Potential Cis-regulatory Elements Research Articles

Integrative ATAC-seq and RNA-seq analysis of myogenic differentiation of ovine skeletal muscle satellite cell

Skeletal muscle satellite cells (SMSCs) play an important role in regulating muscle growth and regeneration. Chromatin accessibility allows physical interactions that synergistically regulate gene expression through enhancers, promoters, insulators, and chromatin binding factors. However, the chromatin accessibility altas and its regulatory role in ovine myoblast differentiation is still unclear. Therefore, ATAC-seq and RNA-seq analysis were performed on ovine SMSCs at the proliferation stage (SCG) and differentiation stage (SCD). 17,460 DARs (differential accessibility regions) and 3732 DEGs (differentially expressed genes) were identified. Based on joint analysis of ATAC-seq and RNA-seq, we revealed that PI3K-Akt, TGF-β and other signaling pathways regulated SMSCs differentiation. We identified two novel candidate genes, FZD5 and MAP2K6, which may affect the proliferation and differentiation of SMSCs. Our data identify potential cis regulatory elements of ovine SMSCs. This study can provide a reference for exploring the mechanisms of the differentiation and regeneration of SMSCs in the future.

Cistrome Data Browser: integrated search, analysisand visualization of chromatin data.

The Cistrome Data Browser is a resource of ChIP-seq, ATAC-seq and DNase-seq data from humans and mice. It provides maps of the genome-wide locations of transcription factors, cofactors, chromatin remodelers, histone post-translational modifications and regions of chromatin accessible to endonuclease activity. Cistrome DB v3.0 contains approximately 45000 human and 44000 mouse samples with about 32000 newly collected datasets compared to the previous release. The Cistrome DB v3.0 user interface is implemented as a single page application that unifies menu driven and data driven search functions and provides an embedded genome browser, which allows users to find and visualize data more effectively. Users can find informative chromatin profiles through keyword, menu, and data-driven search tools. Browser search functions can predict the regulators of query genes as well as the cell type and factor dependent functionality of potential cis-regulatory elements. Cistrome DB v3.0 expands the display of quality control statistics, incorporates sequence logos into motif enrichment displays and includes more expansive sample metadata. Cistrome DB v3.0 is available at http://db3.cistrome.org/browser.

The Landscape of Accessible Chromatin and Developmental Transcriptome Maps Reveal a Genetic Mechanism of Skeletal Muscle Development in Pigs

The epigenetic regulation mechanism of porcine skeletal muscle development relies on the openness of chromatin and is also precisely regulated by transcriptional machinery. However, fewer studies have exploited the temporal changes in gene expression and the landscape of accessible chromatin to reveal the underlying molecular mechanisms controlling muscle development. To address this, skeletal muscle biopsy samples were taken from Landrace pigs at days 0 (D0), 60 (D60), 120 (D120), and 180 (D180) after birth and were then analyzed using RNA-seq and ATAC-seq. The RNA-seq analysis identified 8554 effective differential genes, among which ACBD7, TMEM220, and ATP1A2 were identified as key genes related to the development of porcine skeletal muscle. Some potential cis-regulatory elements identified by ATAC-seq analysis contain binding sites for many transcription factors, including SP1 and EGR1, which are also the predicted transcription factors regulating the expression of ACBD7 genes. Moreover, the omics analyses revealed regulatory regions that become ectopically active after birth during porcine skeletal muscle development after birth and identified 151,245, 53,435, 30,494, and 40,911 peaks. The enriched functional elements are related to the cell cycle, muscle development, and lipid metabolism. In summary, comprehensive high-resolution gene expression maps were developed for the transcriptome and accessible chromatin during postnatal skeletal muscle development in pigs.

Integration of single-cell transcriptome and chromatin accessibility of early gonads development among goats, pigs, macaques, and humans.

