Genes In Human Cell Lines Research Articles

Identify essential genes based on clustering based synthetic minority oversampling technique

Prediction of essential genes in a life organism is one of the central tasks in synthetic biology. Computational predictors are desired because experimental data is often unavailable. Recently, some sequence-based predictors have been constructed to identify essential genes. However, their predictive performance should be further improved. One key problem is how to effectively extract the sequence-based features, which are able to discriminate the essential genes. Another problem is the imbalanced training set. The amount of essential genes in human cell lines is lower than that of non-essential genes. Therefore, predictors trained with such imbalanced training set tend to identify an unseen sequence as a non-essential gene. Here, a new over-sampling strategy was proposed called Clustering based Synthetic Minority Oversampling Technique (CSMOTE) to overcome the imbalanced data issue. Combining CSMOTE with the Z curve, the global features, and Support Vector Machines, a new protocol called iEsGene-CSMOTE was proposed to identify essential genes. The rigorous jackknife cross validation results indicated that iEsGene-CSMOTE is better than the other competing methods. The proposed method outperformed λ-interval Z curve by 35.48% and 11.25% in terms of Sn and BACC, respectively.

Polymorphism in the human arylamine N-acetyltransferase 1 gene 3′-untranslated region determines polyadenylation signal usage

Human arylamine N-acetyltransferase 1 (NAT1) encodes a drug-metabolising enzyme that plays a role in chemical-associated cancer risk, cancer cell survival and mitochondrial function. Its expression and protein activity are regulated by transcriptional, translational, and post-translational processes, including microRNAs such as miR-1290. Several studies have shown the presence of multiple polyadenylation sites in the NAT1 gene. However, their role in NAT1 expression is poorly understood. Here, we have investigated the genetic sequence of the NAT1 gene in human cell lines, peripheral blood mononuclear cells and breast tumour tissue. We identified five potential polyadenylation signals, two of which carry known single nucleotide polymorphism that affect site usage. Cells that are homozygous for adenine at base 1642, the most distal polyadenylation site, use this site whereas those homozygous for cytosine at base 1642 could not. We also found that the presence of adenine at base 1642 is associated with the NAT1*10 haplotype. Because the putative binding site for miR-1290 is located between the last two polyadenylation sites, we hypothesised that cells that do not use the most distal site will be unaffected by miR-1290. However, this was not the case. NAT1 activity was positively correlated with miR-1290, and induction of miR-1290 in SH-SY5Y cells was associated with induction, not inhibition, of NAT1 activity. The use of PolyA1264 or PolyA1642 did not alter NAT1 activity following ectopic expression of a miR-1290 mimic. These results suggest that the role of miR-1290 in the regulation of NAT1 activity is more complex than previously reported.

CRISPR/Cas9 Screening to Identify Conditionally Essential Genes in Human Cell Lines.

Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells. However, most such screens have been performed in vitro with little consideration into how medium composition might affect gene essentiality. This protocol describes a method to use CRISPR/Cas9-based loss-of-function screens to ask how gene essentiality in human cell lines varies with medium composition. First, a single-guide RNA (sgRNA) library is packaged into lentivirus, and an optimal infection titer is determined for the target cells. Following selection, genomic DNA (gDNA) is extracted from an aliquot of the transduced cells. The remaining transduced cells are then screened in at least two distinct cell culture media. At the conclusion of the screening period, gDNA is collected from each cell population. Next, high-throughput sequencing is used to determine sgRNA barcode abundances from the initial and each of the final populations. Finally, an analytical pipeline is used to identify medium-essential candidate genes from these screen results.

Towards understanding the Regulation of Histone H1 Somatic Subtypes with OMICs

Histone H1 is involved in the regulation of chromatin higher-order structure and compaction. In humans, histone H1 is a multigene family with seven subtypes differentially expressed in somatic cells. Which are the regulatory mechanisms that determine the variability of the H1 complement is a long-standing biological question regarding histone H1. We have used a new approach based on the integration of OMICs data to address this issue. We have examined the 3D-chromatin structure, the binding of transcription factors (TFs), and the expression of somatic H1 genes in human cell lines, using data from public repositories, such as ENCODE. Analysis of Hi-C, ChIP-seq, and RNA-seq data, have revealed that transcriptional control has a greater impact on H1 regulation than previously thought. Somatic H1 genes located in topologically associated domains (TADs) show higher expression than in boundary regions. H1 genes are targeted by a variable number of transcription factors including cell cycle-related TFs, and tissue-specific TFs, suggesting a fine-tuned, subtype-specific transcriptional control. We describe, for the first time, that all H1 somatic subtypes are under transcriptional co-regulation. The replication-independent subtypes, which are encoded in different chromosomes isolated from other histone genes, are also co-regulated with the rest of the somatic H1 genes, indicating that transcriptional co-regulation extends beyond the histone cluster. Transcriptional control and transcriptional co-regulation explain, at least in part, the variability of H1 complement, the fluctuations of H1 subtypes during development, and also the compensatory effects observed, in model systems, after perturbation of one or more H1 subtypes.

