miR-23b-5p, miR-139-3p, miR-5123, and miR-7068-3p levels. Importantly, 19 putative targets of these 5 miRNAs are commonly repressed in CBK and CM-RevDKO heart (of which 16 are targeted by let-7c-1-3p). These observations suggest that disruption of the circadian BMAL1–REV-ERBα/β regulatory network in the heart induces distinct miRNAs, whose mRNA targets impact critical cellular functions.

MODL-12. REGULATION OF OLIGODENDROGLIAL PROGENITOR CELL (OPC) CELL FATE IN DIFFUSE MIDLINE GLIOMA (DMG) BY THE HOMEOBOX TRANSCRIPTION FACTOR DLX2

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. 80% of patients with DMG harbor K27M mutations in canonical histone variants H3.3 and H3.1, leading to global epigenetic dysregulation and an oligodendroglial progenitor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. Our lab has previously identified DLX2 transcription factor occupancy of genes required for OPC differentiation in vivo (Olig2, Nkx2.2, Myt1, Plp1), and shown that co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. Likewise, increased expression of Olig2, Nkx2.2, and Plp-1 are observed in a Dlx1/Dlx2 double knockout (DKO) mouse model. Transient overexpression of a Dlx2-GFP construct into cells of a genetically engineered mouse model (GEMM) of DMG resulted in increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, and a reduction in migration, invasion, and colony formation in vitro. Inverse expression of DLX2 and OLIG1/2 was observed in a bulk RNAseq database of 45 human DMG cell lines. We have established a pipeline to evaluate cell fate changes following changes in DLX2 expression levels. OLIG1/2high/DLX2low DMG expressing cell lines underwent stable transfection of a DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone marks (H3K27me3, H3K27ac), K27M, and OPC signature genes will be presented with comparison of altered OPC genes from the DKO mouse as determined by RNA-seq. The manipulation of DLX2 expression in human DMG cells in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.

A monoclonal antibody activating AdipoR for type 2 diabetes and nonalcoholic steatohepatitis.

Adiponectin receptors, AdipoR1 and AdipoR2 are promising targets for the prevention and treatment of metabolic diseases. In this study, we aimed to establish agonistic antibodies against AdipoR1 and AdipoR2 with a long enough half-life to provide a means of improving poor medication adherence associated with preclinical small-molecule AdipoR agonists or existing antidiabetic drugs. Monoclonal antibodies were obtained by immunizing AdipoR knockout mice with human AdipoR-expressing cells. Of the antibodies shown to bind to both, an agonist antibody was obtained, which exhibited adenosine 5'-monophosphate-activated protein kinase-activating properties such as adiponectin and was named AdipoR-activating monoclonal antibody (AdipoRaMab). AdipoRaMab ameliorated glucose intolerance in high-fat diet-fed mice, which was not observed in AdipoR1·AdipoR2 double knockout mice. AdipoRaMab exhibited anti-inflammatory and antifibrotic effects in the nonalcoholic steatohepatitis (NASH) model, indicating its therapeutic potential in diabetes and in NASH. In addition, the results of this study indicated that AdipoRaMab may exert therapeutic effects even in a once-monthly dosing regimen through its humanization.

Functional characterization of the CRY2 circadian clock component variant p.Ser420Phe revealed a new degradation pathway for CRY2

Cryptochromes (CRYs) are essential components of the circadian clock, playing a pivotal role as transcriptional repressors. Despite their significance, the precise mechanisms underlying CRYs' involvement in the circadian clock remain incompletely understood. In this study, we identified a rare CRY2 variant, p.Ser420Phe, from the 1000 Genomes Project and Ensembl database that is located in the functionally important coiled-coil-like helix (CC−helix) region. Functional characterization of this variant at the cellular level revealed that p.Ser420Phe CRY2 had reduced repression activity on CLOCK:BMAL1−driven transcription due to its reduced affinity to the core clock protein PER2 and defective translocation into the nucleus. Intriguingly, the CRY2 variant exhibited an unexpected resistance to degradation via the canonical proteasomal pathway, primarily due to the loss of interactions with E3 ligases (FBXL3 and FBXL21), which suggests Ser-420 of CRY2 is required for the interaction with E3 ligases. Further studies revealed that wild type and CRY2 variant are degraded by the lysosomal-mediated degradation pathway, a mechanism not previously associated with CRY2. Surprisingly, our complementation study with Cry1−/−Cry2−/− double knockout mouse embryonic fibroblast cells indicated that CRY2 variant caused 7 hours shorter circadian period length in contrast to the observed prolonged period length in CRY2−/− cell lines. In summary, this study reveals a hitherto unknown degradation pathway for CRY2, shedding new light on the regulation of circadian rhythm period length.