The early gonads of mammals contain primordial germ cells (PGCs) and somatic cell precursors that are essential for sex determination and gametogenesis. Although it is extensively documented in mice, the development of early gonads in non-rodents remains to be delineated. Because molecular differences between mouse and human gonadal cells have been reported, it warrants the study of the key markers and regulatory features that are conserved or divergent between non-rodent species and human. Here, we integrate single-cell transcriptome and chromatin accessibility analysis to identify regulatory signatures of PGCs and somatic cells in the early gonads of goats, pigs, macaques, and humans. We identify the evolutionarily conserved and species-specific events, including genes expression, signaling pathways, and cell-cell interactions. We also uncover potential cis-regulatory elements and key transcription factors in PGCs and somatic cells. Our datasets provide important resources for better understanding the evolutionary programs of PGCs and gonadal somatic cell development in mammals.

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5′-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

Integration of ATAC-seq and RNA-seq analysis identifies key genes affecting intramuscular fat content in pigs.

Meat quality is one of the most important economic traits in pig breeding and production, and intramuscular fat (IMF) content is the major factor in improving meat quality. The IMF deposition in pigs is influenced by transcriptional regulation, which is dependent on chromatin accessibility. However, how chromatin accessibility plays a regulatory role in IMF deposition in pigs has not been reported. Xidu black is a composite pig breed with excellent meat quality, which is an ideal research object of this study. In this study, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) analysis to identify the accessible chromatin regions and key genes affecting IMF content in Xidu black pig breed with extremely high and low IMF content. First, we identified 21,960 differential accessible chromatin peaks and 297 differentially expressed genes. The motif analysis of differential peaks revealed several potential cis-regulatory elements containing binding sites for transcription factors with potential roles in fat deposition, including Mef2c, CEBP, Fra1, and AP-1. Then, by integrating the ATAC-seq and RNA-seq analysis results, we found 47 genes in the extremely high IMF (IMF_H) group compared with the extremely low IMF (IMF_L) group. For these genes, we observed a significant positive correlation between the differential gene expression and differential ATAC-seq signal (r2 = 0.42). This suggests a causative relationship between chromatin remodeling and the resulting gene expression. We identified several candidate genes (PVALB, THRSP, HOXA9, EEPD1, HOXA10, and PDE4B) that might be associated with fat deposition. Through the PPI analysis, we found that PVALB gene was the top hub gene. In addition, some pathways that might regulate fat cell differentiation and lipid metabolism, such as the PI3K-Akt signaling pathway, MAPK signaling pathway, and calcium signaling pathway, were significantly enriched in the ATAC-seq and RNA-seq analysis. To the best of our knowledge, our study is the first to use ATAC-seq and RNA-seq to examine the mechanism of IMF deposition from a new perspective. Our results provide valuable information for understanding the regulation mechanism of IMF deposition and an important foundation for improving the quality of pork.

Nuclear Factor Kappa B Promotes Ferritin Heavy Chain Expression in Bombyx mori in Response to B. mori Nucleopolyhedrovirus Infection.

Ferritin heavy chain (FerHCH) is a major component of ferritin and plays an important role in maintaining iron homeostasis and redox equilibrium. Our previous studies have demonstrated that the Bombyx mori ferritin heavy chain homolog (BmFerHCH) could respond to B. mori nucleopolyhedrovirus (BmNPV) infection. However, the mechanism by which BmNPV regulates the expression of BmFerHCH remains unclear. In this study, BmFerHCH increased after BmNPV infection and BmNPV infection enhanced nuclear factor kappa B (NF-κB) activity in BmN cells. An NF-κB inhibitor (PDTC) reduced the expression of the virus-induced BmFerHCH in BmN cells, and overexpression of BmRelish (NF-κB) increased the expression of virus-induced BmFerHCH in BmN cells. Furthermore, BmNPV infection enhanced BmFerHCH promoter activity. The potential NF-κB cis-regulatory elements (CREs) in the BmFerHCH promoter were screened by using the JASPAR CORE database, and two effective NF-κB CREs were identified using a dual luciferase reporting system and electrophoretic mobility shift assay (EMSA). BmRelish (NF-κB) bound to NF-κB CREs and promoted the transcription of BmFerHCH. Taken together, BmNPV promotes activation of BmRelish (NF-κB), and activated BmRelish (NF-κB) binds to NF-κB CREs of BmFerHCH promoter to enhance BmFerHCH expression. Our study provides a foundation for future research on the function of BmFerHCH in BmNPV infection.

