Optimization and quality control of genome-wide Hi-C library preparation.

Highest-throughput chromosome conformation capture (Hi-C) is one of the key assays for genome- wide chromatin interaction studies. It is a time-consuming process that involves many steps and many different kinds of reagents, consumables, and equipments. At present, the reproducibility is unsatisfactory. By optimizing the key steps of the Hi-C experiment, such as crosslinking, pretreatment of digestion, inactivation of restriction enzyme, and in situ ligation etc., we established a robust Hi-C procedure and prepared two biological replicates of Hi-C libraries from the GM12878 cells. After preliminary quality control by Sanger sequencing, the two replicates were high-throughput sequenced. The bioinformatics analysis of the raw sequencing data revealed the mapping-ability and pair-mate rate of the raw data were around 90% and 72%, respectively. Additionally, after removal of self-circular ligations and dangling-end products, more than 96% of the valid pairs were reached. Genome-wide interactome profiling shows clear topological associated domains (TADs), which is consistent with previous reports. Further correlation analysis showed that the two biological replicates strongly correlate with each other in terms of both bin coverage and all bin pairs. All these results indicated that the optimized Hi-C procedure is robust and stable, which will be very helpful for the wide applications of the Hi-C assay.

Coordinated regulation of IFITM1, 2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactions.

Interferon-induced transmembrane protein (IFITM) 1, 2 and 3 genes encode a family of interferon (IFN)-induced transmembrane proteins that block entry of a broad spectrum of pathogens. However, the transcriptional regulation of these genes, especially whether there exist any enhancers and their roles during the IFN induction process remain elusive. Here, through public data mining, episomal luciferase reporter assay and in vivo CRISPR-Cas9 genome editing, we identified an IFN-responsive enhancer located 35kb upstream of IFITM3 gene promoter upregulating the IFN-induced expression of IFITM1, 2 and 3 genes. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer. Finally, we showed that in vivo truncation of the enhancer impaired IFN-induced resistance to influenza A virus (IAV) infection. These findings expand our understanding of the mechanisms underlying the transcriptional regulation of IFITM1, 2 and 3 expression and its ability to mediate IFN signaling.

Characterization of the conbercept gene localization in DHFR- amplified CHO cells using fluorescence in situ hybridization.

Chinese-hamster ovary (CHO) cells are most widely used for mammalian protein expression. After integration into the CHO genome, the exogenous gene may be lost in the process of large-scale protein production due to the removal of related selection pressures. Therefore, it is necessary to test its stability in the genome. Conbercept is a fusion protein that specifically binds to the various isoforms of vascular endothelial growth factor (VEGF)-A, VEGF-B, and placental growth factor (PlGF), thereby exerting anti-angiogenic activities. It has been approved for Phase Ⅲ clinical trials in the United States. In this study, fluorescence in situ hybridization (FISH) was used to localize the conbercept gene in dihydrofolatereductase (DHFR)-amplified CHO cell lines. Metaphase FISH showed that genomic integration of the conbercept gene was stable after 4 and 19 passages, and manifested three characteristics: first, the gene locates on one chromosome, rather than a number of chromosomes; second, the gene locates on the longer chromosomes; third, there are many copies located on the same chromosome. At the same time, the copy number of the conbercept gene in the CHO genome and the conbercept protein expression levels are also stable, as verified by qPCR and ELISA assays, respectively. These experiments demonstrated that the conbercept gene remained stable in the genome after 19 passages, and could be actively expressed, which strongly support the mass production and the quality control of conbercept.

Construction of CTCF degradation cell line by CRISPR/Cas9 mediated genome editing.

The CCCTC-binding factor (CTCF) is the main insulator protein described in vertebrates. It plays fundamental roles during diverse cellular processes. CTCF gene knockout mice led to death during embryonic development. To further explore the functions of CTCF, we employed a CRISPR/Cas9-based genome engineering strategy to in-frame insert the mitosis-special degradation domain (MD) of cyclin B into the upstream open reading frame of CTCF gene. Fusion protein is designed to degrade during mitosis leaded by MD. As a control group, mutation of a single arginine (R42A) within the destruction box inactivates the MD leading to constitutive expression of MD*-CTCF. The homozygous clones were obtained via the screening by puromycin when coexpressed with puromycin resistence gene. The protein level of CTCF in MD-CTCF cell line was about 10% of wild-type cells throughout cell cycles by the analyses of Western blotting and immunofluorescence. There was no significant difference between MD*-CTCF cell line and wild type. Flow cytometry results showed prolonged G1 phase in MD-CTCF cell line. Taken together, we demonstrated the feasibility of efficiently inserting MD domain into genome with the CRISPR/Cas9 technology and reported the first CTCF-specific degradation human cell line.

Involvement of aberrant miR-139/Jun feedback loop in human gastric cancer.

Accumulating evidence indicates that some miRNAs could form feedback loops with their targets to fine-tune tissue homeostasis, while disruption of these loops constitutes an essential step towards human tumorigenesis. In this study, we report the identification of a novel negative feedback loop formed between miR-139 and its oncogenic target Jun. In this loop, miR-139 could inhibit Jun expression by targeting a conserved site on its 3'-UTR, whereas Jun could induce miR-139 expression in a dose dependent manner through a distant upstream regulatory element. Interestingly, aberration in this loop was found in human gastric cancer, where miR-139 was down-regulated and inversely correlated with Jun expression. Further functional analysis showed that restored expression of miR-139 in gastric cancer cells significantly induces apoptosis, and inhibits cell migration and proliferation as well as tumour growth through targeting Jun. Thus, our data strongly suggests a role of aberrant miR-139/Jun negative feedback loop in the development of human gastric cancer and miR-139 as a potential therapeutic target for gastric cancer. Given that miR-139 and Jun are deregulated in many cancers, our findings here might have broader implication in other types of human cancers.

[In vivo delivery of siRNA].

RNA interference (RNAi) is a mechanism of posttranscriptional gene silencing mediated by small interfering RNA (siRNA). The ability of synthetic siRNA to silence genes in vivo has made it well suited as therapeutic drug, but the instability and polarity of siRNA and the complexity of in vivo circumstances retarded rapid development of RNAi-based therapies. In this review, a summary of the advances on in vivo siRNA delivery is presented and discussed.