KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres.

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-ß, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.

Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFß signaling through TGFBR1 stabilization.

Pulmonary fibrosis (PF) is a major public health problem with limited therapeutic options. There is a clear need to identify novel mediators of PF to develop effective therapeutics. Here we show that an ER protein disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), is highly upregulated in the lung tissues from both patients with idiopathic pulmonary fibrosis and a mouse model of bleomycin (BLM)-induced PF. Global deletion of Txndc5 markedly reduces the extent of PF and preserves lung function in mice following BLM treatment. Mechanistic investigations demonstrate that TXNDC5 promotes fibrogenesis by enhancing TGFß1 signaling through direct binding with and stabilization of TGFBR1 in lung fibroblasts. Moreover, TGFß1 stimulation is shown to upregulate TXNDC5 via ER stress/ATF6-dependent transcriptional control in lung fibroblasts. Inducing fibroblast-specific deletion of Txndc5 mitigates the progression of BLM-induced PF and lung function deterioration. Targeting TXNDC5, therefore, could be a novel therapeutic approach against PF.

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

RATIONALE: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. OBJECTIVE: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. METHODS AND RESULTS: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor ß1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor ß1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor ß1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor ß1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively. CONCLUSIONS: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against ß agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.