Exploiting Genomic Features to Improve the Prediction of Transcription Factor-Binding Sites in Plants.

The identification of transcription factor (TF) target genes is central in biology. A popular approach is based on the location by pattern matching of potential cis-regulatory elements (CREs). During the last few years, tools integrating next-generation sequencing data have been developed to improve the performance of pattern matching. However, such tools have not yet been comprehensively evaluated in plants. Hence, we developed a new streamlined method aiming at predicting CREs and target genes of plant TFs in specific organs or conditions. Our approach implements a supervised machine learning strategy, which allows decision rule models to be learnt using TF ChIP-chip/seq experimental data. Different layers of genomic features were integrated in predictive models: the position on the gene, the DNA sequence conservation, the chromatin state and various CRE footprints. Among the tested features, the chromatin features were crucial for improving the accuracy of the method. Furthermore, we evaluated the transferability of predictive models across TFs, organs and species. Finally, we validated our method by correctly inferring the target genes of key TFs controlling metabolite biosynthesis at the organ level in Arabidopsis. We developed a tool-Wimtrap-to reproduce our approach in plant species and conditions/organs for which ChIP-chip/seq data are available. Wimtrap is a user-friendly R package that supports an R Shiny web interface and is provided with pre-built models that can be used to quickly get predictions of CREs and TF gene targets in different organs or conditions in Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa and Zea mays.

Abstract 2350: Foxj1 is a new master regulator of activated PI3K pathway pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer mortality within a decade with overall 5-year survival of 8% for all stages combined. Currently, it is well documented that mechanisms driving PDAC progression involve epigenetic and transcriptional rewiring. Here we combined assay for transposase-accessible chromatin using sequencing (ATAC-seq) and enrichment for H3K27 acetylation chromatin immunoprecipitation (H3K27ac ChIP-seq) measures to explore the epigenetic landscape of different mouse primary pancreatic tumor (PPT) cell lines. Methods: Kras-driven (n=36) and PI3K-driven PPT cell lines (n=9) were cultured in DMEM medium (Gibco). DNA was extracted using manufacture protocols (Qiagen, MinElute PCR Purification Kit) then DNA libraries and high-throughput sequencing were performed. Bioinformatics analysis (ROSE2 Python script) was conducted on H3k27ac ChIP-seq data to define super-enhancer (SEs) and SE-associated genes. Then, ATAC-seq data was explored using Coltron Python package to distinguish enriched Transcription Factor (TF) motifs into SEs. Transcriptomic data was used to slim down the list of potential cis-regulatory elements. We developed knockout (ko) PPT cell lines using CRISPR/CAS9 gene editing method to better characterize the role of Foxj1 as a novel potential master regulator in pancreatic cancer. Lastly, immunohistochemistry (IHC) staining for FOXJ1 was conducted on human PDAC cohort. Results: By k-means clustering, we identified 463 SE-associated genes. Many of them are associated with Kras-driven (epithelial or mesenchymal) or PI3K-driven cell lines exclusively. Surprisingly, we found Foxj1 as SE-associated TF exclusively in PI3K-driven PPT cell lines. Consistent with the epigenetic data, transcriptomic analysis confirmed higher expression of Foxj1 in PI3K-driven PPT cell lines. Then, RNA-seq data revealed downregulation of predicted Foxj1 target genes and enhanced EMT and Wnt/β-catenin signatures in Foxj1 ko cells. These data suggest that epithelial properties of PDAC cells are stabilized by Foxj1 activity. Consistent with these results we detect a higher potential of TGFβ treatment to induce mesenchymal features in Foxj1 ko cells. Furthermore, overexpression of β-catenin protein was confirmed by immunofluorescence. Enhanced Wnt/β-catenin signaling could be responsible for the higher proliferation of Foxj1 ko cells as revealed by proliferation assay. Finally, we investigated FOXJ1 protein level in our PDAC human cohort. Interestingly, we found high nuclear FOXJ1 expression in 23% of cases which is linked with better overall survival. Conclusions: In summary, our data revealed Foxj1 as a novel PDAC associated TF with the ability to reduce the cancer aggressiveness blocking epithelial to mesenchymal transition and β-catenin activity elucidating the better prognosis into the FOXJ1 high expressed patients. Citation Format: Andrea Terrasi, Swathi Subramanian, Christine Klement, Sruthi Ramesh, Heike Bollig, Chiara Falcomatà, Katja Steiger, Rupert Öllinger, Dieter Saur, Roland Rad, Maximilian Reichert, Günter Schneider, Gunnar Schotta. Foxj1 is a new master regulator of activated PI3K pathway pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2350.

