Biochimica et Biophysica Acta

Tailoring the CRISPR system to transactivate coagulation gene promoters in normal and mutated contexts Publication date: June 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 6 Author(s): Silvia Pignani, Federico Zappaterra, Elena Barbon, Antonia Follenzi, Matteo Bovolenta, Francesco Bernardi, Alessio Branchini, Mirko Pinotti Abstract Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold and 3-fold; sgRNAF8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.

A non-autonomous role of MKL1 in the activation of hepatic stellate cells Publication date: June 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 6 Author(s): Zilong Li, Ping Li, Yunjie Lu, Donglin Sun, Xiaoying Zhang, Yong Xu Abstract Although hepatic stellate cells (HSC) represent the major source of fibrogenesis in the liver under various pathological conditions, other cell types including hepatic parenchymal cells (hepatocytes) also contribute to HSC activation and hence liver fibrosis. The underlying mechanism, however, is poorly defined. Here we report that hepatocytes exposed to high concentrations of glucose (HG) emit a pro-fibrogenic cue as evidenced by the observation that primary HSCs cultured in conditioned media (CM) collected from hepatocytes exposed to HG up-regulated the production of extracellular matrix (ECM) proteins compared to CM collected from hepatocytes exposed to low glucose. We further identified the pro-fibrogenic cue from hepatocytes to be connective tissue growth factor (CTGF) because either depletion of endogenous CTGF in hepatocytes with siRNA or the addition of a CTGF-specific neutralizing antibody to the CM blunted the pro-fibrogenic effect elicit by HG treatment. Of interest, we discovered that genetic ablation or pharmaceutical inhibition of the transcriptional modulator MKL1 in hepatocytes also abrogated the HG-induced pro-fibrogenic effects. Mechanistically, MKL1 interacted with AP-1 and SMAD3 to trans-activate CTGF in hepatocytes in response to HG treatment. In conclusion, our data suggest that MKL1 contribute to HSC activation in a non-autonomous fashion by promoting CTGF transcription in hepatocytes.

Enhancer long-range contacts: The multi-adaptor protein LDB1 is the tie that binds Publication date: June 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 6 Author(s): Guoyou Liu, Ann Dean Abstract The eukaryotic genome is organized at varying levels into chromosome territories, transcriptional compartments and topologically associating domains (TADs), which are architectural features largely shared between different cell types and across species. In contrast, within TADs, chromatin loops connect enhancers and their target genes to establish unique transcriptomes that distinguish cells and tissues from each other and underlie development and differentiation. How these tissue-specific and temporal stage-specific long-range contacts are formed and maintained is a fundamental question in biology. The widely expressed Lim domain binding 1protein, LDB1, plays a critical role in connecting enhancers and genes by forming complexes with cell-type specificity across diverse developmental pathways including neurogenesis, cardiogenesis, retinogenesis and hematopoiesis. Here we review the multiple roles of LDB1 in cell fate determination and in chromatin loop formation, with an emphasis on mammalian systems, to illuminate how LDB1 functions in normal cells and in diseases such as cancer.

A novel role of U1 snRNP: Splice site selection from a distance Publication date: June 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 6 Author(s): Ravindra N. Singh, Natalia N. Singh Abstract Removal of introns by pre-mRNA splicing is fundamental to gene function in eukaryotes. However, understanding the mechanism by which exon-intron boundaries are defined remains a challenging endeavor. Published reports support that the recruitment of U1 snRNP at the 5′ss marked by GU dinucleotides defines the 5′ss as well as facilitates 3′ss recognition through cross-exon interactions. However, exceptions to this rule exist as U1 snRNP recruited away from the 5′ss retains the capability to define the splice site, where the cleavage takes place. Independent reports employing exon 7 of Survival Motor Neuron (SMN) genes suggest a long-distance effect of U1 snRNP on splice site selection upon U1 snRNP recruitment at target sequences with or without GU dinucleotides. These findings underscore that sequences distinct from the 5′ss may also impact exon definition if U1 snRNP is recruited to them through partial complementarity with the U1 snRNA. In this review we discuss the expanded role of U1 snRNP in splice-site selection due to U1 ability to be recruited at more sites than predicted solely based on GU dinucleotides.

