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The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis. Main points: An unbiased, multiomics comparison between multiple blood cell types identified novel PU.1 cis regulatory elements with specific chromatin signatures. A 35-kb insulated neighborhood forms a territory for lineage-specific interactions involving an 8-kb PU.1 cis regulatory element cluster in 3D chromatin space. [ABSTRACT FROM AUTHOR]

Epithelial-to-mesenchymal transitions (EMTs) and extracellular matrix (ECM) remodeling are distinct yet important processes during carcinoma invasion and metastasis. Transforming growth factor β (TGF-β) and RAS, signaling through SMAD and RAS-responsive element-binding protein 1 (RREB1), jointly trigger expression of EMT and fibrogenic factors as two discrete arms of a common transcriptional response in carcinoma cells. Here, we demonstrate that both arms come together to form a program for lung adenocarcinoma metastasis and identify chromatin determinants tying the expression of the constituent genes to TGF-β and RAS inputs. RREB1 localizes to H4K16acK20ac marks in histone H2A.Z-loaded nucleosomes at enhancers in the fibrogenic genes interleukin-11 (IL11), platelet-derived growth factor-B (PDGFB), and hyaluronan synthase 2 (HAS2), as well as the EMT transcription factor SNAI1 , priming these enhancers for activation by a SMAD4-INO80 nucleosome remodeling complex in response to TGF-β. These regulatory properties segregate the fibrogenic EMT program from RAS-independent TGF-β gene responses and illuminate the operation and vulnerabilities of a bifunctional program that promotes metastatic outgrowth. [Display omitted] • TGF-β and RAS drive expression of EMT and fibrogenic genes that jointly fuel metastasis • Epigenetic determinants segregate different programs within a global TGF-β response • RAS/MAPK-regulated RREB1 primes enhancers for activation by SMAD-recruited INO80 • Targeting RREB1 selectively inhibits LUAD metastasis During carcinoma metastasis, malignant progenitors undergo a TGF-β-dependent EMT associated with fibroblast activation and extracellular matrix remodeling in the tumor microenvironment. RAS-activated RREB1 primes enhancers of EMT and fibrogenic genes in lung adenocarcinoma cells for activation by chromatin remodeling complexes that the TGF-β/SMAD pathway recruits to these enhancers. Inhibiting RREB1 disables this pro-metastatic process. [ABSTRACT FROM AUTHOR]

Currently, routine diagnostics for spinocerebellar ataxia (SCA) look for polyQ repeat expansions and conventional variations affecting the proteins encoded by known SCA genes. However, ~40% of the patients still remain without a genetic diagnosis after routine tests. Increasing evidence suggests that variations in the enhancer regions of genes involved in neurodegenerative disorders can also cause disease. Since the enhancers of SCA genes are not yet known, it remains to be determined whether variations in these regions are a cause of SCA. In this pilot project, we aimed to identify the enhancers of the SCA genes ATXN1, ATXN3, TBP and ITPR1 in the human cerebellum using 4C-seq, publicly available datasets, reciprocal 4C-seq, and luciferase assays. We then screened these enhancers for copy number variants (CNVs) in a cohort of genetically undiagnosed SCA patients. We identified two active enhancers for each of the four SCA genes. CNV analysis did not reveal any CNVs in the enhancers of the four SCA genes in the genetically undiagnosed SCA patients. However, in one patient, we noted a CNV deletion with an unknown clinical significance near one of the ITPR1 enhancers. These results not only reveal elements involved in SCA gene regulation but can also lead to the discovery of novel SCA-causing genetic variants. As enhancer variations are being increasingly recognized as a cause of brain disorders, screening the enhancers of ATXN1, ATXN3, TBP and ITPR1 for variations other than CNVs and identifying and screening enhancers of other SCA genes might elucidate the genetic cause in undiagnosed patients. [ABSTRACT FROM AUTHOR]

