Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is highly expressed in smokers, but little is known about the molecular mechanism of UCHL1 in airway epithelium and its possible role in affecting extracellular matrix (ECM) remodelling in the underlying submucosa. Since cigarette smoking is a major cause of lung diseases, we studied its effect on UCHL1 expression and DNA methylation patterns in human bronchial epithelial cells, obtained after laser capture micro-dissection (LCM) or isolated from residual tracheal/main stem bronchial tissue. Targeted regulation of UCHL1 expression via CRISPR/dCas9 based-epigenetic editing was used to explore the function of UCHL1 in lung epithelium. Our results show that cigarette smoke extract (CSE) stimulated the expression of UCHL1 in vitro. The methylation status of the UCHL1 gene was negatively associated with UCHL1 transcription in LCM-obtained airway epithelium at specific sites. Treatment with a UCHL1 inhibitor showed that the TGF-ß1-induced upregulation of the ECM gene COL1A1 can be prevented by the inhibition of UCHL1 activity in cell lines. Furthermore, upon downregulation of UCHL1 by epigenetic editing using CRISPR/dCas-EZH2, mRNA expression of COL1A1 and fibronectin was reduced. In conclusion, we confirmed higher UCHL1 expression in current smokers compared to non- and ex-smokers, and induced downregulation of UCHL1 by epigenetic editing. The subsequent repression of genes encoding ECM proteins suggest a role for UCHL1 as a therapeutic target in fibrosis-related disease.

Functional Validation of the Putative Oncogenic Activity of PLAU.

Plasminogen activator, urokinase (PLAU) is involved in cell migration, proliferation and tissue remodeling. PLAU upregulation is associated with an increase in aggressiveness, metastasis, and invasion of several cancer types, including breast cancer. In patients, this translates into decreased sensitivity to hormonal treatment, and poor prognosis. These clinical findings have led to the examination of PLAU as a biomarker for predicting breast cancer prognosis and therapy responses. In this study, we investigated the functional ability of PLAU to act as an oncogene in breast cancers by modulating its expression using CRISPR-deactivated Cas9 (CRISPR-dCas9) tools. Different effector domains (e.g., transcription modulators (VP64, KRAB)) alone or in combination with epigenetic writers (DNMT3A/3L, MSssI) were fused to dCas9 and targeted to the PLAU promoter. In MDA-MB-231 cells characterized by high PLAU expression downregulation of PLAU expression by CRISPR-dCas9-DNMT3A/3L-KRAB, resulted in decreased cell proliferation. Conversely, CRISPR-dCas9-VP64 induced PLAU upregulation in low PLAU expressing MCF-7 cells and significantly increased aggressiveness and invasion. In conclusion, modulation of PLAU expression affected metastatic related properties of breast cancer cells, thus further validating its oncogenic activity in breast cancer cells.

Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli.

Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFß1 Activation.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.

I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive ß-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased ß-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

Siculibacillus lacustris gen. nov., sp. nov., a new rosette-forming bacterium isolated from a freshwater crater lake (Lake St. Ana, Romania).

A new aerobic alphaproteobacterium, strain SA-279T, was isolated from a water sample of a crater lake. The 16S rRNA gene sequence analysis revealed that strain SA-279T formed a distinct lineage within the family Ancalomicrobiaceae and shared the highest pairwise similarity values with Pinisolibacterravus E9T (96.4 %) and Ancalomicrobiumadetum NBRC 102456T (94.2 %). Cells of strain SA-279T were rod-shaped, motile, oxidase and catalase positive, and capable of forming rosettes. Its predominant fatty acids were C18 : 1ω7c (69.0 %) and C16 : 1ω7c (22.7 %), the major respiratory quinone was Q-10, and the main polar lipids were phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, phosphatidylglycerol, an unidentified aminophospholipid and an unidentified lipid. The G+C content of the genomic DNA of strain SA-279T was 69.2 mol%. On the basis of the phenotypic, chemotaxonomic and molecular data, strain SA-279T is considered to represent a new genus and species within the family Ancalomicrobiaceae, for which the name Siculibacillus lacustris gen. nov., sp. nov. is proposed. The type strain is SA-279T (=DSM 29840T=JCM 31761T).

Establishment of Cell Lines Stably Expressing dCas9-Fusions to Address Kinetics of Epigenetic Editing.

Epigenetic editing is a promising approach to modulate the local chromatin environment of target genes with the ultimate goal of stable gene expression reprogramming. Epigenetic editing tools minimally consist of a DNA-binding domain and an effector domain. The CRISPR/dCas9 platform, where mutations in the nuclease domains render the Cas9 protein inactive, is widely used to guide epigenetic effectors to their intended genomic loci. Its flexible nature, simple use, and relatively low cost have revolutionized the research field of epigenetic editing. Although effective expression modulation is readily achieved, only a few studies have addressed the maintenance of the induced effects on endogenous loci. Here, we describe a detailed protocol to engineer cells that stably express the CRISPR/dCas9-effectors. The protocol involves modification of published dCas9-based plasmid vectors for easy transfer of the effector domain between the vector designed for transient transfection and the vector used for establishing cell lines stably expressing the dCas9-effector fusion protein. Transient transfection of the dCas9-effector-producing cells with sgRNA-expressing plasmids allows studying of the maintenance of epigenetic editing. Targeting various genes in different chromatin contexts and/or co-targeting multiple CRISPR/dCas9-effectors can be used to unravel rules underlying maintained gene expression reprogramming.