Abstract 4802: Functional analysis of cancer associated mitochondrial genome alterations

Abstract It is well documented that a variety of alterations can occur in the mitochondrial genome during human tumor development. These alterations often correlate with cancer phenotypes like treatment resistance and metastasis. Alterations to the mitochondrial genome may therefore be a biomarker for early detection, prognosis and/or predictor of therapeutic sensitivity. However, a method to easily and quickly create isogenic populations of human cells with mitochondrial genome specific alterations is not available. To combat this, we have developed a technology using transient expression of mitochondria targeted TAL domain nickases and TAL endonucleases (TALENs). This the first reliable method to introduce specific mutations into the mitochondrial genome of mammalian cells. We have efficiently induced mtDNA deletions in zebrafish embryos, human and mouse cell lines. In mouse NIH3T3 fibroblasts, we deleted a mitochondrial genome region corresponding to the same disease sequence seen in the human Kearns-Sayre Syndrome (KSS). The deletion resulted in a measurable phenotype including decreased mitochondria basal respiration, ATP production and maximal respiration. We are expanding this work by engineering targeted and precise mtDNA deletions in conjunction with known cancer initiating nuclear mutations in mammalian cells, as well as systemically in mice. These tools will allow us to determine the effect mitochondrial alterations have on the transformation of cancer cells harboring known cancer causing nuclear mutations. We are also developing an inducible system for expressing mitoTALENs, making it possible to create “conditional” loss of function mutations in mitochondrial genes in human cell lines, including induced pluripotent stem cells (iPSCs). This inducible model system will be highly useful in settings where the mitochondrial deletion may cause a selective disadvantage during early cellular differentiation. Citation Format: Neil Otto. Functional analysis of cancer associated mitochondrial genome alterations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4802.

Inhibition of histone acetyltransferase GCN5 extends lifespan in both yeast and human cell lines.

Histone acetyltransferases (HATs) are important enzymes that transfer acetyl groups onto histones and thereby regulate both gene expression and chromosomal structures. Previous work has shown that the activation of sirtuins, which are histone deacetylases, can extend lifespan. This suggests that inhibiting HATs may have a similar beneficial effect. In the present study, we utilized a range of HAT inhibitors or heterozygous Gcn5 and Ngg1 mutants to demonstrate marked yeast life extension. In human cell lines, HAT inhibitors and selective RNAi‐mediated Gcn5 or Ngg1 knockdown reduced the levels of aging markers and promoted proliferation in senescent cells. Furthermore, this observed lifespan extension was associated with the acetylation of histone H3 rather than that of H4. Specifically, it was dependent upon H3K9Ac and H3K18Ac modifications. We also found that the ability of caloric restriction to prolong lifespan is Gcn5‐, Ngg1‐, H3K9‐, and H3K18‐dependent. Transcriptome analysis revealed that these changes were similar to those associated with heat shock and were inversely correlated with the gene expression profiles of aged yeast and aged worms. Through a bioinformatic analysis, we also found that HAT inhibition activated subtelomeric genes in human cell lines. Together, our results suggest that inhibiting the HAT Gcn5 may be an effective means of increasing longevity.

A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene.

The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.

GC Content of Early Metazoan Genes and Its Impact on Gene Expression Levels in Mammalian Cell Lines.

With the genomes available for many animal clades, including the early-branching metazoans, one can readily study the functional conservation of genes across a diversity of animal lineages. Ectopic expression of an animal protein in, for instance, a mammalian cell line is a generally used strategy in structure–function analysis. However, this might turn out to be problematic in case of distantly related species. Here we analyzed the GC content of the coding sequences of basal animals and show its impact on gene expression levels in human cell lines, and, importantly, how this expression efficiency can be improved. Optimization of the GC3 content in the coding sequences of cadherin, alpha-catenin, and paracaspase of Trichoplax adhaerens dramatically increased the expression of these basal animal genes in human cell lines.

Schisandrol B protects against cholestatic liver injury through pregnane X receptors.