Aberrant DNA methylation plays a crucial role in breast cancer progression by regulating gene expression. However, the regulatory pattern of DNA methylation in long noncoding RNAs (lncRNAs) for breast cancer remains unclear. In this study, we integrated gene expression, DNA methylation, and clinical data from breast cancer patients included in The Cancer Genome Atlas (TCGA) database. We examined DNA methylation distribution across various lncRNA categories, revealing distinct methylation characteristics. Through genome-wide correlation analysis, we identified the CpG sites located in lncRNAs and the distally associated CpG sites of lncRNAs. Functional genome enrichment analysis, conducted through the integration of ENCODE ChIP-seq data, revealed that differentially methylated CpG sites (DMCs) in lncRNAs were mostly located in promoter regions, while distally associated DMCs primarily acted on enhancer regions. By integrating Hi-C data, we found that DMCs in enhancer and promoter regions were closely associated with the changes in three-dimensional chromatin structures by affecting the formation of enhancer–promoter loops. Furthermore, through Cox regression analysis and three machine learning models, we identified 11 key methylation-driven lncRNAs (DIO3OS, ELOVL2-AS1, MIAT, LINC00536, C9orf163, AC105398.1, LINC02178, MILIP, HID1-AS1, KCNH1-IT1, and TMEM220-AS1) that were associated with the survival of breast cancer patients and constructed a prognostic risk scoring model, which demonstrated strong prognostic performance. These findings enhance our understanding of DNA methylation's role in lncRNA regulation in breast cancer and provide potential biomarkers for diagnosis. [ABSTRACT FROM AUTHOR]

The mammalian p57 protein is a member of the CIP/KIP family of cyclin‐dependent kinase inhibitors and plays an essential role in the development of multiple tissues during embryogenesis as well as in the maintenance of tissue stem cells in adults. Although several transcription factors have been implicated in regulating the p57 gene, cis‐elements such as enhancers that regulate its expression have remained ill‐defined. Here we identify a candidate enhancer for the mouse p57 gene (Cdkn1c) within an intron of the Kcnq1 locus by 4C‐seq analysis in mouse embryonic stem cells (mESCs). Deletion of this putative enhancer region with the CRISPR‐Cas9 system or its suppression by CRISPR interference resulted in a marked attenuation of Cdkn1c expression in differentiating mESCs. Our results thus suggest that this region may serve as an enhancer for the p57 gene during early mouse embryogenesis. [ABSTRACT FROM AUTHOR]

There is evidence that transcription factor (TF) encoding genes, which temporally control development in multiple cell types, can have tens of enhancers that regulate their expression. The NR2F1 TF developmentally promotes caudal and ventral cortical regional fates. Here, we epigenomically compared the activity of Nr2f1's enhancers during mouse cortical development with their activity in a transgenic assay. We identified at least six that are likely to be important in prenatal cortical development, with three harboring de novo mutants identified in ASD individuals. We chose to study the function of two of the most robust enhancers by deleting them singly or together. We found that they have distinct and overlapping functions in driving Nr2f1's regional and laminar expression in the developing cortex. Thus, these two enhancers, probably in combination with the others that we defined epigenetically, precisely tune Nr2f1's regional, cell type, and temporal expression during corticogenesis. [ABSTRACT FROM AUTHOR]

G6PC2 encodes a glucose-6-phosphatase catalytic subunit that opposes the action of glucokinase in pancreatic islets, thereby modulating the sensitivity of insulin and glucagon secretion to glucose. In mice, G6pc2 is expressed at ~20-fold higher levels in ß-cells than in a-cells, whereas in humans G6PC2 is expressed at only ~5-fold higher levels in ß-cells. We therefore hypothesize that G6PC2 likely influences glucagon secretion to a greater degree in humans. With a view to generating a humanized mouse that recapitulates augmented G6PC2 expression levels in a-cells, we sought to identify the genomic regions that confer differential mouse G6pc2 expression in a-cells versus ß-cells as well as the evolutionary changes that have altered this ratio in humans. Studies in islet-derived cell lines suggest that the elevated G6pc2 expression in mouse ß-cells versus a-cells is mainly due to a difference in the relative activity of the proximal G6pc2 promoter in these cell types. Similarly, the smaller difference in G6PC2 expression between a-cells and ß-cells in humans is potentially explained by a change in relative proximal G6PC2 promoter activity. However, we show that both glucocorticoid levels and multiple differences in the relative activity of eight transcriptional enhancers between mice and humans likely contribute to differential G6PC2 expression. Finally, we show that a mouse-specific non-coding RNA, Gm13613, whose expression is controlled by G6pc2 enhancer I, does not regulate G6pc2 expression, indicating that altered expression of Gm13613 in a humanized mouse that contains both the human promoter and enhancers should not affect G6PC2 function. [ABSTRACT FROM AUTHOR]

Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer–promoter (E‐P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward‐ and outward‐oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD‐boundary‐crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long‐distance E‐P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA‐mediated TAD boundary function via R‐loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E‐P interactions is developmentally regulated. [ABSTRACT FROM AUTHOR]