Increased 12/15-lipoxygenase by disturbed flow promotes endothelial dysfunction and the development of atherosclerosis

Abstract Introduction Atherosclerosis is predisposed at bifurcations, branch points and arterial curvatures where the blood flow is disturbed. Deposition and oxidative modification of low-density lipoprotein (LDL) in the subendothelial intima is an early event in the pathogenesis of atherosclerosis. However, the effect of disturbed flow (d-flow) on LDL oxidation and the underlying mechanisms remain unclear. Purpose In this study, we investigated the role of 12/15-lipoxygenase (LOX) in LDL oxidation in endothelial cells (EC) and the development of atherosclerosis at d-flow sites. Methods En face immunofluorescence was performed to analyze the expression of 12/15-LOX in EC under exposure to different flow patterns. Protein expression and LDL oxidation was studied in vitro by using 2 different flow apparatuses. Animal model of d-flow was established by partial ligation of the carotid artery. Results En face staining exhibited a significant increase in 12/15-LOX protein expression in EC in areas exposed to d-flow than those exposed to steady flow (s-flow). Carotid partial ligation in ApoE knockout mice led to substantially increased deposition of oxidized LDL in the subendothelial intima and formation of atherosclerotic plaques in the carotid artery, whereas these detrimental effects by d-flow were markedly attenuated in ApoE/12/15-LOX double knockout mice. In cultured EC, d-flow generated by a reversal flow pump markedly promoted the expression of 12/15-LOX and translocation of the protein onto the cell membrane. Inhibition of 12/15-LOX in EC, either by knockdown with its specific siRNA or a pharmacological inhibitor, suppressed production of 15s-HETE and LDL oxidation in response to d-flow. Mechanically, we found d-flow induced the expression of 12/15-LOX by activating a specific responsive element in the 12/15-LOX promoter through recruiting a shear stress-sensitive transcriptional factor SREBP2. Chromatin immunoprecipitation further confirmed the interaction of SREBP2 with the promoter of 12/15-LOX upon d-flow exposure. Conclusions These data define an essential role of 12/15-LOX in promoting the pathogenesis of atherosclerosis under d-flow by increasing LDL oxidation in EC through SREBP2 signaling.

RNA surveillance by ADAR1 is required for vascular endothelial resilience

Abstract Introduction Long dsRNAs are recognised as danger-associated molecular patterns by the cytosolic innate immune sensors inducing thus innate immune responses. RNA editing is a post-transcriptional modification induced by the binding of Adenosine Deaminases Acting on RNA (ADARs) to double-stranded RNA structures (dsRNAs). We have recently reported that ADAR1 is the main RNA editor in endothelial cells (ECs) but the role of ADAR1 in vascular homeostatic processes remains yet elusive. Methods Genetic deletion strategies were employed by crossing mice carrying a conditional (floxed) Adar1 , Ifh1 or Tlr3 allele with either a Tie2-Cre or with a tamoxifen-inducible VE-Cadherin-CreERT2 mouse line. Primary human and murine vascular EC culture assays, dsRNA metabolism studies, gene-silencing and expression techniques, immunohistochemistry and confocal microscopy were used to assess the EC-specific ADAR1 effects. Results EC-specific ADAR1 deletion resulted in prenatal embryonic lethality evidencing an absolute requirement of EC-ADAR1 in life. Inducible EC-ADAR1 ablation in adult mice elicited premature sudden death due to a widespread lung vascular leakage and pleural effusion, reflecting a compromised EC barrier function. Mechanistically, silencing of endothelial ADAR1 disrupted long-to-short dsRNA metabolism witnessed by the striking accumulation of cytoplasmic long dsRNAs. Subsequently, the cytoplasmic accumulation of endogenous long dsRNAs induced aberrant levels of interferon-β and activation of innate immune system. Finally, activation of the cytosolic dsRNA sensors inflicted the dissociation of β-catenin from VE-cadherin in EC junctions, while, co-silencing of the dsRNA sensor, MDA5, was sufficient to restore the integrity of the ADAR1-deficient endothelium. While double EC-specific ADAR1 and TLR3 did not fully rescue the lethality of the EC-ADAR1 animals, in vivo experiments using double EC-specific ADAR1 and MDA5 KO mice fully rescued the phenotype. Validation experiments using murine vascular ECs isolated from the mouse strains of our studies confirmed our mechanistic and phenotypic findings. Conclusion Endogenous suppression of double-stranded RNA-induced MDA5 autoinflammatory signaling by the RNA editor ADAR1 is essential for the maintenance of vascular integrity in adult mice.