Investigating chromatin accessibility during development and differentiation by ATAC-sequencing to guide the identification of cis-regulatory elements.

Mapping accessible chromatin across time scales can give insights into its dynamic nature, for example during cellular differentiation and tissue or organism development. Analysis of such data can be utilised to identify functional cis-regulatory elements (CRE) and transcription factor binding sites and, when combined with transcriptomics, can reveal gene regulatory networks (GRNs) of expressed genes. Chromatin accessibility mapping is a powerful approach and can be performed using ATAC-sequencing (ATAC-seq), whereby Tn5 transposase inserts sequencing adaptors into genomic DNA to identify differentially accessible regions of chromatin in different cell populations. It requires low sample input and can be performed and analysed relatively quickly compared with other methods. The data generated from ATAC-seq, along with other genomic approaches, can help uncover chromatin packaging and potential cis-regulatory elements that may be responsible for gene expression. Here, we describe the ATAC-seq approach and give examples from mainly vertebrate embryonic development, where such datasets have identified the highly dynamic nature of chromatin, with differing landscapes between cellular precursors for different lineages.

Time-Course Transcriptional and Chromatin Accessibility Profiling Reveals Genes Associated With Asymmetrical Gonadal Development in Chicken Embryos.

In birds, male gonads form on both sides whereas most females develop asymmetric gonads. Multiple early lines of evidence suggested that the right gonad fails to develop into a functional ovary, mainly due to differential expression of PITX2 in the gonadal epithelium. Despite some advances in recent years, the molecular mechanisms underlying asymmetric gonadal development remain unclear. Here, using bulk analysis of whole gonads, we established a relatively detailed profile of four representative stages of chicken gonadal development at the transcriptional and chromatin levels. We revealed that many candidate genes were significantly enriched in morphogenesis, meiosis and subcellular structure formation, which may be responsible for asymmetric gonadal development. Further chromatin accessibility analysis suggested that the transcriptional activities of the candidate genes might be regulated by nearby open chromatin regions, which may act as transcription factor (TF) binding sites and potential cis-regulatory elements. We found that LHX9 was a promising TF that bound to the left-biased peaks of many cell cycle-related genes. In summary, this study provides distinctive insights into the potential molecular basis underlying the asymmetric development of chicken gonads.

Comparative Genome Analysis Reveals Cis-Regulatory Elements on Gene-Sized Chromosomes of Ciliated Protists.

Gene-sized chromosomes are a distinct feature of the macronuclear genome in ciliated protists known as spirotrichs. These nanochromosomes are often only several kilobase pairs long and contain a coding region for a single gene. However, the ways in which transcription is regulated on nanochromosomes is still largely unknown. Here, we generated macronuclear genome assemblies for two species of Pseudokeronopsis ciliates to better understand transcription regulation on gene-sized chromosomes. We searched within the short subtelomeric regions for potential cis-regulatory elements and identified distinct AT-rich sequences conserved in both species, at both the 5’ and 3’ end of each gene. We further acquired transcriptomic data for these species, which showed the 5’ cis-regulatory element is associated with active gene expression. Gene family evolution analysis suggests nanochromosomes in spirotrichs may originated approximately 900 million years ago. Together our comparative genomic analyses reveal novel insights into the biological roles of cis-regulatory elements on gene-sized chromosomes.

