Expression Of Neighboring Genes Research Articles

DeepDoublet identifies neighboring cell-dependent gene expression

Cells interact with each other for proper function and homeostasis. Often, co-expression of ligand-receptor pairs from the single-cell RNAseq (scRNAseq) has been used to identify interacting cell types. Recently, RNA sequencing of physically interacting multi-cells has been used to identify interacting cell types without relying on co-expression of ligand-receptor pairs. This opens a new avenue to study the expression of interacting cell types. We present DeepDoublet, a deep-learning-based tool to decompose the transcriptome of physically interacting two cells (or doublet) into two sets of transcriptome. Applying DeepDoublet to the doublets of hepatocyte and liver endothelial cells (LECs), we successfully decomposed into the transcriptome of each cell type. Especially, DeepDoublet identified specific expression of hepatocytes when they are interacting with LECs. Among them was Angptl3 which has a role in blood vessel formation. DeepDoublet is a tool to identify neighboring cell-dependent gene expression.

ERBB2 comprehensive profiling and prognostication in Stage III Colon Cancer: Findings from PETACC8 and IDEA-France cohorts

ERBB2-pathway activation, through amplification or activating mutations, represents a new target for colon cancer (CC) treatment. We compared molecular methods to the gold-standard for assessing ERBB2 status and determined the prognostic value of ERBB2 amplification, mutations, and expression using data from two phase III trials involving nearly 3,000 stage III CC patients. In the PETACC8 trial, IHC/FISH, DNA, and RNA analysis were performed on 1813, 1719, and 1733 samples, respectively. In the IDEA-France trial, DNA and RNAseq were performed on 1129 and 1263 samples. The breast cancer SCAN-B cohort (n=3409) served as an external reference. A new molecular ERBB2-amplified status was defined using ERBB2 NGS-score, RNAseq expression, and clustering based on ERBB2 neighboring gene expression. Concordance between diagnostic techniques and the association between time-to-recurrence (TTR) and ERBB2-status were evaluated. The prevalence of the molecular ERBB2-amplified group was 1.85% in PETACC8 and 1.5% in IDEA-France, with a concordance of 0.81 (95% CI [0.70-0.92]) with the gold-standard IHC/FISH method in PETACC8. A non-linear relationship was observed between TTR and ERBB2-expression, with extreme groups showing a less favorable prognosis (P<.0001) in both colon and breast cancers. Patients with molecular ERBB2-amplified status or mutations had the poorest prognosis, followed by low-expression and intermediate-expression groups (3-year TTR 67.0%, 71.2%, and 77.9%, respectively). In multivariate analysis, the low-expression group had a significantly shorter TTR (HR=1.28; 95%CI [1.07-1.52]). The molecular definition of ERBB2-status could represent a cost-effective alternative in stage III CC. ERBB2 alterations and low RNA expression significantly reduce TTR, highlighting the complex role of ERBB2.

Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.

Multiple genetic variants at the SLC30A8 locus affect local super-enhancer activity and influence pancreatic β-cell survival and function.

Variants at the SLC30A8 locus are associated with type 2 diabetes (T2D) risk. The lead variant, rs13266634, encodes an amino acid change, Arg325Trp (R325W), at the C-terminus of the secretory granule-enriched zinc transporter, ZnT8. Although this protein-coding variant was previously thought to be the sole driver of T2D risk at this locus, recent studies have provided evidence for lowered expression of SLC30A8 mRNA in protective allele carriers. In the present study, we examined multiple variants that influence SLC30A8 allele-specific expression. Epigenomic mapping has previously identified an islet-selective enhancer cluster at the SLC30A8 locus, hosting multiple T2D risk and cASE associations, which is spatially associated with the SLC30A8 promoter and additional neighboring genes. Here, we show that deletion of variant-bearing enhancer regions using CRISPR-Cas9 in human-derived EndoC-βH3 cells lowers the expression of SLC30A8 and several neighboring genes and improves glucose-stimulated insulin secretion. While downregulation of SLC30A8 had no effect on beta cell survival, loss of UTP23, RAD21, or MED30 markedly reduced cell viability. Although eQTL or cASE analyses in human islets did not support the association between these additional genes and diabetes risk, the transcriptional regulator JQ1 lowered the expression of multiple genes at the SLC30A8 locus and enhanced stimulated insulin secretion.