Currently, ursodeoxycholic acid and obeticholic acid are the only two FDA-approved drugs for cholestatic liver diseases. Thus, new therapeutic approaches need to be developed. Here we have evaluated the anti-cholestasis effects of Schisandrol B (SolB), a bioactive compound isolated from Schisandra sphenanthera. Hepatoprotective effect of SolB against intrahepatic cholestasis, induced by lithocholic acid (LCA), was evaluated in mice. Metabolomic analysis and gene analysis were used to assess involvement of pregnane X receptor (PXR). Molecular docking, cell-based reporter gene analysis and knockout mice were used to demonstrate the critical role of the PXR pathway in the anti-cholestasis effects of SolB. SolB protected against LCA-induced intrahepatic cholestasis. Furthermore, therapeutic treatment with SolB decreased mortality in cholestatic mice. Metabolomics and gene analysis showed that SolB accelerated metabolism of bile acids, promoted bile acid efflux into the intestine, and induced hepatic expression of the PXR-target genes Cyp3a11, Ugt1a1, and Oatp2, which are involved in bile acid homeostasis. Mechanistic studies showed that SolB activated human PXR and up-regulated PXR target genes in human cell lines. Additionally, SolB did not protect Pxr-null mice from liver injury induced by intrahepatic cholestasis, thus providing genetic evidence that the effect of SolB was PXR-dependent. These findings provide direct evidence for the hepatoprotective effects of SolB against cholestasis by activating PXR. Therefore, SolB may provide a new and effective approach to the prevention and treatment of cholestatic liver diseases.

Novel functional roles of caspase-related genes in the regulation of apoptosis and autophagy.

Caspases, a family of cysteine proteases, cleave substrates and play significant roles in apoptosis, autophagy, and development. Recently, our group identified 72 genes that interact with Death Caspase-1 (DCP-1) proteins in Drosophila by genetic screening of 15,000 EP lines. However, the cellular functions and molecular mechanisms of the screened genes, such as their involvement in apoptosis and autophagy, are poorly understood in mammalian cells. In order to study the functional characterizations of the genes in human cells, we investigated 16 full-length human genes in mammalian expression vectors and tested their effects on apoptosis and autophagy in human cell lines. Our studies revealed that ALFY, BIRC4, and TAK1 induced autophagy, while SEC61A2, N-PAC, BIRC4, WIPI1, and FALZ increased apoptotic cell death. BIRC4 was involved in both autophagy and apoptosis. Western blot analysis and luciferase reporter activity indicated that ALFY, BIRC4, PDGFA, and TAK1 act in a p53-dependent manner, whereas CPSF1, SEC61A2, N-PAC, and WIPI1 appear to be p53-independent. Overexpression of BIRC4 and TAK1 caused upregulation of p53 and accumulation of its target proteins as well as an increase in p53 mRNA levels, suggesting that these genes are involved in p53 transcription and expression of its target genes followed by p53 protein accumulation. In conclusion, apoptosis and/or autophagy mediated by BIRC4 and TAK1 may be regulated by p53 and caspase activity. These novel findings may provide valuable information that will aid in a better understanding of the roles of caspase-related genes in human cell lines and be useful for the process of drug discovery.

Essential Human Genes

Three studies in Science and Cell identify ∼2,000 essential genes in human cell lines using CRISPR-Cas9 and retroviral gene-trap genetic screens.

Meta-Analysis of Large-Scale Toxicogenomic Data Finds Neuronal Regeneration Related Protein and Cathepsin D to Be Novel Biomarkers of Drug-Induced Toxicity.

Undesirable toxicity is one of the main reasons for withdrawing drugs from the market or eliminating them as candidates in clinical trials. Although numerous studies have attempted to identify biomarkers capable of predicting pharmacotoxicity, few have attempted to discover robust biomarkers that are coherent across various species and experimental settings. To identify such biomarkers, we conducted meta-analyses of massive gene expression profiles for 6,567 in vivo rat samples and 453 compounds. After applying rigorous feature reduction procedures, our analyses identified 18 genes to be related with toxicity upon comparisons of untreated versus treated and innocuous versus toxic specimens of kidney, liver and heart tissue. We then independently validated these genes in human cell lines. In doing so, we found several of these genes to be coherently regulated in both in vivo rat specimens and in human cell lines. Specifically, mRNA expression of neuronal regeneration-related protein was robustly down-regulated in both liver and kidney cells, while mRNA expression of cathepsin D was commonly up-regulated in liver cells after exposure to toxic concentrations of chemical compounds. Use of these novel toxicity biomarkers may enhance the efficiency of screening for safe lead compounds in early-phase drug development prior to animal testing.

