SgRNA Expression of CRIPSR-Cas9 System Based on MiRNA Polycistrons as a Versatile Tool to Manipulate Multiple and Tissue-Specific Genome Editing.

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter. Effective expression and further processing of functional miRNAs and sgRNAs were achieved. The redundant nucleotides adjacent to sgRNA were degraded, and 5'- cap structure was responsible for the compromised nuclease capacity of sgRNA: Cas9 complex. Furthermore, this strategy fulfilled conducting multiplex genome editing, as well as executing neural- specific genome editing and enhancing the proportion of homologous recombination via inhibiting NHEJ pathway by shRNA. In summary, we designed a new construction for efficient expression of sgRNAs with miRNAs (shRNAs) by virtue of RNA polymerase II promoters, which will spur the development of safer, more controllable/regulable and powerful CRISPR/Cas9 system-mediated genome editing in a wide variety of further biomedical applications.

Trigeminal star-like platinum complexes induce cancer cell senescence through quadruplex-mediated telomere dysfunction.

Two trigeminal star-like platinum complexes were synthesized to induce the formation of human telomere G-quadruplex (hTel G4) with extremely high selectivity and affinity. The induced hTel G4 activates strong telomeric DNA damage response (TDDR), resulting in telomere dysfunction and cell senescence.

Conformation Selective Antibody Enables Genome Profiling and Leads to Discovery of Parallel G-Quadruplex in Human Telomeres.

G-quadruplexes are specialized secondary structures in nucleic acids that possess significant conformational polymorphisms. The precise G-quadruplex conformations in vivo and their relevance to biological functions remain controversial and unclear, especially for telomeric G-quadruplexes. Here, we report a novel single-chain variable fragment (scFv) antibody, D1, with high binding selectivity for parallel G-quadruplexes in vitro and in vivo. Genome-wide chromatin immunoprecipitation using D1 and deep-sequencing revealed the consensus sequence for parallel G-quadruplex formation, which is characterized by G-rich sequence with a short loop size (<3 nt). By using D1, telomeric parallel G-quadruplex was identified and its formation was regulated by small molecular ligands targeting and telomere replication. Together, parallel G-quadruplex specific antibody D1 was found to be a valuable tool for determination of G-quadruplex and its conformation, which will prompt further studies on the structure of G-quadruplex and its biological implication in vivo.

Protection against doxorubicin-induced myocardial dysfunction in mice by cardiac-specific expression of carboxyl terminus of hsp70-interacting protein.

Carboxyl terminus of Hsp70-interacting protein (CHIP) is a critical ubiquitin ligase/cochaperone to reduce cardiac oxidative stress, inflammation, cardiomyocyte apoptosis and autophage etc. However, it is unclear whether overexpression of CHIP in the heart would exert protective effects against DOX-induced cardiomyopathy. Cardiac-specific CHIP transgenic (CHIP-TG) mice and the wild-type (WT) littermates were treated with DOX or saline. DOX-induced cardiac atrophy, dysfunction, inflammation, oxidative stress and cardiomyocyte apoptosis were significantly attenuated in CHIP-TG mice. CHIP-TG mice also showed higher survival rate than that of WT mice (40% versus 10%) after 10-day administration of DOX. In contrast, knockdown of CHIP by siRNA in vitro further enhanced DOX-induced cardiotoxic effects. Global gene microarray assay revealed that after DOX-treatment, differentially expressed genes between WT and CHIP-TG mice were mainly involved in apoptosis, atrophy, immune/inflammation and oxidative stress. Mechanistically, CHIP directly promotes ubiquitin-mediated degradation of p53 and SHP-1, which results in activation of ERK1/2 and STAT3 pathways thereby ameliorating DOX-induced cardiac toxicity.

CHIP enhances angiogenesis and restores cardiac function after infarction in transgenic mice.