Abstract 135: The Noncoding Haplotype Region Of Blood Pressure-associated Single Nucleotide Polymorphism Rs1173771 Regulates The Expression Of Natriuretic Peptide Receptor C

Genome-wide association studies have identified 1,071 loci containing 26,585 single nucleotide polymorphisms (SNPs) as associated with blood pressure (BP). These loci together explain more than 25% of BP heritability. The BP-associated SNP rs1173771 has been associated with NPR3 expression in human tissues. NPR3 encodes natriuretic peptide receptor C (NPR-C). The haplotype in linkage disequilibrium (LD) with rs1173771 contains 11 SNPs spanning a noncoding genomic region of more than 17 kbp. The haplotype region is located 126 kbp from the transcription start site of NPR3 . We used genome editing to examine the effect of the noncoding haplotype region in LD with rs1173771 on gene expression. We employed a CRISPR/Cas9 mediated genetic engineering approach to delete the 17 kbp haplotype region in a human induced pluripotent stem cell (iPSC) line. Two clones of iPSCs with homozygous deletion of the 17 kbp region were obtained after screening 50 single cell clones. The genomic modification was confirmed by PCR, Sanger sequencing, and whole genome sequencing. Immunofluorescence staining for pluripotency markers indicated that pluripotency was retained following the genome editing procedures. Each edited iPSC clone and the unedited iPSC line were differentiated into endothelial cells (iECs) in 3 independent rounds of differentiation. Real-time PCR analysis showed a 1.59±0.17-fold upregulation of NPR3 mRNA in iECs with the haplotype region deletion compared to unedited iECs (n=11 and 7, p&lt;0.05). The expression of other neighboring genes ( SUB1 and TARS ) and a distal gene ( MRPS30 ) was not significantly affected by the deletion. The results provide definitive evidence that the noncoding haplotype region in LD with BP-associated SNP rs1173771 regulates the expression of natriuretic peptide receptor C in iECs.

Genomic features underlie the co-option of SVA transposons as cis-regulatory elements in human pluripotent stem cells.

Domestication of transposable elements (TEs) into functional cis-regulatory elements is a widespread phenomenon. However, the mechanisms behind why some TEs are co-opted as functional enhancers while others are not are underappreciated. SINE-VNTR-Alus (SVAs) are the youngest group of transposons in the human genome, where ~3,700 copies are annotated, nearly half of which are human-specific. Many studies indicate that SVAs are among the most frequently co-opted TEs in human gene regulation, but the mechanisms underlying such processes have not yet been thoroughly investigated. Here, we leveraged CRISPR-interference (CRISPRi), computational and functional genomics to elucidate the genomic features that underlie SVA domestication into human stem-cell gene regulation. We found that ~750 SVAs are co-opted as functional cis-regulatory elements in human induced pluripotent stem cells. These SVAs are significantly closer to genes and harbor more transcription factor binding sites than non-co-opted SVAs. We show that a long DNA motif composed of flanking YY1/2 and OCT4 binding sites is enriched in the co-opted SVAs and that these two transcription factors bind consecutively on the TE sequence. We used CRISPRi to epigenetically repress active SVAs in stem cell-like NCCIT cells. Epigenetic perturbation of active SVAs strongly attenuated YY1/OCT4 binding and influenced neighboring gene expression. Ultimately, SVA repression resulted in ~3,000 differentially expressed genes, 131 of which were the nearest gene to an annotated SVA. In summary, we demonstrated that SVAs modulate human gene expression, and uncovered that location and sequence composition contribute to SVA domestication into gene regulatory networks.

Mouse strain-specific polymorphic provirus functions as cis-regulatory element leading to epigenomic and transcriptomic variations

Polymorphic integrations of endogenous retroviruses (ERVs) have been previously detected in mouse and human genomes. While most are inert, a subset can influence the activity of the host genes. However, the molecular mechanism underlying how such elements affect the epigenome and transcriptome and their roles in driving intra-specific variation remain unclear. Here, by utilizing wildtype murine embryonic stem cells (mESCs) derived from distinct genetic backgrounds, we discover a polymorphic MMERGLN (GLN) element capable of regulating H3K27ac enrichment and transcription of neighboring loci. We demonstrate that this polymorphic element can enhance the neighboring Klhdc4 gene expression in cis, which alters the activity of downstream stress response genes. These results suggest that the polymorphic ERV-derived cis-regulatory element contributes to differential phenotypes from stimuli between mouse strains. Moreover, we identify thousands of potential polymorphic ERVs in mESCs, a subset of which show an association between proviral activity and nearby chromatin states and transcription. Overall, our findings elucidate the mechanism of how polymorphic ERVs can shape the epigenome and transcriptional networks that give rise to phenotypic divergence between individuals.

