Xyloglucan endotransglucosylase/hydrolase (XTH) has been recognized as a cell wall-modifying enzyme, participating in the diverse physiological roles. From water-stressed hot pepper plants, we isolated three different cDNA clones (pCaXTH1, pCaXTH2, and pCaXTH3) that encode XTH homologs. RT-PCR analysis showed that three CaXTH mRNAs were concomitantly induced by a broad spectrum of abiotic stresses, including drought, high salinity and cold temperature, and in response to stress hormone ethylene, suggesting their role in the early events in the abiotic-related defense response. Transgenic Arabidopsis plants that constitutively expressed the CaXTH3 gene under the control of the CaMV 35S promoter exhibited abnormal leaf morphology; the transgenic leaves showed variable degrees of twisting and bending along the edges, resulting in a severely wrinkled leaf shape. Microscopic analysis showed that 35S-CaXTH3 leaves had increased numbers of small-sized cells, resulting in disordered, highly populated mesophyll cells in each dorsoventral layer, and appeared to contain a limited amount of starch. In addition, the 35S-CaXTH3 transgenic plants displayed markedly improved tolerance to severe water deficit, and to lesser extent to high salinity in comparison with the wild-type plants. These results indicate that CaXTH3 is functional in heterologous Arabidopsis cells, thereby effectively altering cell growth and also the response to abiotic stresses. Although the physiological function of CaXTHs is not yet clear, there are several possibilities for their involvement in a subset of physiological responses to counteract dehydration and high salinity stresses in transgenic Arabidopsis plants.
BTF3 (betaNAC) was originally isolated as a general transcription factor required for RNA polymerase II-dependent transcription, and later found to be a beta-subunit of nascent-polypeptide-associated complex that has been implicated in regulating protein localization during translation. In this study, virus-induced gene silencing of NbBTF3 encoding a Nicotiana benthamiana homolog of human BTF3 caused leaf yellowing and abnormal leaf morphology without altering the overall growth of the plant. The NbBTF3 gene is constitutively expressed and the NbBTF3-GFP fusion protein is primarily targeted to the nucleus. At the cellular level, downregulation of NbBTF3 expression reduced the chloroplast sizes and chlorophyll contents. The affected cells produced excessive amounts of reactive oxygen species. Furthermore, the transcript level of various plastid- and mitochondria-encoded genes was severely reduced in the NbBTF3-depleted leaf cells. These findings indicate that depletion of NbBTF3 activity preferentially affected development and/or physiology of chloroplasts and mitochondria in plants, possibly by hampering efficient translocation of the nascent organellar proteins into the organelles.
Ethylene induced an increase in the level of 1‐aminocyclopropane‐1‐carboxylate oxidase (VR‐ACO1) transcript in mung bean hypocotyls. Time course study revealed that the level of the VR‐ACO1 mRNA in excised mung bean hypocotyls increased after 2‐h ethylene treatment and much higher mRNA levels were observed thereafter, while the basal level of transcript was slightly decreased in control tissues. The in vivo ACC oxidase activity increased from 31 nl g−1 h−1 at zero time to maximum activity of 62 nl g−1 h−1 at 10‐h ethylene treatment. Polyclonal antibody against VR‐ACO1 protein expressed in E. coli cells was generated. Immunoblot analysis using the resulting antiserum showed that the induction pattern of ACC oxidase polypeptide was in parallel with that of enzyme activity during the incubation with ethylene. Thus, ethylene increases the levels of the VR‐ACO1 mRNA and ACC oxidase protein as well as enzyme activity in mung bean hypocotyls. The abundance of the VR‐ACO1 transcript and protein was also induced in all parts of light‐grown mung bean seedlings by ethylene treatment. However, both the basal levels and the magnitudes of ethylene‐induction of VR‐ACO1 were markedly different in different tissues, with the roots being the most sensitive to ethylene. These results suggest that the different part of mung bean seedlings has a distinct potential to respond to ethylene with regard to ACC oxidase gene activation. Furthermore, our data suggest that the induction of VR‐ACO1 by ethylene is subject not only to transcriptional control but also to posttranscriptional control in hypocotyls, whereas ethylene regulates the VR‐ACO1 gene expression mainly at the transcriptional level in roots. The possible molecular mechanism of regulation of ACC oxidase gene expression by ethylene and its significance are discussed.