Integrated Chromatin Accessibility and Transcriptome Landscapes of 5-Fluorouracil-Resistant Colon Cancer Cells.

Background: 5-Fluorouracil (5-FU) is one of the most effective and widely used chemotherapeutic drugs in the treatment of colon cancer, yet chemoresistance is a common feature of colon cancer treatment, resulting in poor prognosis and short survival. Dynamic reprogramming of chromatin accessibility is crucial for proper regulation of gene transcription associated with cancer drug resistance by providing the gene regulatory machinery with rapid access to the open genomic DNA. Methods: Here, we explored the global chromatin accessibility and transcription changes by the assay for transposase-accessible chromatin using sequencing (ATAC-seq) in combination with transcriptome sequencing of both parental and 5-FU-resistant HCT15 cells, followed by integrative analysis to better understand the regulatory network underlying 5-FU resistance in colon cancer cells. Results: A total of 3,175 differentially expressed mRNAs (DEGs), lncRNAs (DELs), and miRNAs (DEMs) related to 5-FU resistance were identified, including significantly upregulated IL33, H19, and miR-17-5p; the downregulated AKR1B10, LINC01012, and miR-125b-5p; and chromatin modifiers such as INO80C, HDAC6, and KDM5A. The construction of the ceRNA regulatory network revealed that H19, HOXA11-AS, and NEAT1 might function as ceRNAs associated with 5-FU resistance in HCT15 cells. Moreover, 9,868 differentially accessible regions (DARs) were obtained, which were positively (r = 0.58) correlated with their nearest DEGs and DELs. The upregulated genes related to 4,937 hyper-accessible regions were significantly enriched in signaling pathways of MAPK, FOX, and WNT, while the 4,931 hypo-accessible regions were considered to be involved in declined biosynthesis of amino acids and nucleotide sugars, signaling pathways of Notch, and HIF-1. Analyses of the DAR sequences revealed that besides the AP-1 family, the TF motifs of FOX and KLF family members were highly enriched in hyper- and hypo-accessible regions, respectively. Finally, we obtained several critical TFs and their potential targets associated with DARs and 5-FU resistance, including FOXA1 and KLF3. Conclusion: These data provided clear insights and valuable resources for an improved understanding of the non-genetic landscape of 5-FU-resistant colon cancer cells based on chromatin accessibility and transcript levels, which allowed for genome-wide detection of TF binding sites, potential cis-regulatory elements and therapeutic targets.

Prospects and challenges of epigenomics in crop improvement.

The advent of high-throughput epigenome mapping techniques has ushered in a new era of multiomics with powerful tools now available to map and record genomic output at different levels. Integrating the different components of the epigenome from these multiomics measures allows investigations of cis-regulatory elements on a genome-scale. Mapping of chromatin state, chromatin accessibility dynamics, and higher-order chromatin structure enables a new level of understanding of cell fate determination, identity and function in normal growth and development, disease resistance, and yield. In this paper, the recent advances in epigenomics research of rice, maize, and wheat are reviewed, and the development trends of epigenomics of major crops in the coming years are projected. We highlight the role of epigenomics in regulating growth and development and identifying potential distal cis-regulatory elements in three major crops, and discuss the prospects and challenges for new epigenetics-mediated breeding technologies in crop improvement. In this review, we summarize and analyze recent epigenomic advances in three major crops epigenomics and discuss possibilities and challenges for future research in the field.