Expression of Retroelements in Cervical Cancer and Their Interplay with HPV Infection and Host Gene Expression.

Simple SummaryRetroelements are endogenous DNA elements present in the human genome. They have a key role in tumorigenesis and cancer progression. Several studies have reported their expression as biomarkers and immunotherapeutic targets to cancer. Their expression can be induced by epigenetic alteration and virus infections. Retroelement overexpression has been shown in distinct types of cancer, such as breast cancer, melanoma, renal cell carcinoma and prostate cancer. However, studies on other specific types of cancer, such as in cervical cancer, are scarce. Here we explored the expression of retroelements in cervical cancer as well as their interplay with HPV infection and their association with expression of neighboring genes. We find expression of retroelements specific to cervical cancer, associated with tumor histological type and HPV infection, as a new target as biomarkers and for immune therapy approaches.Retroelements are expressed in diverse types of cancer and are related to tumorigenesis and to cancer progression. We characterized the expression of retroelements in cervical cancer and explored their interplay with HPV infection and their association with expression of neighboring genes. Forty biopsies of invasive cervical carcinoma (squamous cell carcinomas and adenocarcinomas) with genotyped HPV were selected and analyzed for human endogenous retrovirus (HERV) and long interspersed nuclear element 1 (L1) expression through RNA-seq data. We found 8060 retroelements expressed in the samples and a negative correlation of DNA methyltransferase 1 expression with the two most expressed L1 elements. A total of 103 retroelements were found differentially expressed between tumor histological types and between HPV types, including several HERV families (HERV-K, HERV-H, HERV-E, HERV-I and HERV-L). The comparison between HPV mono- and co-infections showed the highest proportion of differentially expressed L1 elements. The location of retroelements affected neighboring gene expression, such as shown for the interleukin-20 gene family. Three HERVs and seven L1 were located close to this gene family and two L1 showed a positive association with IL20RB expression. This study describes the expression of retroelements in cervical cancer and shows their association with HPV status and host gene expression.

Cis-Repression of Foxq1 Expression Affects Foxf2-Mediated Gene Expression in Palate Development.

Disruption of FOXF2, encoding a member of the Forkhead family transcription factors, has been associated with cleft palate in humans and mice. FOXF2 is located in a conserved gene cluster containing FOXQ1, FOXF2, and FOXC1. We found that expression of Foxq1 is dramatically upregulated in the embryonic palatal mesenchyme in Foxf2–/– mouse embryos. We show here that the Foxf2 promoter-deletion mutation caused dramatically increased expression of the cis-linked Foxq1 allele but had little effect on the Foxq1 allele in trans. We analyzed effects of the Foxf2 mutation on the expression of other neighboring genes and compared those effects with the chromatin domain structure and recently identified enhancer-promoter associations as well as H3K27ac ChIP-seq data. We show that the Foxf2 mutation resulted in significantly increased expression of the Foxq1 and Exoc2 genes located in the same topologically associated domain with Foxf2 but not the expression of the Foxc1 and Gmds genes located in the adjacent chromatin domain. We inactivated the Foxq1 gene in mice homozygous for a Foxf2 conditional allele using CRISPR genome editing and generated (Foxf2/Foxq1)+/– mice with loss-of-function mutations in Foxf2 and Foxq1 in cis. Whereas the (Foxf2/Foxq1)–/– mice exhibited cleft palate at birth similar as in the Foxf2–/– mice, systematic expression analyses of a large number of Foxf2-dependent genes revealed that the (Foxf2/Foxq1)–/– embryos exhibited distinct effects on the domain-specific expression of several important genes, including Foxf1, Shox2, and Spon1, in the developing palatal shelves compared with Foxf2–/– embryos. These results identify a novel cis-regulatory effect of the Foxf2 mutation and demonstrate that cis-regulation of Foxq1 contributed to alterations in palatal gene expression in Foxf2–/– embryos. These results have important implications for interpretation of results and mechanisms from studies of promoter- or gene-deletion alleles. In addition, the unique mouse lines generated in this study provide a valuable resource for understanding the cross-regulation and combinatorial functions of the Foxf2 and Foxq1 genes in development and disease.