Neuronal activity-regulated gene expression plays a crucial role in sculpting neural circuits that underpin adaptive brain function. Transcriptional enhancers are now recognized as key components of gene regulation that orchestrate spatiotemporally precise patterns of gene transcription. We propose that the dynamics of enhancer activation uniquely position these genomic elements to finely tune activity-dependent cellular plasticity. Enhancer specificity and modularity can be exploited to gain selective genetic access to specific cell states, and the precise modulation of target gene expression within restricted cellular contexts enabled by targeted enhancer manipulation allows for fine-grained evaluation of gene function. Mounting evidence also suggests that enduring stimulus-induced changes in enhancer states can modify target gene activation upon restimulation, thereby contributing to a form of cell-wide metaplasticity. We advocate for focused exploration of activity-dependent enhancer function to gain new insight into the mechanisms underlying brain plasticity and cognitive dysfunction. Neuronal activity-responsive gene expression regulates neural circuit plasticity. Griffith et al. review the critical role of cis -regulatory enhancer elements in orchestrating gene expression programs and highlight the promise of enhancer-focused studies to advance understanding of neural plasticity mechanisms. [ABSTRACT FROM AUTHOR]

Background Genome-wide association studies have identified several nonfunctional tag single-nucleotide polymorphisms (SNPs) associated with severe malaria. We hypothesized that causal SNPs could play a significant role in severe malaria by altering promoter or enhancer activity. Here, we sought to identify such regulatory SNPs. Methods SNPs in linkage disequilibrium with tagSNPs associated with severe malaria were identified and were further annotated using FUMA. Then, SNPs were prioritized using the integrative weighted scoring method to identify regulatory ones. Gene reporter assays were performed to assess the regulatory effect of a region containing candidates. The association between SNPs and severe malaria was assessed using logistic regression models in a Senegalese cohort. Results Among 418 SNPs, the best candidates were rs116525449 and rs79644959, which were in full disequilibrium between them, and located within the ARL14 promoter. Our gene reporter assay results revealed that the region containing the SNPs exhibited cell-specific promoter or enhancer activity, while the SNPs influenced promoter activity. We detected an association between severe malaria and those 2 SNPs using the overdominance model and we replicated the association of severe malaria with the tagSNP rs116423146. Conclusions We suggest that these SNPs regulate ARL14 expression in immune cells and the presentation of antigens to T lymphocytes, thus influencing severe malaria development. [ABSTRACT FROM AUTHOR]

Background: X-linked acrogigantism (X-LAG; MIM: 300942) is a severe form of pituitary gigantism caused by chromosome Xq26.3 duplications involving GPR101. X-LAG-associated duplications disrupt the integrity of the topologically associating domain (TAD) containing GPR101 and lead to the formation of a neo-TAD that drives pituitary GPR101 misexpression and gigantism. As X-LAG is fully penetrant and heritable, duplications involving GPR101 identified on prenatal screening studies, like amniocentesis, can pose an interpretation challenge for medical geneticists and raise important concerns for patients and families. Therefore, providing robust information on the functional genomic impact of such duplications has important research and clinical value with respect to gene regulation and triplosensitivity traits. Methods: We employed 4C/HiC-seq as a clinical tool to determine the functional impact of incidentally discovered GPR101 duplications on TAD integrity in three families. After defining duplications and breakpoints around GPR101 by clinical-grade and high-density aCGH, we constructed 4C/HiC chromatin contact maps for our study population and compared them with normal and active (X-LAG) controls. Results: We showed that duplications involving GPR101 that preserved the centromeric invariant TAD boundary did not generate a pathogenic neo-TAD and that ectopic enhancers were not adopted. This allowed us to discount presumptive/suspected X-LAG diagnoses and GPR101 misexpression, obviating the need for intensive clinical follow-up. Conclusions: This study highlights the importance of TAD boundaries and chromatin interactions in determining the functional impact of copy number variants and provides proof-of-concept for using 4C/HiC-seq as a clinical tool to acquire crucial information for genetic counseling and to support clinical decision-making in cases of suspected TADopathies. [ABSTRACT FROM AUTHOR]

Histone modifications, known as histone marks, are pivotal in regulating gene expression within cells. The vast array of potential combinations of histone marks presents a considerable challenge in decoding the regulatory mechanisms solely through biological experimental approaches. To overcome this challenge, we have developed a method called CatLearning. It utilizes a modified convolutional neural network architecture with a specialized adaptation Residual Network to quantitatively interpret histone marks and predict gene expression. This architecture integrates long-range histone information up to 500Kb and learns chromatin interaction features without 3D information. By using only one histone mark, CatLearning achieves a high level of accuracy. Furthermore, CatLearning predicts gene expression by simulating changes in histone modifications at enhancers and throughout the genome. These findings help comprehend the architecture of histone marks and develop diagnostic and therapeutic targets for diseases with epigenetic changes. [ABSTRACT FROM AUTHOR]

Abstract The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis.