Translational repression of cyclin D3 by a stable G-quadruplex in its 5′ UTR: implications for cell cycle regulation

cyclin D3 (CCND3) is one of the three D-type cyclins that regulate the G1/S phase transition of the cell cycle. Expression of CCND3 is observed in nearly all proliferating cells; however, the presence of high levels of CCND3 has been linked to a poor prognosis for several types of cancer. Therefore, further mechanistic studies on the regulation of CCND3 expression are urgently needed to provide therapeutic implications. In this study, we report that a conserved RNA G-quadruplex-forming sequence (hereafter CRQ), located in the 5′ UTR of mammalian CCND3 mRNA, is able to fold into an extremely stable, intramolecular, parallel G-quadruplex in vitro. The CRQ G-quadruplex dramatically reduces the activity of a reporter gene in human cell lines, but it has little impact on its mRNA level, indicating a translational repression. Moreover, the CRQ sequence in its natural context inhibits translation of CCND3. Disruption of the G-quadruplex structure by G/U-mutation or deletion results in an elevated expression of CCND3 and an increased phosphorylation of Rb, a downstream target of CCND3, which promotes progression of cells through the G1 phase. Our results add to the growing understanding of the regulation of CCND3 expression and provide a potential therapeutic target for cancer treatment.

DNA methylation profiles of protease nexin 1 (SERPINE2) gene in human cell lines

To investigated whether epigenetic mechanisms contribute to the variable expression of variable protease nexin1(PN-1) encoded by the SERPINE2 gene in different cell types. Working with 5 human cell lines, we determined the CpG methylation status within two CpG islands in the SERPINE2 gene by bisulphate sequencing and the PN-1 mRNA level by Q-RT PCR. A CpG island spanning the transcription initiation site showed little methylation in 3 of the cell lines and substantial methylation in 2 of the cell lines. A CpG island covering the translation starting site showed full methylation in all investigated cell lines. Methylation within the CpG island was not randomly distributed, but showed accumulation at specific sites. However, we were not able to distinguish any patterns which related the methylation frequency to the gene expression level. Inhibition of CpG methylation with 5-aza-2'-deoxycytidine led to a several fold increase in PN-1 mRNA levels, but based on the results on CpG methylation in the CpG island spanning the transcript, the effect is most likely indirect. We have carefully mapped the CpG methylation pattern in two CpG islands in the 5' part of the SERPINE2 gene without finding any obvious inverse correlation between methylation frequency and expression level.

Have No Fear, MitoQ 10 Is Here

The physical manifestation of a trait is a constant interplay between one’s genetic makeup and environmental factors. This notion has been substantiated by nutrigenomic studies demonstrating the benefits of nutritional supplements along the continuum between health and disease.1 More importantly, these studies have revealed the plasticity by which the genetic substrate can interact with various environmental components to either exacerbate or mitigate the manifestation of a disease process in those genetically predisposed. Focusing on cardiovascular disease, a vast literature has demonstrated that this disease process is associated with impaired energy production, increased oxidative stress, and cell calcium overload. To these ends, both clinical and animal studies have demonstrated nutrient deficiencies, integral to these processes, to be associated with cardiovascular disease and that these deficiencies represent independent predictors of increased morbidity and mortality.2,3 One cellular component that has received considerable attention regarding function and nutrient supplementation is the mitochondria. Mitochondria are responsible for cellular bioenergetics via oxidative phosphorylation. However, the perpetual transfer of electrons from one molecule to another within the mitochondria renders this organelle a major site for the genesis of reactive oxygen species (ROS).4 Although the mitochondria have antioxidant defenses,4 the disequilibrium between ROS production and ROS neutralization paves the way for disease manifestation. ROS damage to mitochondrial proteins, lipids, and DNA poses …

Regulation of ERBB2 by oestrogen receptor–PAX2 determines response to tamoxifen

Crosstalk between the oestrogen receptor (ER) and ERBB2/HER-2 pathways has long been implicated in breast cancer aetiology and drug response, yet no direct connection at a transcriptional level has been shown. Here we show that oestrogen-ER and tamoxifen-ER complexes directly repress ERBB2 transcription by means of a cis-regulatory element within the ERBB2 gene in human cell lines. We implicate the paired box 2 gene product (PAX2), in a previously unrecognized role, as a crucial mediator of ER repression of ERBB2 by the anti-cancer drug tamoxifen. We show that PAX2 and the ER co-activator AIB-1/SRC-3 compete for binding and regulation of ERBB2 transcription, the outcome of which determines tamoxifen response in breast cancer cells. The repression of ERBB2 by ER-PAX2 links these two breast cancer subtypes and suggests that aggressive ERBB2-positive tumours can originate from ER-positive luminal tumours by circumventing this repressive mechanism. These data provide mechanistic insight into the molecular basis of endocrine resistance in breast cancer.