Circular exonic RNAs: when RNA structure meets topology Publication date: Available online 15 May 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Dmitri D. Pervouchine Abstract Although RNA circularization was first documented in the 1990s, the extent to which it occurs was not known until recent advances in high-throughput sequencing enabled the widespread identification of circular RNAs (circRNAs). Despite this, many aspects of circRNA biogenesis, structure, and function yet remain obscure. This review focuses on circular exonic RNAs, a subclass of circRNAs that are generated through backsplicing. Here, I hypothesize that RNA secondary structure can be the common factor that promotes both exon skipping and spliceosomal RNA circularization, and that backsplicing of double-stranded regions could generate topologically linked circRNA molecules. CircRNAs manifest themselves by the presence of tail-to-head exon junctions, which were previously attributed to post-transcriptional exon permutation and repetition. I revisit these observations and argue that backsplicing does not automatically imply RNA circularization because tail-to-head exon junctions give only local information about transcript architecture and, therefore, they are in principle insufficient to determine globally circular topology.

DISC1 promotes translation maintenance during sodium arsenite-induced oxidative stress Publication date: Available online 7 May 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Francisco Fuentes-Villalobos, Carlos Farkas, Sebastián Riquelme-Barrios, Marisol E. Armijo, Ricardo Soto-Rifo, Roxana Pincheira, Ariel F. Castro Abstract Variation in Disrupted-in-Schizophrenia 1 (DISC1) increases the risk for neurodegenerative diseases, schizophrenia, and other mental disorders. However, the functions of DISC1 associated with the development of these diseases remain unclear. DISC1 has been reported to inhibit Akt/mTORC1 signaling, a major regulator of translation, and recent studies indicate that DISC1 could exert a direct role in translational regulation. Here, we present evidence of a novel role of DISC1 in the maintenance of protein synthesis during oxidative stress. In order to investigate DISC1 function independently of Akt/mTORC1, we used Tsc2−/− cells, where mTORC1 activation is independent of Akt. DISC1 knockdown enhanced inhibition of protein synthesis in cells treated with sodium arsenite (SA), an oxidative agent used for studying stress granules (SGs) dynamics and translational control. N-acetyl-cysteine inhibited the effect of DISC1, suggesting that DISC1 affects translation in response to oxidative stress. DISC1 decreased SGs number in SA-treated cells, but resided outside SGs and maintained protein synthesis independently of a proper SG nucleation. DISC1-dependent stimulation of translation in SA-treated cells was supported by its interaction with eIF3h, a component of the canonical translation initiation machinery. Consistent with a role in the homeostatic maintenance of translation, DISC1 knockdown or overexpression decreased cell viability after SA exposure. Our data suggest that DISC1 is a relevant component of the cellular response to stress, maintaining certain levels of translation and preserving cell integrity. This novel function of DISC1 might be involved in its association with pathologies affecting tissues frequently exposed to oxidative stress.

PTBP1 enhances exon11a skipping in Mena pre-mRNA to promote migration and invasion in lung carcinoma cells Publication date: Available online 7 May 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Shuaiguang Li, Lianghua Shen, Luyuan Huang, Sijia Lei, Xingdong Cai, Mason Breitzig, Bin Zhang, Annan Yang, Wenzuo Ji, Meiyan Huang, Qing Zheng, Hanxiao Sun, Feng Wang Abstract Alternative splicing (AS) events occur in the majority of human genes. AS in a single gene can give rise to different functions among multiple isoforms. Human ortholog of mammalian enabled (Mena) is a conserved regulator of actin dynamics that plays an important role in metastasis. Mena has been shown to have multiple splice variants in human tumor cells due to AS. However, the mechanism mediated Mena AS has not been elucidated. Here we showed that polypyrimidine tract-binding protein 1 (PTBP1) could modulate Mena AS. First, PTBP1 levels were elevated in metastatic lung cancer cells as well as during epithelial-mesenchymal transition (EMT) process. Then, knockdown of PTBP1 using shRNA inhibited migration and invasion of lung carcinoma cells and decreased the Mena exon11a skipping, whereas overexpression of PTBP1 had the opposite effects. The results of RNA pull-down assays and mutation analyses demonstrated that PTBP1 functionally targeted and physically interacted with polypyrimidine sequences on both upstream intron11 (TTTTCCCCTT) and downstream intron11a (TTTTTTTTTCTTT). In addition, the results of migration and invasion assays as well as detection of filopodia revealed that the effect of PTBP1 was reversed by knockdown of Mena but not Mena11a+. Overexpressed MenaΔ11a also rescued the PTBP1-induced migration and invasion. Taken together, our study provides a novel mechanism that PTBP1 modulates Mena exon11a skipping, and indicates that PTBP1 depends on the level of Mena11a− to promote lung cancer cells migration and invasion. The regulation of Mena AS may be a potential prognostic marker and a promising target for treatment of lung carcinoma.