Multitissue Circadian Proteome Atlas of WT and Per1−/−/Per2−/− Mice

The molecular basis of circadian rhythm, driven by core clock genes such as Per1/2, has been investigated on the transcriptome level, but not comprehensively on the proteome level. Here we quantified over 11,000 proteins expressed in eight types of tissues over 46h with an interval of 2h, using WT and Per1/Per2 double knockout mouse models. The multitissue circadian proteome landscape of WT mice shows tissue-specific patterns and reflects circadian anticipatory phenomena, which are less obvious on the transcript level. In most peripheral tissues of double knockout mice, reduced protein cyclers are identified when compared with those in WT mice. In addition, PER1/2 contributes to controlling the anticipation of the circadian rhythm, modulating tissue-specific cyclers as well as key pathways including nucleotide excision repair. Severe intertissue temporal dissonance of circadian proteome has been observed in the absence of Per1 and Per2. The γ-aminobutyric acid might modulate some of these temporally correlated cyclers in WT mice. Our study deepens our understanding of rhythmic proteins across multiple tissues and provides valuable insights into chronochemotherapy. The data are accessible at https://prot-rhythm.prottalks.com/.

Abstract 17310: Epsins Promote Atherosclerosis Through Vascular Smooth Muscle Phenotypic Modulation

Hypothesis: VSMC play crucial roles in atherosclerosis via phenotypic switching. The trans-differentiation of VSMC into other cell types might contribute to atherosclerotic lesion development, progression, and the subsequent diseases such as myocardial infarction or stroke. Epsins, a family of endocytic adaptors, are crucial for atherosclerosis development and progression; yet, the role of epsins in VSMC phenotypic modulation is unknown. Methods and Results: To decipher the role of VSMC epsins in regulating atheroma development and progression, we created WT and SMC-specific inducible epsin1/2 double knockout (SMC-iDKO) mice on ApoE -/- background fed a western diet. Using single-cell RNA-seq analysis, we found VSMC and EC population significantly changed in SMC-iDKO/ApoE -/- mice compared to ApoE -/- mice. Using immunofluorescent and FACS analysis, we observed that both VSMC and EC marker genes expressions were up-regulated in DKO-VSMCs indicating that DKO-SMCs were maintaining their SMC phenotype and may further trans-differentiating into ECs via a process called MEndoT, mesenchymal to endothelial transition. Given the critical role of KLF4 in regulating VSMCs phenotype during atheroma progression, we observed diminished KLF4 expression in DKO-SMCs compared to WT treated with oxLDL. Furthermore, we found that epsins interacted with KLF4 with Epsin-UIM domain and their interaction improved KLF4 stability and promoted KLF4 to transfer into nuclei. In this study, we found that epsins were highly expressed and positively correlated with the lesion severity in VSMCs in atherosclerotic. Using immunofluorescent and Oil Red O stainings, we observed reduced lipid accumulation, lesion size and macrophage infiltration but elevated VSMCs in the cap of lesions in SMC-iDKO/ApoE -/- mice compared with ApoE -/- mice. Conclusions: In conclusions, we demonstrated an unexpected role of epsins in regulating phenotypic switching by repressing SM-contractile and EC marker genes expression through an epigenetic regulatory mechanism. Our data suggest that epsins may be a therapeutic target for treating occlusive vascular diseases, and uncover regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

Abstract 12159: Heart Generation via Blastocyst Complementation in Mesp1/2-Deficient Mice

Introduction: Heart transplantation is the only curative option available for patients with advanced heart failure. However, donor organ shortage and graft rejection remain critical challenges in heart transplantation. Blastocyst complementation using pluripotent stem cells (PSCs) can be used to generate organs such as the pancreas and kidneys in animal models with abnormalities in essential developmental genes. Nonetheless, whether functional adult hearts can be generated with blastocyst complementation remains unclear, and it is unknown if parenchyma, blood vessels, and stroma can be generated concomitantly from PSCs using blastocyst complementation to avoid graft rejection. Methods: We generated functional hearts in acardiac Mesp1 and Mesp2 double-knockout (Mesp1/2-DKO) mice via blastocyst complementation using mouse and rat PSCs. Results: The generated adult murine hearts were structurally and functionally normal and restored embryonic lethality in Mesp1/2-DKO mice. All four cardiovascular lineages, including cardiomyocytes, vascular endothelial cells, smooth muscle cells, and cardiac fibroblasts, were virtually entirely derived from exogenous PSCs in the myocardium. Exogenous rat PSCs also generated rat-derived xenogeneic hearts in Mesp1/2-DKO mice via interspecies blastocyst complementation. Conclusions: Blastocyst complementation is a viable technique for generating hearts derived from PSCs and may represent significant progress toward generating rejection-free hearts.