Large DNA fragment deletion in lncRNA77580 regulates neighboring gene expression in soybean (Glycine max)

Long non-coding RNAs (lncRNAs) affect gene expressions via a wide range of mechanisms and are considered important regulators of numerous essential biological processes, including abiotic stress responses. However, the biological functions of most lncRNAs are yet to be determined. Moreover, to date, no effective methods have been developed to study the function of plant lncRNAs. We previously discovered a salt stress-related lncRNA, lncRNA77580 in soybean (Glycine max L.). In this study, we cloned the full-length lncRNA77580 and found that it shows nuclear-specific localisation. Furthermore, we employed CRISPR/Cas9 technology to induce large DNA fragment deletions in lncRNA77580 in soybean using a dual-single guide RNA/Cas9 design. As a result, we obtained deletion mutant soybean roots with targeted genomic fragment deletion in lncRNA77580. Deletion and overexpression of lncRNA77580 were found to alter the expression of several neighboring protein-coding genes associated with the response to salt stress. The longer the deleted DNA fragment in lncRNA77580, the greater the influence on the expression of lncRNA77580 itself and neighboring genes. Collectively, the findings of this study revealed that large DNA fragment deletion in lncRNAs using the CRISPR/Cas9 system is a powerful method to obtain functional mutations of soybean lncRNAs that could benefit future research on lncRNA function in soybean.

LncRNA STXBP5-AS1 suppresses stem cell-like properties of pancreatic cancer by epigenetically inhibiting neighboring androglobin gene expression

Previous studies suggest the tumor suppressor role of long non-coding RNA (lncRNA) STXBP5-AS1 in cervical and gastric cancer, but its expression pattern and functional mechanism are still elusive in pancreatic cancer (PC). Relative expression of STXBP5-AS1 in PC both in vivo and in vitro was analyzed by real-time PCR. IC50 of Gemcitabine was determined by the MTT assay. Cell proliferation in response to drug treatment was investigated by colony formation assay. Cell apoptosis was measured by both caspase-3 activity and Annexin V/PI staining. Cell invasion capacity was scored by the transwell assay in vitro, and lung metastasis was examined with the tail vein injection assay. Cell stemness was determined in vitro by sphere formation and marker profiling, respectively, and in vivo by limited dilution of xenograft tumor incidence. Subcellular localization of STXBP5-AS1 was analyzed with fractionation PCR. Association between STXBP5-AS1 and EZH2 was investigated by RNA-immunoprecipitation. The binding of EZH2 on ADGB promoter was analyzed by chromatin immunoprecipitation. The methylation was quantified by bisulfite sequencing. We showed downregulation of STXBP5-AS1 in PC associated with poor prognosis. Ectopic STXBP5-AS1 inhibited chemoresistance and metastasis of PC cells. In addition, STXBP5-AS1 compromised stemness of PC cells. Mechanistically, STXBP5-AS1 potently recruited EZH2 and epigenetically regulated neighboring ADGB transcription, which predominantly mediated the inhibitory effects of STXBP5-AS1 on stem cell-like properties of PC cells. Our study highlights the importance of the STXBP5-EZH2-ADGB axis in chemoresistance and stem cell-like properties of PC.

Chromatin-enriched RNAs mark active and repressive cis-regulation: An analysis of nuclear RNA-seq.

Long noncoding RNAs (lncRNAs) localize in the cell nucleus and influence gene expression through a variety of molecular mechanisms. Chromatin-enriched RNAs (cheRNAs) are a unique class of lncRNAs that are tightly bound to chromatin and putatively function to locally cis-activate gene transcription. CheRNAs can be identified by biochemical fractionation of nuclear RNA followed by RNA sequencing, but until now, a rigorous analytic pipeline for nuclear RNA-seq has been lacking. In this study, we survey four computational strategies for nuclear RNA-seq data analysis and develop a new pipeline, Tuxedo-ch, which outperforms other approaches. Tuxedo-ch assembles a more complete transcriptome and identifies cheRNA with higher accuracy than other approaches. We used Tuxedo-ch to analyze benchmark datasets of K562 cells and further characterize the genomic features of intergenic cheRNA (icheRNA) and their similarity to enhancer RNAs (eRNAs). We quantify the transcriptional correlation of icheRNA and adjacent genes and show that icheRNA is more positively associated with neighboring gene expression than eRNA or cap analysis of gene expression (CAGE) signals. We also explore two novel genomic associations of cheRNA, which indicate that cheRNAs may function to promote or repress gene expression in a context-dependent manner. IcheRNA loci with significant levels of H3K9me3 modifications are associated with active enhancers, consistent with the hypothesis that enhancers are derived from ancient mobile elements. In contrast, antisense cheRNA (as-cheRNA) may play a role in local gene repression, possibly through local RNA:DNA:DNA triple-helix formation.