HSF1 phosphorylation by cyclosporin A confers hyperthermia sensitivity through suppression of HSP expression Publication date: Available online 3 May 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms Author(s): Jingyu Shao, Beibei Han, Pengxiu Cao, Bingwei Zhang, Ming Liu, Danyu Li, Nan Zhou, Qiang Hao, Xianglin Duan, Yanzhong Chang, Akira Nakai, Yumei Fan, Ke Tan Abstract Heat shock leads to the activation of heat shock factor 1 (HSF1) and up-regulation of a number of heat shock proteins (HSPs). Cyclosporin A (CsA) is an immunosuppressant that has revolutionized organ transplantation in clinical medicine. However, the roles and regulatory mechanisms of CsA on the HSP expression remain largely unknown. Here, we found that CsA pretreatment prevented the induction of HSPs during heat shock by enhancing the phosphorylation of Ser303 and Ser307 in HSF1 which inhibited HSF1 transcriptional activity. Inhibition of ERK1/2, GSK3β and CK2 ameliorated CsA-induced down-regulation of HSP expressions and up-regulation of HSF1 phosphorylation. CsA impeded HSF1-SSBP1 complex formation, HSF1 nuclear translocation and recruitment to the HSP70 promoter. Due to the low expression of HSPs, CsA treatment clearly caused cell death during proteotoxic stresses. These results indicated that CsA suppressed the induction of HSPs during heat shock through regulation of the phosphorylation and nuclear translocation of HSF1. Our study could provide a conceptual framework for the development of novel strategies for combination therapy utilizing hyperthermia or chemotherapy and CsA treatment.

Editorial Board Publication date: May 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 5 Author(s):

MiR-15/16 mediate crosstalk between the MAPK and Wnt/β-catenin pathways during hepatocyte differentiation from amniotic epithelial cells Publication date: May 2019 Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1862, Issue 5 Author(s): Chunyu Bai, Hongwei Zhang, Xiangyang Zhang, Wancai Yang, Xiangchen Li, Yuhua Gao Abstract MiR-15/16 play an important role in liver development and hepatocyte differentiation, but the mechanisms by which these miRNAs regulate their targets and downstream genes to influence cell fate are poorly understood. In this study, we showed up-regulation of miR-15/16 during HGF- and FGF4-induced hepatocyte differentiation from amniotic epithelial cells (AECs). To elucidate the role of miR-15/16 and their targets in hepatocyte differentiation, we investigated the roles of miR-15/16 in both the MAPK and Wnt/β-catenin pathways, which were predicted to be involved in miR-15/16 signaling. Our results demonstrated that the transcription of miR-15/16 was enhanced by c-Fos, c-Jun, and CREB, important elements of the MAPK pathway, and miR-15/16 in turn directly targeted adenomatous polyposis coli (APC) protein, a major member of the β-catenin degradation complex. MiR-15/16 destroyed these degradation complexes to activate β-catenin, and the activated β-catenin combined with LEF/TCF7L1 to form a transcriptional complex that enhanced transcription of hepatocyte nuclear factor 4 alpha (HNF4α). HNF4α also bound the promoter region of miR-15/16 and promoted its transcription, thereby forming a regulatory circuit to promote the differentiation of AECs into hepatocytes. Endogenous miRNAs are, therefore, involved in hepatocyte differentiation from AECs and should be considered during the development of an effective hepatocyte transplant therapy for liver damage.