Isolation and functional assessment of common, polymorphic variants of the B-MYB proto-oncogene associated with a reduced cancer risk

The B-MYB proto-oncogene is a transcription factor belonging to the MYB family that is frequently overexpressed or amplified in different types of human malignancies. While it is suspected that B-MYB plays a role in human cancer, there is still no direct evidence of its causative role. Looking for mutations of the B-MYB gene in human cell lines and primary cancer samples, we frequently isolated two nonsynonymous B-MYB polymorphic variants (rs2070235 and rs11556379). Compared to the wild-type protein, the B-MYB isoforms display altered conformation, impaired regulation of target genes and decreased antiapoptotic activity, suggesting that they are hypomorphic variants of the major allele. Importantly, the B-MYB polymorphisms are common; rs2070235 and rs11556379 are found, depending on the ethnic background, in 10-50% of human subjects. We postulated that, if B-MYB activity is important for transformation, the presence of common, hypomorphic variants might modify cancer risk. Indeed, the B-MYB polymorphisms are underrepresented in 419 cancer patients compared to 230 controls (odds ratio 0.53; (95%) confidence interval 0.385-0.755; P=0.001). This data imply that a large fraction of the human population is carrier of B-MYB alleles that might be associated with a reduced risk of developing neoplastic disease.

Mouse Double Minute 2 Associates with Chromatin in the Presence of p53 and Is Released to Facilitate Activation of Transcription

The tumor suppressor p53 is a potent transcription factor of which the ability to mediate transcription is inhibited through an interaction with the oncoprotein mouse double minute 2 (Mdm2). The present study has tested the hypothesis that Mdm2 inhibits the p53 response in normally growing cells by binding to chromatin-associated p53. Using chromatin immunoprecipitation, we show that Mdm2 localizes with p53 at its responsive elements on the waf1 and mdm2 genes in human cell lines expressing p53, but not in cell lines lacking p53 expression, indicating that Mdm2 is recruited to regions of DNA in a p53-dependent manner. Interestingly, our results show a decrease of Mdm2 protein associated with p53-responsive elements on the waf1 and mdm2 genes when p53-induced transcription is activated either by DNA damage or through controlled overexpression of p53. Rapid activation of p53 transcriptional activity before increasing p53 protein levels was observed with addition of either small-molecule inhibitors to disrupt the p53-Mdm2 interaction or small interfering RNA to mdm2. These findings indicate Mdm2 transiently localizes with p53 at responsive elements and suggest that latent p53 results from the recruitment of Mdm2 to chromatin.

Roles of PSF protein and VL30 RNA in reversible gene regulation

The mammalian protein PSF contains a DNA-binding domain (DBD) that coordinately represses multiple oncogenic genes in human cell lines, indicating a role for PSF as a human tumor-suppressor protein. PSF also contains two RNA-binding domains (RBD) that form a complex with a noncoding VL30 retroelement RNA, releasing PSF from a gene and reversing repression. Thus, the DBD and RBD in PSF are linked by a mechanism of reversible gene regulation involving a noncoding RNA. This mechanism also could apply to other regulatory proteins that contain both DBD and RBD. The mouse genome has multiple copies of VL30 retroelements that are developmentally regulated, and mouse cells contain VL30 RNAs that have normal and pathological roles in gene regulation. Human chromosome 11 has a VL30 retroelement, and a VL30 EST was identified in human blastocyst cells, indicating that the PSF-VL30 RNA regulatory mechanism also could function in human cells.

Functional studies of the 5'-untranslated region of human 5-HT4 receptor mRNA.

The serotonin 5-HT4 receptor (where 5-HT stands for 5-hydroxy-tryptamine) is a member of the seven transmembrane-spanning G-protein-coupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In the present study, we isolated and characterized the 5'-flanking region of the h5-HT4 (human 5-HT4) receptor. We demonstrate the existence of a novel exon that corresponds to the 5'-untranslated region of the h5-HT4 receptor gene. RNase protection analysis and reverse transcriptase-PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT4 receptor gene is located at -3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT4 receptor was characterized. The promoter region lacks TATA and CAAT canonical motifs in the appropriate location, but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the (-3299/-3050) gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region (-220/-61) located in the long 5'-untranslated region. Mutational analysis, gel shift and transfection assays identified an Nkx2.5 (NK2-transcription-factor-related 5)-like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT4 receptor gene expression involving distinct promoters and non-coding exons.