Viral vectors are among the main approaches currently used in studies for executing gene delivery to target cells. During the past decades of active studies in gene therapy, including viruses, adenoviruses (Ads), lentiviruses (LVs), and adeno-associated viruses (AAVs), have received the most attention among the biological approaches where potentially successful outcomes are recorded for numerous genetic conditions. The success of delivery methods, however, remains unsatisfactory. Using some additives that can improve transgene expression, transfection efficiency, viral particle production, and transduction efficiency is considered as a solution to overcoming the limitations of gene delivery approaches. These additives include caffeine, histone deacetylase (HDAC) inhibitors like sodium butyrate and valproic acid, and polycationic agents like polybrene and protamine sulfate. In this review article, we present an overview of viral vector-mediated retinal gene therapies and the application of some enhancers used to improve the outcomes of gene delivery. Three routes of administrating viral vectors into ocular compartments are employed for the delivery of target genes into impacted cells, and some additives have shown enhanced efficiency of gene delivery in retinal cells. The current study places a special focus on the viral vectors and enhancers used for gene therapies of inherited retinal diseases.How to cite: Najafi S, Rahimpour A, Ahmadieh H, et al. The significance of chemical transfection/transduction enhancers in promoting the viral vectors-assisted gene delivery approaches: A focus on potentials for inherited retinal diseases. Electron J Biotechnol 2024;72. https://doi.org/10.1016/j.ejbt.2024.07.005.

Long or Post COVID-19 is a condition of collected symptoms persisted after recovery from COVID-19. Host genetic factors play a crucial role in developing Long COVID-19, and GWAS studies identified several SNPs/genes in various ethnic populations. In African-American population two SNPS, rs10999901 (C>T, p = 3.6E-08, OR = 1.39, MAF-0,27, GRCH38, chr10:71584799 bp) and rs1868001 (G>A, p = 6.7E-09, OR = 1.40, MAF-0.46, GRCH38, chr10:71587815 bp) and in Hispanic population, rs3759084 (A>C, p = 9.7E-09, OR = 1.56, MAF-0.17, chr12: 81,110,156 bp) are strongly associated with Long COVID-19. All these three SNPs reside in noncoding regions implying their regulatory function in the genome. In silico dissection suggests that rs10999901 and rs1868001 physically interact with the CDH23 and C10orf105 genes. Both SNPs act as distant enhancers and bind with several transcription factors (TFs). Further, rs10999901 SNP is a CpG that is methylated in CD4++ T cells and monocytes and loses its methylation due to transition from C>T. rs3759084 is located in the promoter (− 687 bp) of MYF5, acts as a distant enhancer, and physically interacts with PTPRQ. These results offer plausible explanations for their association and provide the basis for experiments to dissect the development of symptoms of Long COVID-19. [ABSTRACT FROM AUTHOR]

Abstract Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.

Abstract Background Recognition of enhancer–promoter Interactions (EPIs) is crucial for human development. EPIs in the genome play a key role in regulating transcription. However, experimental approaches for classifying EPIs are too expensive in terms of effort, time, and resources. Therefore, more and more studies are being done on developing computational techniques, particularly using deep learning and other machine learning techniques, to address such problems. Unfortunately, the majority of current computational methods are based on convolutional neural networks, recurrent neural networks, or a combination of them, which don’t take into consideration contextual details and the long-range interactions between the enhancer and promoter sequences. A new transformer-based model called EPI-Trans is presented in this study to overcome the aforementioned limitations. The multi-head attention mechanism in the transformer model automatically learns features that represent the long interrelationships between enhancer and promoter sequences. Furthermore, a generic model is created with transferability that can be utilized as a pre-trained model for various cell lines. Moreover, the parameters of the generic model are fine-tuned using a particular cell line dataset to improve performance. Results Based on the results obtained from six benchmark cell lines, the average AUROC for the specific, generic, and best models is 94.2%, 95%, and 95.7%, while the average AUPR is 80.5%, 66.1%, and 79.6% respectively. Conclusions This study proposed a transformer-based deep learning model for EPI prediction. The comparative results on certain cell lines show that EPI-Trans outperforms other cutting-edge techniques and can provide superior performance on the challenge of recognizing EPI.