Inhibition of the Hypoxia-Inducible Factor 1α-Induced Cardiospecific HERNA1 Enhance-Templated RNA Protects From Heart Disease.

Enhancers are genomic regulatory elements conferring spatiotemporal and signal-dependent control of gene expression. Recent evidence suggests that enhancers can generate noncoding enhancer RNAs, but their (patho)biological functions remain largely elusive. We performed chromatin immunoprecipitation-coupled sequencing of histone marks combined with RNA sequencing of left ventricular biopsies from experimental and genetic mouse models of human cardiac hypertrophy to identify transcripts revealing enhancer localization, conservation with the human genome, and hypoxia-inducible factor 1α dependence. The most promising candidate, hypoxia-inducible enhancer RNA ( HERNA)1, was further examined by investigating its capacity to modulate neighboring coding gene expression by binding to their gene promoters by using chromatin isolation by RNA purification and λN-BoxB tethering-based reporter assays. The role of HERNA1 and its neighboring genes for pathological stress-induced growth and contractile dysfunction, and the therapeutic potential of HERNA1 inhibition was studied in gapmer-mediated loss-of-function studies in vitro using human induced pluripotent stem cell-derived cardiomyocytes and various in vivo models of human pathological cardiac hypertrophy. HERNA1 is robustly induced on pathological stress. Production of HERNA1 is initiated by direct hypoxia-inducible factor 1α binding to a hypoxia-response element in the histoneH3-lysine27acetylation marks-enriched promoter of the enhancer and confers hypoxia responsiveness to nearby genes including synaptotagmin XVII, a member of the family of membrane-trafficking and Ca2+-sensing proteins and SMG1, encoding a phosphatidylinositol 3-kinase-related kinase. Consequently, a substrate of SMG1, ATP-dependent RNA helicase upframeshift 1, is hyperphoshorylated in a HERNA1- and SMG1-dependent manner. In vitro and in vivo inactivation of SMG1 and SYT17 revealed overlapping and distinct roles in modulating cardiac hypertrophy. Finally, in vivo administration of antisense oligonucleotides targeting HERNA1 protected mice from stress-induced pathological hypertrophy. The inhibition of HERNA1 postdisease development reversed left ventricular growth and dysfunction, resulting in increased overall survival. HERNA1 is a novel heart-specific noncoding RNA with key regulatory functions in modulating the growth, metabolic, and contractile gene program in disease, and reveals a molecular target amenable to therapeutic exploitation.

The epigenetic regulation of HsMar1, a human DNA transposon

BackgroundBoth classes of transposable elements (DNA and RNA) are tightly regulated at the transcriptional level leading to the inactivation of transposition via epigenetic mechanisms. Due to the high copies number of these elements, the hypothesis has emerged that their regulation can coordinate a regulatory network of genes. Herein, we investigated whether transposition regulation of HsMar1, a human DNA transposon, differs in presence or absence of endogenous HsMar1 copies. In the case where HsMar1 transposition is regulated, the number of repetitive DNA sequences issued by HsMar1 and distributed in the human genome makes HsMar1 a good candidate to regulate neighboring gene expression by epigenetic mechanisms.ResultsA recombinant active HsMar1 copy was inserted in HeLa (human) and CHO (hamster) cells and its genomic excision monitored. We show that HsMar1 excision is blocked in HeLa cells, whereas CHO cells are competent to promote HsMar1 excision. We demonstrate that de novo HsMar1 insertions in HeLa cells (human) undergo rapid silencing by cytosine methylation and apposition of H3K9me3 marks, whereas de novo HsMar1 insertions in CHO cells (hamster) are not repressed and enriched in H3K4me3 modifications. The overall analysis of HsMar1 endogenous copies in HeLa cells indicates that neither full-length endogenous inactive copies nor their Inverted Terminal Repeats seem to be specifically silenced, and are, in contrast, devoid of epigenetic marks. Finally, the setmar gene, derived from HsMar1, presents H3K4me3 modifications as expected for a human housekeeping gene.ConclusionsOur work highlights that de novo and old HsMar1 are not similarly regulated by epigenetic mechanisms. Old HsMar1 are generally detected as lacking epigenetic marks, irrespective their localisation relative to the genes. Considering the putative existence of a network associating HsMar1 old copies and SETMAR, two non-mutually exclusive hypotheses are proposed: active and inactive HsMar1 copies are not similarly regulated or/and regulations concern only few loci (and few genes) that cannot be detected at the whole genome level.