BACKGROUND: Carboxyl terminus of Hsp70-interacting protein (CHIP) is a chaperone/ubiquitin ligase that plays an important role in stress-induced apoptosis. However, the effect of CHIP on angiogenesis, cardiac function and survival 4 weeks after myocardial infarction (MI) remain to be explored. METHODS: Wild-type (WT) and transgenic mice (TG) with cardiac-specific overexpression of CHIP were used for coronary artery ligation. The cardiac function, cardiomyocyte apoptosis, inflammation and angiogenesis were examined by echocardiography, histological analysis, real-time PCR and Western blot analysis. RESULTS: At 4 weeks of after coronary artery ligation, echocardiography demonstrated that cardiac remodeling and dysfunction were prevented in TG mice compared with WT mice. The infarct size, cardiomyocyte apoptosis and inflammation were significantly reduced in TG mice than in WT mice. The survival rate after MI in TG mice was higher than that of WT mice. Furthermore, the levels of p53 protein was markedly decreased, but the expression of HIF-1α and VEGF, and the formation of capillary and arteriole after MI were significantly enhanced in TG mice compared with WT mice. CONCLUSION: We report the first in vivo evidence that CHIP enhances angiogenesis, inhibits inflammation, restores cardiac function, and improves survival at 4 weeks after MI. The present study expands on previous results and defines a novel mechanism. Thus, increased CHIP level may provide a novel therapeutic approach for left ventricular dysfunction after MI.

Carboxyl terminus of heat shock protein 70-interacting protein inhibits angiotensin II-induced cardiac remodeling.

BACKGROUND: The carboxyl terminus of heat shock protein 70-interacting protein (CHIP), an E3 ligase/chaperone, was found to protect cardiomyocytes against apoptosis induced by ischemic injury; however, the functional role of CHIP in remodeling induced by angiotensin II (Ang II) remains unclear. METHODS: We generated CHIP-overexpressed transgenic (TG) mice infused with Ang II (1,500 ng/kg/min) or saline for days or small interfering RNA (siRNA) knockdown of neonatal rat cardiomyocytes. Heart sections were stained with hematoxylin and eosin, Masson trichrome, TdT-mediated dUTP nick-end labeling (TUNEL) staining, and immunohistochemistry, and the levels of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) were measured by western blot analysis. RESULTS: Seven days after Ang II infusion, cardiac-specific overexpression of CHIP significantly enhanced cardiac contractile performance in mice and attenuated cardiac apoptosis, fibrosis, and inflammation: the number of TUNEL-positive cells, fibrotic areas, macrophage infiltration, and the expression of interleukin-1ß (IL-1ß), IL-6, monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) in heart tissues were decreased as compared with wild-type (WT) mice (all P < 0.05). In contrast, CHIP siRNA knockdown markedly increased Ang II-induced apoptosis and the expression of proinflammatory cytokines, as compared with siRNA control. The mechanisms underlying these beneficial actions were associated with CHIP-mediated inhibition of NF-κB and MAPK (p38 and JNK) pathways. CONCLUSIONS: CHIP plays an important role in regulating Ang II-triggered hypertensive cardiac apoptosis, inflammation, and fibrosis.

Stability of F-box protein atrogin-1 is regulated by p38 mitogen-activated protein kinase pathway in cardiac H9c2 cells.

BACKGROUND: Atrogin-1/MAFbx is a major atrophy-related E3 ubiquitin ligase that functions as a negative regulator of cardiac hypertrophy. The mRNA expression of atrogin-1 is induced by oxidative stress via p38 mitogen-activated protein kinase (p38 MAPK). However, the molecular mechanisms that regulate the stability of atrogin-1 protein remain unclear. METHODS: 293T and cardiac H9c2 cells were transfected with plasmids as indicated. The in vivo and in vitro ubiquitination assay and pulse-chase analysis were performed to detect the ubiquitination and stability of atrogin-1. The protein levels were measured by Western blot analysis. RESULTS: We found that atrogin-1 underwent ubiquitin-mediated degradation by proteasome. The F-box motif of atrogin-1 and Skp1-Cul1-Roc1-F-box (SCF) complex are required for ubiquitination and degradation of atrogin-1. Furthermore, p38 MAPK signaling plays critical roles in regulating the ubiquitination and degradation of atrogin-1 as well as serum starvation-induced expression of atrogin-1 and reduction of H9c2 cell size. CONCLUSION: These findings may define a new mechanism for regulating the stability of atrogin-1 partially by p38 MAPK signaling.