Methyl-CpG Binding Domain Protein MBD2 Is a Targetable Vulnerability in TET2 Mutant Myeloid Neoplasia

Background & Significance. Epigenetic dysregulation leading to acquired disorganization of CpG methylation (mCpG) is a hallmark of cancer in general and myeloid neoplasia (MN) in particular. TET dioxygenases (TET1, TET2 and TET3) progressively oxidize 5-methyl cytosine (mC) to facilitate CpG demethylation, a process critical to efficient transcription, lineage differentiation, and proliferation. Loss of function TET2 mutation ( TET2 MT), the predominant TET in HSPCs, isone of the most common genetic lesions in MN and often occurs early in MDS/AML as a founder lesion. It is frequently observed in clonal hematopoiesis of indeterminate potential (CHIP) 1,2, a prodromal condition linked with increased risk of MN, coronary heart disease 3, and pulmonary arterial hypertension 4. TET2 MT results in abnormal accumulation of mC in the genome resulting in loss of lineage plasticity due to transcriptional repression of differentiation-promoting genes and tumor suppressor genes (TSGs), contributing to a volatile pre-neoplastic state. Indeed, the accumulation of mC at otherwise unmethylated promoter- and enhancer-associated CpGs is a hallmark cancer 2,5. The epigenetic reader mCpG binding domain protein 2 (MBD2) specifically recognizes mCpG, thereby modulating regional gene transcription 6. This modulation occurs through the recruitment of co-repressor complexes which deliver enzymatic machinery to reorganize chromatin into compact, inaccessible, and stably silenced regions. This tightly regulated process, required for normal development and cell lineage differentiation, is hijacked by malignant cells to maintain a pro-oncogenic transcriptional program. Therefore, MBD2 inhibition may restore the normal hematopoiesis in TET2 MT HSPCs and may represent a selective therapeutic target. Methods. Genetic and epigenetic in vitro and in vivo model systems of clonal evolution including competitive repopulation assays and CRISPR-engineered cells coupled with NGS and biochemical analysis were used to establish the functional relevance of MBD2 in the clonal evolution of TET2 MT HSPCs. In addition, gene knockout or knockdown (ko/kd) were used to establish MBD2 as a novel therapeutic vulnerability in TET2 MT MN. Results. Here we report that loss of TET activity, either by TET2 MT or by neomorphic IDH1/2 MT, selectively enriches promoter/enhancer mCpG contributing to malignant evolution of TET2 MT HSPCs that creates sensitivity to MBD2 inhibition. TET2 MT creates regional accumulation of mC that recruits the MBD2-NuRd complex, resulting in silencing of TSGs as demonstrated via MBD2 chromatin immunoprecipitation assay. MBD2 ko/kd in TET2 MT cells (SIG-M5, OCIAML-5) or isogenic CRISPR-engineered leukemic cells (THP1, K562) resulted in the termination of the malignant program as reflected in a complete loss of colony forming ability in vitro. Orthotropic transplant of SIG-M5 cells (10 5 cells/mice) created leukemia in SGM3-NSGS mice with a maximum survival of 45 days, however, MBD2 ko in transplanted cells resulted in 100% disease-free survival of these mice. To further test if Mbd2 loss is sufficient to restore normal hematopoiesis and if it can abrogate the proliferative advantage of Tet2 -/- HSPCs, we developed a Tet2 -/-;Mbd2 -/- double ko (DKO) murine model by crossing Tet2 -/-with Mbd2 -/- mice. The DKO mice developed normally with restored hematopoiesis within the myeloid lineage compared to Tet2 -/-. Most importantly, the proliferative advantage of Tet2 -/-HSPCs was ablated, as reflected in the mutant fraction of total WBC and the restoration of CD11b + myeloid cells following Mbd2 loss in competitive bone marrow repopulation assays ( Fig. A).This restoration was mainly driven by the transcriptional rescue of differentiation-promoting genes which were silenced by promoter methylation in Tet2 -/- HSPCs ( Fig. B). Discussion & Conclusion. Here, for the first time, we demonstrate that MBD2 is required for maintaining the undifferentiated state of TET2 MT HSPCs with myeloid skewing. We provide several lines of evidence in human cells as well as in murine models that MBD2 is a targetable vulnerability in TET2 MT-associated leukemia. Our current findings provide a rationale for development of novel and potent non-cytotoxic therapeutic agents targeting MBD2 and their application in a biomarker-driven fashion for a large proportion of leukemias.