LncGata6-controlled stemness in regeneration and cancer.

Long-noncodingRNAs (lncRNAs) have been proposed as a critical regulator of various cellularprocesses including cell fate decision and cell proliferation (1,2).Mechanistically, lncRNAs control various gene expressions by recruiting thechromatin modifiers in cis and trans modes (1). Transcription oflncRNAs itself also can activate or suppress the neighboring gene expression bythe transcriptional interference through regulatory sequences (1,3). lncRNAsare also involved in a wide range of protein and RNA regulatory functions, suchas scaffoldings of proteins and RNAs, protein and RNA decoys, and functionalmicro-peptides production (1). Aberrant regulation of lncRNA-mediated cellularprocesses is implicated in human disease including cancer (4,5). The recenttechnologic advance including the single-cell RNA sequencing (scRNA-seq), andgene editing, genetically engineered mouse (GEM) models has made it feasible toelucidate the various roles of lncRNAs in the normal tissue physiology and thepathologic conditions including tumorigenesis and metastasis (2,6).

Constant conflict between Gypsy LTR retrotransposons and CHH methylation within a stress-adapted mangrove genome.

The evolutionary dynamics of the conflict between transposable elements (TEs) and their host genome remain elusive. This conflict will be intense in stress-adapted plants as stress can often reactivate TEs. Mangroves reduce TE load convergently in their adaptation to intertidal environments and thus provide a unique opportunity to address the host-TE conflict and its interaction with stress adaptation. Using the mangrove Rhizophora apiculata as a model, we investigated methylation and short interfering RNA (siRNA) targeting patterns in relation to the abundance and age of long terminal repeat (LTR) retrotransposons. We also examined the distance of LTR retrotransposons to genes, the impact on neighboring gene expression and population frequencies. We found differential accumulation amongst classes of LTR retrotransposons despite high overall methylation levels. This can be attributed to 24-nucleotide siRNA-mediated CHH methylation preferentially targeting Gypsy elements, particularly in their LTR regions. Old Gypsy elements possess unusually abundant siRNAs which show cross-mapping to young copies. Gypsy elements appear to be closer to genes and under stronger purifying selection than other classes. Our results suggest a continuous host-TE battle masked by the TE load reduction in R.apiculata. This conflict may enable mangroves, such as R.apiculata, to maintain genetic diversity and thus evolutionary potential during stress adaptation.

Screen and Verification for Transgene Integration Sites in Pigs

Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and without potential non-coding RNAs. After CRISPR/Cas9-mediated site-specific integration on Pifs501, we detected high EGFP expression in different pig cell types and ubiquitous EGFP expression in diverse tissues of transgenic pigs without adversely affecting 600 kb neighboring gene expression. Promoters integrated on Pifs501 exhibit hypomethylated modification, which suggest a permissive epigenetic status of this locus. We establish a versatile master cell line on Pifs501, which allows us to achieve site-specific exchange of EGFP to Follistatin with Cre/loxP system conveniently. Through in vitro and in vivo functional assays, we demonstrate the effectiveness of this screening method, and take Pifs501 as a potential site for transgene insertion in pigs. We anticipate that Pifs501 will have useful applications in pig genome engineering, though the identification of genomic safe harbor should over long-term various functional studies.