In silico analysis and expression profiling of S-domain receptor-like kinases (SD-RLKs) under different abiotic stresses in Arabidopsis thaliana

BackgroundS-domain receptor-like kinases (SD-RLKs) are an important and multi-gene subfamily of plant receptor-like/pelle kinases (RLKs), which are known to play a significant role in the development and immune responses of Arabidopsis thaliana. The conserved cysteine residues in the extracellular domain of SD-RLKs make them interesting candidates for sensing reactive oxygen species (ROS), assisting oxidative stress mitigation and associated signaling pathways during abiotic stresses. However, how closely SD-RLKs are interrelated to abiotic stress mitigation and signaling remains unknown in A. thaliana.ResultsThis study was initiated by examining the chromosomal localization, phylogeny, sequence and differential expression analyses of 37 SD-RLK genes using publicly accessible microarray datasets under cold, osmotic stress, genotoxic stress, drought, salt, UV-B, heat and wounding. Out of 37 SD-RLKs, 12 genes displayed differential expression patterns in both the root and the shoot tissues. Promoter structure analysis suggested that these 12 SD-RLK genes harbour several potential cis-regulatory elements (CREs), which are involved in regulating multiple abiotic stress responses. Based on these observations, we investigated the expression patterns of 12 selected SD-RLKs under ozone, wounding, oxidative (methyl viologen), UV-B, cold, and light stress at different time points using semi-qRT-PCR. Of these 12 SD-SRKs, the genes At1g61360, At1g61460, At1g61380, and At4g27300 emerged as potential candidates that maintain their expression in most of the stress treatments till exposure for 12 h. Expression patterns of these four genes were further verified under similar stress treatments using qRT-PCR. The expression analysis indicated that the gene At1g61360, At1g61380, and At1g61460 were mostly up-regulated, whereas the expression of At4g27300 either up- or down-regulated in these conditions.ConclusionsTo summarize, the computational analysis and differential transcript accumulation of SD-RLKs under various abiotic stresses suggested their association with abiotic stress tolerance and related signaling in A. thaliana. We believe that a further detailed study will decipher the specific role of these representative SD-RLKs in abiotic stress mitigation vis-a-vis signaling pathways in A. thaliana.

Genome-Wide Analysis of Allele-Specific Expression Genes in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction. Pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common pediatric hematological malignancy and it remains an important cause of morbidity and mortality in children. In this study, we performed an allele-specific expression (ASE) analysis of pediatric BCP ALL with the aim to investigate the relationship between cis-regulatory mutations and gene expression patterns.Materials and methods. Twenty-two high hyperdiploid ALL, twenty ETV6/RUNX1-positive ALL, seven TCF3/PBX1-positive ALL and twenty-eight genetically unclassified BCP ALL (“B-other”) were subjected to whole genome sequencing, SNP array analysis and RNA sequencing. The binomial test was applied to estimate the allelic bias of heterozygous exonic single nucleotide variants (SNVs) in the RNA sequencing data against the genomic data. Allelic ratios >2 or <0.5, and P values <0.05 were used to identify allele-specific expression protein-coding genes.Results. We identified 12,693 expressed genes, of which 9,672 (76%) had heterozygous exonic SNVs (informative genes), in multiple BCP ALL samples (n>2) in 77 of the investigated samples. Genes with ASE were distributed evenly across the autosomal chromosomes in the different subtypes with a range of 30 - 165 ASE genes per case (median number, 86). We found that 630 (6.5%) genes displayed ASE in multiple BCP ALL samples (n>2), of which only eight autosomal genes had monoallelic expression in more than two investigated samples. This suggests that ASE and monoallelic expression are relatively rare in BCP ALL. Gene enrichment analyses showed that pathways involving negative regulation of natural killer cell-mediated cytotoxicity and cell proliferation were enriched, indicating that ASE events possibly were associated with the cell proliferation and leukemia progression in BCP ALL. Furthermore, the hematopoiesis pathway was also enriched in ASE genes that showed high allelic expression bias (allelic ratios >2.5), suggesting that ASE genes might be associated with leukemia development. Somatic genomic aberrations that could cause ASE were also investigated in this study. All informative cases with TCF3/PBX1 rearrangement (n=4) showed monoallelic expression of the PBX1 gene, likely associated with the PBX1 truncation caused by the fusion. Additionally, CHP1, located in 15q15.1, displayed ASE in one case with an inversion involving that chromosome band, indicating a potential cis-acting element in the inversion region that regulated the CHP1 gene expression. Notably, PAX5 displayed various patterns of ASE in BCP ALL. One of three cases with PAX5/ZCCHC7 gene rearrangements displayed PAX5 ASE while the other two did not, indicating a potential uncovered cis-regulatory element around the PAX5/ZCCHC7 breakpoints. Furthermore, two cases with no PAX5 gene rearrangement displayed monoallelic expression of the PAX5 gene, suggesting that there are additional epigenetic alterations were also involved in the regulation of PAX5 gene expression in BCP ALL.Conclusions. In this study, we have characterized genes displaying ASE in childhood BCP ALL. Our data provide new insight into pathogenesis of BCP ALL and may be used to identify novel targets for treatment. DisclosuresNo relevant conflicts of interest to declare.