The Influence of TET2 and/or DNMT3A Knockout on the 5hmC Levels and Growth Characteristics of Modified Human Primary CD4 T Cells after Activation

Angioimmunoblastic T-cell lymphoma (AITL) has a unique immunohistological profile featuring malignant CD4+ T follicular helper cells (Tfh) inter-mixed with an extensive infiltrate of multiple lineages of immune cells. The AITL tumor microenvironment (TME) classically features small or large B cells (variably Epstein-Barr Virus (EBV) positive), reactive CD8+ T-cells, plasma cells and proliferative high endothelial venules (HEVs). Common genetic mutations have been identified within the malignant Tfh clone in AITL including epigenetic modifiers TET2, DNMT3A, and IDH2. DNMT3A mutations in AITL occur at a frequency of 20-38.5%and most of the DNMT3A mutations occur together with TET2 mutations. Tet2/ Dnmt3a double-knockout mice show synergistic effects on the expression of lineage-specifying transcription factor genes via epigenetic mechanisms. However, it is still not clear how DNMT3A mutations enhance the effect of TET2 mutations on promoting AITL development. To investigate how TET2 and DNMT3A mutations interact in AITL development, we first generated TET2 and/or DNMT3A knockout human primary CD4-T cells with CRISPR-Cas9 mediated genome editing. TET proteins could oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmc) in replication- independent DNA demethylation. Unmethylated cytosine base can be methylated by the DNA methylation machinery (DNMT3A/B) to form 5mC, which in turn provides the substrate for TET enzymes to produce 5hmC. TET and DNMT family members are thus needed to be dynamically modulate the epigenome to maintain cellular homeostasis or alter cellular function or development. We found that in response to CD3/CD28 stimulation, WT CD4-T cells exhibited division mediated dilutional reduction in 5 hmC which was later reconstitution. However, in TET2 or DKO cells, this process was compromised, showing delayed global 5hmc levels recovery (Fig. 1A). We then investigated whether the changes in methylation dynamics would alter cellular function. When using CD3/CD28 beads stimulation (10 days/cycle), we found that after two cycles of stimulation, these CD4-T cells with different gene editing started showing distinct behaviors with proliferation and apoptosis when exposed to CD3/CD28 beads again. The TET2/ DNMT3A double knockout (DKO) CD4-T cells showed more rapid response in cell division and higher proliferation rate than the other three groups (Fig. 1B). Compared with WT cells, consistently higher apoptotic cells were found in DNMT3A-KO group, but not with either TET2-KO or DKO cells (Fig. 1C). Interestingly, withdraw of beads at the end of the 10 days cycle, DKO cells kept higher baseline proliferation level than WT cells (Fig. 1D). We then used autologous or allogeneic EBV transformed B-cell for stimulation of the CD4 T-cells after one or two cycles of CD3/28 beads stimulation, and different response kinetics were observed among these prior activated CD4-T cells with TET2 and/or DNMT3A mutations. DKO cells showed more rapid response in cell division and elevated percentages of replicated cells to either autologous or allogeneic B-cells compared with WT cells (Fig. 1E). An interesting aspect of this B/T interaction is that, even the autologous B-cells could activate a large percentage of the T-cell population, particularly in the DKO cells, so it is not likely due to specific antigen recognition but rather a non-specific stimulation by the B-cells, probably via one or more co-stimulatory molecules (ICOSL, CD86 or 4-1BBL). This may be relevant to in vivo evolution of the mutated T-cells with B-cells/activated B-cells providing support.

SETD2 deficiency promotes renal fibrosis through the TGF-β/Smad signalling pathway in the absence of VHL.