Regulation of Notch Signaling by Src Kinase

Notch signaling is a form of intercellular communication which plays pivotal roles at various stages in development and disease. The Notch receptor is a single‐pass transmembrane receptor which undergoes proteolytic processing upon binding with a ligand extending from an adjacent cell's membrane. This ligation and subsequent cleavage liberates the transcriptionally active intracellular domain of Notch from the full length receptor. The emancipated Notch intracellular domain undergoes nuclear translocation where it facilitates target gene transcription. Through this series of molecular events, cells can enact changes in a neighboring cell's gene expression patterns. It is now being revealed that Notch signaling has roles which extend beyond juxtacrine signal transduction. Work in our laboratory focuses on the mechanisms by which Notch signaling integrates cell‐cell stimuli with extracellular cues. In a recent publication from our laboratory, we reported that integrin signaling is capable coordinating the Notch pathway. Here, we reveal that the regulation of Notch by integrins is carried out by Src family kinases working downstream of integrins. This is achieved through the phosphorylation of tyrosine residues, within the Notch intracellular domain, by Src. While serine/threonine phosphorylation of the Notch receptor has been well characterized and is essential to important regulatory events, the tyrosine phosphorylation of the Notch receptor is a relatively unexplored area of research. We have identified four tyrosine residues within the intracellular domain of Notch1 that are phosphorylated by Src. In order to observe the functional consequences of Src induced phosphorylation of Notch1, mutagenesis was performed at these sites. We have found that phosphorylation at these residues has implications for Notch1 intracellular domain stability and transcriptional activity. In addition to this work, we have investigated a number of polyphenolic compounds which are capable of binding integrins and regulating the Notch pathway. We report on the Notch induced ramifications of cellular stimulation with resveratrol, and related polyphenols, that alter Notch target gene transcription. Having established these results, we turn our attention towards the importance of Notch‐Src interactions and polyphenolic regulation of Notch signaling as it pertains to vascular biology.Support or Funding InformationNIH 2R15GM102852‐02, NIH 2R15GM102852‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA-Seq Mouse Brain Regions Expression Data Analysis: Focus on ApoE Functional Network

ApoE expression status was proved to be a highly specific marker of energy metabolism rate in the brain. Along with its neighbor, Translocase of Outer Mitochondrial Membrane 40 kDa (TOMM40) which is involved in mitochondrial metabolism, the corresponding genomic region constitutes the neuroenergetic hotspot. Using RNA-Seq data from a murine model of chronic stress a significant positive expression coordination of seven neighboring genes in ApoE locus in five brain regions was observed. ApoE maintains one of the highest absolute expression values genome-wide, implying that ApoE can be the driver of the neighboring gene expression alteration observed under stressful loads. Notably, we revealed the highly statistically significant increase of ApoE expression in the hypothalamus of chronically aggressive (FDR < 0.007) and defeated (FDR < 0.001) mice compared to the control. Correlation analysis revealed a close association of ApoE and proopiomelanocortin (Pomc) gene expression profiles implying the putative neuroendocrine stress response background of ApoE expression elevation therein.

Effects of Type 1 Diabetes Risk Alleles on Immune Cell Gene Expression.

Genetic studies have identified 61 variants associated with the risk of developing Type 1 Diabetes (T1D). The functions of most of the non-HLA (Human Leukocyte Antigen) genetic variants remain unknown. We found that only 16 of these risk variants could potentially be linked to a protein-coding change. Therefore, we investigated whether these variants affected susceptibility by regulating changes in gene expression. To do so, we examined whole transcriptome profiles of 600 samples from the Type 1 Diabetes Genetics Consortium (T1DGC). These comprised four different immune cell types (Epstein-Barr virus (EBV)-transformed B cells, either basal or after stimulation; and cluster of differentiation (CD)4+ and CD8+ T cells). Many of the T1D-associated risk variants regulated expression of either neighboring (cis-) or distant (trans-) genes. In brief, 24 of the non-HLA T1D variants affected the expression of 31 nearby genes (cis) while 25 affected 38 distant genes (trans). The effects were highly significant (False Discovery Rate p < 0.001). In addition, we searched in public databases for expression effects of T1D single nucleotide polymorphisms (SNPs) in other immune cell types such as CD14+ monocytes, lipopolysaccharide (LPS) stimulated monocytes, and CD19+ B cells. In this paper, we review the (expression quantitative trait loci (eQTLs) associated with each of the 60 T1D variants and provide a summary of the genes impacted by T1D risk alleles in various immune cells. We then review the methodological steps involved in analyzing the function of genome wide association studies (GWAS)-identified variants, with emphasis on those affecting gene expression. We also discuss recent advancements in the methodologies and their advantages. We conclude by suggesting future study designs that will aid in the study of T1D risk variants.