Abstract Background Enhancers are important gene regulatory elements that promote the expression of critical genes in development and disease. Aberrant enhancer can modulate cancer risk and activate oncogenes that lead to the occurrence of various cancers. However, the underlying mechanism of most enhancers in cancer remains unclear. Here, we aim to explore the function and mechanism of a crucial enhancer in melanoma. Methods Multi-omics data were applied to identify an enhancer (enh17) involved in melanoma progression. To evaluate the function of enh17, CRISPR/Cas9 technology were applied to knockout enh17 in melanoma cell line A375. RNA-seq, ChIP-seq and Hi-C data analysis integrated with luciferase reporter assay were performed to identify the potential target gene of enh17. Functional experiments were conducted to further validate the function of the target gene ETV4. Multi-omics data integrated with CUT&Tag sequencing were performed to validate the binding profile of the inferred transcription factor STAT3. Results An enhancer, named enh17 here, was found to be aberrantly activated and involved in melanoma progression. CRISPR/Cas9-mediated deletion of enh17 inhibited cell proliferation, migration, and tumor growth of melanoma both in vitro and in vivo. Mechanistically, we identified ETV4 as a target gene regulated by enh17, and functional experiments further support ETV4 as a target gene that is involved in cancer-associated phenotypes. In addition, STAT3 acts as a transcription factor binding with enh17 to regulate the transcription of ETV4. Conclusions Our findings revealed that enh17 plays an oncogenic role and promotes tumor progression in melanoma, and its transcriptional regulatory mechanisms were fully elucidated, which may open a promising window for melanoma prevention and treatment.

The current work assesses the single-cylinder diesel engine fuelled with orange peel oil. The orange oil was extracted from waste orange peels by steam distillation technique. To enhance the ignition patterns of OPO, an innovative approach has been tried by adding Di-tetra-butyl-peroxide as a cetane improver. Modified fuels are prepared with a diverse volume percentage of DTBP in OPO, and the properties were computed and compared with diesel. 10 and 20% by volume of DTBP is blended with 90 and 80% by volume of OPO and termed as 10DTBP90OPO and 20DTBP80OPO correspondingly. The valuation of modified fuel was performed in a research diesel engine at different loads. Test results specify that the combustion patterns, namely the peak in-cylinder pressure and net heat release rate, are enhanced for OPO by adding DTBP, nevertheless found inferior to diesel. Comparable trends are observed for modified fuels toward performance parameters. 0.8, 0.4, 0.7 g/kWh and 1.1% drop in CO, HC, NOX and smoke correspondingly were observed for 20DTBP80OPO than OPO at full load. Adding 20% volume of DTBP in OPO efficiently improves the engine's emission and performance patterns with enhanced combustion parameters. Hence, the outcome of this work suggests that the fuel generated from orange peel oil shall be declared a feasible diesel substitute. [ABSTRACT FROM AUTHOR]

The myeloma overexpressed gene (MYEOV) has been proposed to be a protooncogene due to high RNA transcript levels found in multiple cancers, including myeloma, breast, lung, pancreas and esophageal cancer. The presence of an open reading frame (ORF) in humans and other primates suggests protein-coding potential. Yet, we still lack evidence of a functional MYEOV protein. It remains undetermined how MYEOV overexpression affects cancerous tissues. In this work, we show that MYEOV has likely originated and may still function as an enhancer, regulating CCND1 and LTO1. Firstly, MYEOV 3' enhancer activity was confirmed in humans using publicly available ATAC-STARR-seq data, performed on B-cell-derived GM12878 cells. We detected enhancer histone marks H3K4me1 and H3K27ac overlapping MYEOV in multiple healthy human tissues, which include B cells, liver and lung tissue. The analysis of 3D genome datasets revealed chromatin interactions between a MYEOV-3'-putative enhancer and the proto-oncogene CCND1. BLAST searches and multi-sequence alignment results showed that DNA sequence from this human enhancer element is conserved from the amphibians/amniotes divergence, with a 273 bp conserved region also found in all mammals, and even in chickens, where it is consistently located near the corresponding CCND1 orthologues. Furthermore, we observed conservation of an active enhancer state in the MYEOV orthologues of four non-human primates, dogs, rats, and mice. When studying this homologous region in mice, where the ORF of MYEOV is absent, we not only observed an enhancer chromatin state but also found interactions between the mouse enhancer homolog and Ccnd1 using 3D-genome interaction data. This is similar to the interaction observed in humans and, interestingly, coincides with CTCF binding sites in both species. Taken together, this suggests that MYEOV is a primatespecific gene with a de novo ORF that originated at an evolutionarily older enhancer region. This deeply conserved putative enhancer element could regulate CCND1 in both humans and mice, opening the possibility of studying MYEOV regulatory functions in cancer using non-primate animal models. [ABSTRACT FROM AUTHOR]

