Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLINDEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18-StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies

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.</p

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.</p

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.</p

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.</p

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.</p

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.</p

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-β1-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 <i>PLAU</i>

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

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