A cis-regulatory element promoting increased transcription at low temperature in cultured ectothermic Drosophila cells

BackgroundTemperature change affects the myriad of concurrent cellular processes in a non-uniform, disruptive manner. While endothermic organisms minimize the challenge of ambient temperature variation by keeping the core body temperature constant, cells of many ectothermic species maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation in ectotherms are still poorly understood. At the transcriptional level, the heat shock response has been analyzed extensively. The opposite, the response to sub-optimal temperature, has received lesser attention in particular in animal species. The tissue specificity of transcriptional responses to cool temperature has not been addressed and it is not clear whether a prominent general response occurs. Cis-regulatory elements (CREs), which mediate increased transcription at cool temperature, and responsible transcription factors are largely unknown.ResultsThe ectotherm Drosophila melanogaster with a presumed temperature optimum around 25 °C was used for transcriptomic analyses of effects of temperatures at the lower end of the readily tolerated range (14–29 °C). Comparative analyses with adult flies and cell culture lines indicated a striking degree of cell-type specificity in the transcriptional response to cool. To identify potential cis-regulatory elements (CREs) for transcriptional upregulation at cool temperature, we analyzed temperature effects on DNA accessibility in chromatin of S2R+ cells. Candidate cis-regulatory elements (CREs) were evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. This CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D.ConclusionBeyond a rich data resource for future analyses of transcriptional control within the readily tolerated range of an ectothermic animal, a novel reporter assay permitting quantitative characterization of CRE temperature dependence was developed. Our identification and functional dissection of the pst_E1 enhancer demonstrate the utility of resources and assay. The functional characterization of this CoolUp enhancer provides initial mechanistic insights into transcriptional upregulation induced by a shift to temperatures at the lower end of the readily tolerated range.

Acaudina molpadioides mediates lipid uptake by suppressing PCSK9 transcription and increasing LDL receptor in human liver cells.