Renal fibrosis is the final development pathway and the most common pathological manifestation of chronic kidney disease. Epigenetic alteration is a significant intrinsic factor contributing to the development of renal fibrosis. SET domain-containing 2 (SETD2) is the sole histone H3K36 trimethyltransferase, catalysing H3K36 trimethylation. There is evidence that SETD2-mediated epigenetic alterations are implicated in many diseases. However, it is unclear what role SETD2 plays in the development of renal fibrosis. Kidney tissues from mice as well as HK2 cells were used as research subjects. Clinical databases of patients with renal fibrosis were analysed to investigate whether SETD2 expression is reduced in the occurrence of renal fibrosis. SETD2 and Von Hippel-Lindau (VHL) double-knockout mice were used to further investigate the role of SETD2 in renal fibrosis. Renal tubular epithelial cells isolated from mice were used for RNA sequencing and chromatin immunoprecipitation sequencing to search for molecular signalling pathways and key molecules leading to renal fibrosis in mice. Molecular and cell biology experiments were conducted to analyse and validate the role of SETD2 in the development of renal fibrosis. Finally, rescue experiments were performed to determine the molecular mechanism of SETD2 deficiency in the development of renal fibrosis. SETD2 deficiency leads to severe renal fibrosis in VHL-deficient mice. Mechanically, SETD2 maintains the transcriptional level of Smad7, a negative feedback factor of the transforming growth factor-β (TGF-β)/Smad signalling pathway, thereby preventing the activation of the TGF-β/Smad signalling pathway. Deletion of SETD2 leads to reduced Smad7 expression, which results in activation of the TGF-β/Smad signalling pathway and ultimately renal fibrosis in the absence of VHL. Our findings reveal the role of SETD2-mediated H3K36me3 of Smad7 in regulating the TGF-β/Smad signalling pathway in renal fibrogenesis and provide an innovative insight into SETD2 as a potential therapeutic target for the treatment of renal fibrosis.

The macrophage STING-YAP axis controls hepatic steatosis by promoting the autophagic degradation of lipid droplets.

The hallmark of NAFLD or hepatic steatosis is characterized by lipid droplet (LD) accumulation in hepatocytes. Autophagy may have profound effects on lipid metabolism and innate immune response. However, how innate immune activation may regulate the autophagic degradation of intracellular LDs remains elusive. A mouse model of a high-fat diet-induced NASH was used in the myeloid-specific stimulator of interferon genes (STING) knockout or STING/yes-associated protein (YAP) double knockout mice. Liver injury, lipid accumulation, lipid droplet proteins, autophagic genes, chromatin immunoprecipitation coupled with massively parallel sequencing, and RNA-Seq were assessed in vivo and in vitro . We found that high-fat diet-induced oxidative stress activates STING and YAP pathways in hepatic macrophages. The acrophage STING deficiency (myeloid-specific STING knockout) enhances nuclear YAP activity, reduces lipid accumulation, and increases autophagy-related proteins ATG5, ATG7, and light chain 3B but diminishes LD protein perilipin 2 expression. However, disruption of STING and YAP (myeloid STING and YAP double knockout) increases serum alanine aminotransferase and triglyceride levels and reduces β-fatty acid oxidation gene expression but augments perilipin 2 levels, exacerbating high-fat diet-induced lipid deposition. Chromatin immunoprecipitation coupled with massively parallel sequencing reveals that macrophage YAP targets transmembrane protein 205 and activates AMP-activated protein kinase α, which interacts with hepatocyte mitofusin 2 and induces protein disulfide isomerase activation. Protein disulfide isomerase activates hypoxia-inducible factor-1α signaling, increases autophagosome colocalization with LDs, and promotes the degradation of perilipin 2 by interacting with chaperone-mediated autophagy chaperone HSC70. The macrophage STING-YAP axis controls hepatic steatosis by reprogramming lipid metabolism in a transmembrane protein 205/mitofusin 2/protein disulfide isomerase-dependent pathway. These findings highlight the regulatory mechanism of the macrophage STING-driven YAP activity on lipid control.

Transgenic mouse models support a protective role of type I IFN response in SARS-CoV-2 infection-related lung immunopathology and neuroinvasion

Type I interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infection remains perplexing. Here, we develop two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1-/-) and the other with dampened IFN-I response (hACE2; Ifnar1-/-), to comprehend the role of IFN-I response. We report that hACE2; Irgm1-/- mice are resistant to lethal SARS-CoV-2 infection. In contrast, a severe SARS-CoV-2 infection along with immune cell infiltration, cytokine storm, and enhanced pathology is observed in the lungs and brain of hACE2; Ifnar1-/- mice. The hACE2; Irgm1-/-Ifnar1-/- double-knockout mice display loss of the protective phenotype observed in hACE2; Irgm1-/- mice, suggesting that heightened IFN-I response accounts for the observed immunity. Taking the results together, we demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target against SARS-CoV-2.