high-throughput-screening-for-chemical-modulators-post

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27(KIP1), an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27(KIP1) expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5'UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF-encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27(KIP1) expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27(KIP1) activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27(KIP1) activity can also be modulated by an uORF and mutations affecting uORF could change p27(KIP1) expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.

A novel mutation in the upstream open reading frame of the CDKN1B gene causes a MEN4 phenotype.

The plasma prekallikrein gene is expressed in many different human tissues at distinctly different levels and therefore tissue-specific control of the gene transcription is likely. In this study we demonstrate that transcription of the plasma prekallikrein gene can be initiated at multiple sites, for which at least four different promoters are utilized. A comparison of the genomic and mRNA sequences of mouse plasma prekallikrein revealed that the sequence segment that was formerly regarded as the first exon of the mouse plasma prekallikrein gene consists of three exons, with the first exon localized 14.2 kbp upstream of the translation start. For the rat and human plasma prekallikrein genes, in silico analysis suggested an analogous exon-intron organization. Determination of the transcription start sites showed that in both mouse and human, the proximal and distal regions could be utilized for transcription initiation; however, the proximal region is preferred. A deletion mutation analysis of the proximal promoter region using a 1.7-kbp segment revealed a strong activating region immediately upstream of the known mRNA, followed by both a modest repressor and an enhancer region.

Expression of the plasma prekallikrein gene: utilization of multiple transcription start sites and alternative promoter regions.

The transcription start sites (TSSs) of the human plasma prekallikrein gene (KLKB1) determined in RNA from kidney are mostly localized within intron 1 and exon 2, suggesting that the region encompassing exon 1, intron 1, and exon 2 comprises an alternative promoter. Reporter gene analyses in HepG2 and immortalized human kidney epithelial cells confirmed a significant transcriptional activity of this putative promoter region. However, when the TSSs recruited for transcription of the reporter gene were determined, only a few transcripts starting within the insert were detected, whereas the majority of TSSs were located in the vector backbone up to about 2000 bp upstream of the insert. Further reporter gene studies with deletion mutants of the fragment exon 1-intron 1-exon 2 revealed that the 3'-terminal 13-bp segment of intron 1 is sufficient to promote transcriptional activity and induce upstream displacement of the TSS. We conclude that the 13-bp segment represents a cis-acting element which can displace TSSs by provoking the recruitment of alternative promoters and/or by masking intergenic transcription terminating signals.

Identification of a cis-acting element which assists in the recruitment of alternative transcription start sites/promoters.

Integration of genome-wide profiles of DNA copy number alterations (CNAs) and gene expression variations (GEVs) could provide combined power to the identification of driver genes and gene networks in tumors. Here we merge matched genome and transcriptome microarray analyses from neuroblastoma samples to derive correlation patterns of CNAs and GEVs, irrespective of their genomic location. Neuroblastoma correlation patterns are strongly asymmetrical, being on average 10 CNAs linked to 1 GEV, and show the widespread prevalence of long range covariance. Functional enrichment and network analysis of the genes covarying with CNAs consistently point to a major cell function, the regulation of mitotic spindle assembly. Moreover, elevated expression of 14 key genes promoting this function is strongly associated to high-risk neuroblastomas with 1p loss and MYCN amplification in a set of 410 tumor samples (P < 0.00001). Independent CNA/GEV profiling on neuroblastoma cell lines shows that increased levels of expression of these genes are linked to 1p loss. By this approach, we reveal a convergence of clustered neuroblastoma CNAs toward increased expression of a group of prognostic and functionally cooperating genes. We therefore propose gain of function of the spindle assembly machinery as a lesion potentially offering new targets for therapy of high-risk neuroblastoma.

Segmental chromosome aberrations converge on overexpression of mitotic spindle regulatory genes in high-risk neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of infancy. Amplification of MYCN oncogene is found in approximately 20 % of all neuroblastoma patients and correlates with advanced disease stages, rapid tumor progression, and poor prognosis, making this gene an obvious therapeutic target. However, being a transcriptional factor MYCN is difficult for pharmacological targeting, and there are currently no clinical trials aiming MYCN protein directly. Here we describe an alternative approach to address deregulated MYCN expression. In particular, we focus on the role of a 3' untranslated region (3'UTR) of the MYCN gene in the modulation of its mRNA fate and identification of compounds able to affect it. The luciferase reporter construct with the full length MYCN 3'UTR was generated and subsequently integrated in the CHP134 neuroblastoma cell line. After validation, the assay was used to screen a 2000 compound library. Molecules affecting luciferase activity were checked for reproducibility and counter-screened for promoter effects and cytotoxic activity resulting in selection of four hits. We propose this cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms. Identification of such compounds could potentially result in development of clinically relevant therapeutics for various diseases including neuroblastoma.

A cell-based high-throughput screen addressing 3'UTR-dependent regulation of the MYCN gene.

Iron is an essential cellular nutrient, being a critical cofactor of several proteins involved in cell growth and replication. Compared with normal cells, neoplastic cells have been shown to require a greater amount of iron, thus laying the basis for the promising anticancer activity of iron chelators. In this work, we evaluated the effects of molecules with iron chelation activity on neuroblastoma (NB) cell lines. Of the 17 iron chelators tested, six reduced cell viability of two NB cell lines with an inhibition of growth of 50% below 10 µM; four of the six molecules-ciclopirox olamine (CPX), piroctone, 8-hydroxyquinoline, and deferasirox-were also shown to efficiently chelate intracellular iron within minutes after addition. Effects on cell viability of one of the compounds, CPX, were indeed dependent on chelation of intracellular iron and mediated by both G0/G1 cell cycle block and induction of apoptosis. By combined transcriptome and translatome profiling we identified early translational downregulation of several members of the heat shock protein group as a specific effect of CPX treatment. We functionally confirmed iron-dependent depletion of HSP90 and its client proteins at pharmacologically achievable concentrations of CPX, and we extended this effect to piroctone, 8-hydroxyquinoline, and deferasirox. Given the documented sensitivity of NB cells to HSP90 inhibition, we propose CPX and other iron chelators as investigational antitumor agents in NB therapy.

Article	Year
A novel mutation in the upstream open reading frame of the CDKN1B gene causes a MEN4 phenotype. PLoS genetics\| PubMed ID: 23555276	2013
Expression of the plasma prekallikrein gene: utilization of multiple transcription start sites and alternative promoter regions. Biological chemistry\| PubMed ID: 15843153	2005
Identification of a cis-acting element which assists in the recruitment of alternative transcription start sites/promoters. Gene\| PubMed ID: 19344755	2009
Segmental chromosome aberrations converge on overexpression of mitotic spindle regulatory genes in high-risk neuroblastoma. Genes, chromosomes & cancer\| PubMed ID: 22337647	2012
A cell-based high-throughput screen addressing 3'UTR-dependent regulation of the MYCN gene. Molecular biotechnology\| PubMed ID: 24515800	2014
Translational Downregulation of HSP90 Expression by Iron Chelators in Neuroblastoma Cells. Molecular pharmacology\| PubMed ID: 25564462	2015

Viktoryia Sidarovich

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