Acaudina molpadioides has been long used as traditional medicinal resources and reported to demonstrate various important bioactivities such as anticoagulation, antithrombosis, anti-hyperglycemia and anticancer. However, its lipid lowering activity is yet to be fully explored. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that enhances the lysosomal degradation of hepatic low density lipoprotein receptor (LDLR) resulting in excessive accumulation of the plasma levels of LDL-cholesterols (LDL-C) which subsequently accelerate atherosclerosis. In the present study, A. molpadioides fractions were subjected to promoter-reporter luciferase assay to determine its role as PCSK9 inhibitors. It was found both fractions (EFA and EFB) reduced the transcriptional activity of PCSK9 promoter. Among the seven 5′end deletion constructs of PCSK9 promoter, fragments D1 (−1,711/−94), D3 (−709/−94) and D4 (−440/−94), were suppressed in the presence of both fractions whereas D2 (−1,214/−94), and, D6 (−351/−94) as well as D7 (−335/−94) were inhibited only by EFA and EFB, respectively. Further transcription factor binding sites prediction using MatInspector software discovered various potential cis-regulatory elements namely, PPAR, KLFs, RBPJ-kappa and SREBP that may potentially be involved in ameliorating the transcriptional activity of PCSK9. Immunofluorescence staining was used to evaluate the effects of both fractions on LDL-C and LDLR. Results showed that levels of LDL-C uptake in EFA-treated cells were 69.1% followed by EFB at 32.6%, as compared to untreated control after 24 h treatment. The LDLR protein distribution was induced by 62.41% and 32.2%, which corresponded to an increase in LDL-C uptake in both EFA and EFB treatment, respectively. Hence, the inhibition of PCSK9 by bioactive compounds in EFA and EFB could be another promising therapeutic agent in reducing the cholesterol levels and atherosclerosis by targeting PCSK9.

Two FD homologs from London plane (Platanus acerifolia) are associated with floral initiation and flower morphology

The flowering-time gene FD encodes a bZIP transcription factor that interacts with FLOWERING LOCUS T (FT) to induce flowering in Arabidopsis. Previous research has identified two FT homologs of Platanus acerifolia, PaFT and PaFTL, which each have different expression patterns and are involved in diverse developmental processes. However, it is not known whether such FT/FD complexes participate in the flowering processes in P. acerifolia. Therefore, we isolated two closely related FD homologs, PaFDL1 and PaFDL2, and investigated their functions through the analysis of expression profiles, transgenic phenotypes, their interactions with different FT proteins, and potential cis-regulatory elements in their promoters. The PaFDL genes were found to display their maximal expression levels during the stage of floral transition, and subsequent expression patterns were also seen to be related to inflorescence developmental stage. In addition, both PaFDL1 and PaFDL2 were found to be subject to post-transcriptional alternative splicing, each gene producing two transcript forms. Transgenic tobacco overexpressing each of the four resulting transcript types displayed accelerated floral initiation and produced abnormal flowers. The results suggested that the complete PaFDL proteins may interact with different PaFT/PaFTL proteins in order to fulfill both conservative and diverse functions in floral initiation and floral development.

Novel functional variants in the Notch pathway and survival of Chinese colorectal cancer.

Notch signaling pathway plays crucial roles in progression of colorectal cancer (CRC), likely affecting overall survival (OS). In a two-stage survival analysis of 1116 CRC patients in East China, we found that one locus at MINAR1 out of 133 genes in the Notch signaling pathway was significantly associated with OS (P < 1 × 10-6 , false discovery rate < 0.01). This locus containing seven single-nucleotide polymorphisms (SNPs) in high linkage disequilibrium (R2 = 1) is located on chromosome 15, of which the MINAR1 rs72430409 G allele was associated with a greater death risk (HR = 1.98, 95% CI = 1.55-2.54, P = 6.8 × 10-8 ). Further analysis of ChIP-sequencing data from the encyclopedia of DNA Elements showed that rs72430409 and rs72630408 were potential cis-regulatory elements for the MINAR1 promoter. Additional expression quantitative trait loci analysis revealed that rs72430409 G>A and rs72630408 A>G were correlated with increased MINAR1 expression levels in both blood cells and colon tissues. Dual luciferase assays revealed that the rs72430409 A allele increased MINAR1 promoter activity. The Cancer Genome Atlas data showed that expression levels of MINAR1 in CRC samples were significantly higher than that in normal colorectal tissue and that high expression of MINAR1 was associated with a shortened OS, likely via activating the epithelial mesenchymal transition (EMT) pathway as shown in the gene-set enrichment analysis. In vitro, RNAi-mediated silencing of MINAR1 led to decreased migration and proliferation in CRC cancer cells, and MINAR1 silencing could downregulate the expression of key effector genes in EMT and glycolysis. Larger cohort studies and further experiments are needed to validate our findings.