Introduction: Genome-wide association studies (GWAS) along with expression quantitative trait loci (eQTL) have identified hundreds of genetic variants and target genes in prostate cancer (prCa). Although genetic predisposition has mainly been described in prostate cancer (PrCa), functional characterization of these risk loci remains a challenge.
Methods: Low multiplicity of infection creates single lentiviral integrated cell population, which enable us to evaluate biological significance of steric hindrance at certain SNP sites in large scale. To screen for regulatory SNP, we designed a guide RNA library to target 2166 potential functional SNP sites with CRISPOR software. We performed negative screening in dCas9-KRAB stable prostate cell lines and applied RIGOR program to discover the SNPs that are essential for cell proliferation. We further validated regulatory role of selected SNPs using luciferase reporter assay, ChIP-qPCR and CRISPR-based SNP editing in prostate cells.
Results: After gRNA interfering for 21 days, we performed RIGOR analysis and identified 153 proliferation-essential SNPs, covered by one or multiple prostate cancer cell lines. Intersection analysis showed that these SNPs tended to reside in 5'-UTR and intron regions. To characterize regulatory role of these SNPs, we performed functional analysis in a SNP rs60 since prostate cells containing guide RNAs targeting rs60 were significantly depleted (FDR<0.05). Pooled GWAS SNP analysis showed that the G allele of this SNP exhibit 10% higher PrCa risk than A allele. eQTL analysis showed that the G allele is associated with an increased expression of RUVBL1 (FDR=3.3E-13). To further characterize the SNP-gene association, we performed CRISPR-based RNA interference analysis that targeted the SNP site. We observed significant inhibition of the RUVBL1 gene expression in prostate cell lines. We also applied xCas9 adenine base editor and successfully converted rs60 A into G allele in multiple clones of BPH1, DU145 and PC3 cell line. Consequently, we observed RUVBL1 expression increase after switching A to G allele at this locus. We performed ChIP-qPCR and confirmed rs60 flanking region was highly H3K4me3 modified, suggesting a transactivation role of this loci in regulating RUVBL1 expression. When stably knocked down RUVBL1 expression by shRNA we found significant proliferation inhibition in multiple prostate cell lines. Gene set enrichment analysis showed an enrichment of MYC target genes with RUVBL1 expression in TCGA prostate cancer cohorts. Furthermore, increased RUVBL1 expression was associated with poor progression-free survival (p<0.01) in TCGA datasets.
Conclusion: CRISPRi-SNPs-seq is a powerful screening tool to identify regulatory SNPs essential for cell proliferation. In combination with in-depth functional assays, the technology will facilitate discovery of regulatory variants and their genes responsible for disease risk.
Citation Format: Yijun Tian, Jong A. Park, Liang Wang. CRISPRi-SNPs-seq identified regulatory loci conferring prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 446.
High mobility group A protein-2 (HMGA2) is an architectural transcription factor that binds to the A/T-rich DNA minor groove and is responsible for regulating transcriptional activity of multiple genes indirectly through chromatin change and assembling enhanceosome. HMGA2 is overexpressed in multiple tumor types, suggesting its involvement in cancer initiation and progression, thus, making it an ideal candidate for cancer diagnostic and prognostic. We performed a systematic review to examine the role of HMGA2 as a universal tumor cancer diagnostic and prognostic marker. We used Reporting Recommendations for Tumor Marker Prognostic Studies to systematically search OvidMedline, PubMed, and the Cochrane Library for English language studies, published between 1995 and June 2019. Meta-analysis provided pooled risk estimates and their 95% confidence intervals (CIs) for an association between overall survival and recurrence of cancers for studies with available estimates. We identified 42 eligible studies with a total of 5123 tumor samples in 15 types of cancer. The pooled percentage of HMGA2 gene expression in tumor samples was 65.14%. Meta-analysis showed that cancer patients with HMGA2 positive have significantly reduced survival, compared to patients without HMGA2 gene [pooled-hazard ratio (HR) = 1.85, 95% CI 1.48–2.22]. There was a positive association between cancer patients with HMGA2 overexpression and cancer recurrence though this association did not reach significance (pooled-HR = 1.44, 95% CI 0.80–2.07). Overexpression of HMGA2 was found in 15 types of cancer. There was an association between HMGA2 overexpression with reduced survival of cancer patients. HMGA2 is thus considered a promising universal tumor marker for prognostics.
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