Coronaviruses constitute a global threat to human and animal health. It is essential to investigate the long-distance RNA-RNA interactions that approximate remote regulatory elements in strategies, including genome circularization, discontinuous transcription, and transcriptional enhancers, aimed at the rapid replication of their large genomes, pathogenicity, and immune evasion. Based on the primary sequences and modeled RNA-RNA interactions of two experimentally defined coronaviral enhancers, we detected via an in silico primary and secondary structural analysis potential enhancers in various coronaviruses, from the phylogenetically ancient avian infectious bronchitis virus (IBV) to the recently emerged SARS-CoV-2. These potential enhancers possess a core duplex-forming region that could transition between closed and open states, as molecular switches directed by viral or host factors. The duplex open state would pair with remote sequences in the viral genome and modulate the expression of downstream crucial genes involved in viral replication and host immune evasion. Consistently, variations in the predicted IBV enhancer region or its distant targets coincide with cases of viral attenuation, possibly driven by decreased open reading frame (ORF)3a immune evasion protein expression. If validated experimentally, the annotated enhancer sequences could inform structural prediction tools and antiviral interventions. [ABSTRACT FROM AUTHOR]

MDS1 and EVI1 complex locus (MECOM), a transcription factor encoding several variants, has been implicated in progression of ovarian cancer. The function of regulatory regions in regulating MECOM expression in ovarian cancer is not fully understood. In this study, MECOM expression was evaluated in ovarian cancer cell lines treated with bromodomain and extraterminal (BET) inhibitor JQ-1. Oncogenic phenotypes were assayed using assays of CCK-8, colony formation, wound-healing and transwell. Oncogenic phenotypes were estimated in stable sgRNA-transfected OVCAR3 cell lines. Xenograft mouse model was assayed via subcutaneous injection of enhancer-deleted OVCAR3 cell lines. The results displayed that expression of MECOM is downregulated in cell lines treated with JQ-1. Data from published ChIP-sequencing (H3K27Ac) in 3 ovarian cancer cell lines displayed a potential enhancer around the first exon. mRNA and protein expression were downregulated in OVCAR3 cells after deletion of the MECOM enhancer. Similarly, oncogenic phenotypes both in cells and in the xenograft mouse model were significantly attenuated. This study demonstrates that JQ-1 can inhibit the expression of MECOM and tumorigenesis. Deletion of the enhancer activity of MECOM has an indispensable role in inhibiting ovarian cancer progress, which sheds light on a promising opportunity for ovarian cancer treatment through the application of this non-coding DNA deletion. [ABSTRACT FROM AUTHOR]

Drosophila Hey is a basic helix–loop–helix–orange (bHLH-O) protein with an important role in the establishment of distinct identities of postmitotic cells. We have previously identified Hey as a transcriptional target and effector of Notch signalling during the asymmetric division of neuronal progenitors, generating neurons of two types, and we have shown that Notch-dependent expression of Hey also marks a subpopulation of the newborn enteroendocrine (EE) cells in the midgut primordium of the embryo. Here, we investigate the transcriptional regulation of Hey in neuronal and intestinal tissues. We isolated two genomic regions upstream of the promoter (HeyUP) and in the second intron (HeyIN2) of the Hey gene, based on the presence of binding motifs for Su(H), the transcription factor that mediates Notch activity. We found that both regions can direct the overlapping expression patterns of reporter transgenes recapitulating endogenous Hey expression. Moreover, we showed that while HeyIN2 represents a Notch-dependent enhancer, HeyUP confers both Notch-dependent and independent transcriptional regulation. We induced mutations that removed the Su(H) binding motifs in either region and then studied the enhancer functionality in the respective Hey mutant lines. Our results provide direct evidence that although both enhancers support Notch-dependent regulation of the Hey gene, their role is redundant, as a Hey loss-of-function lethal phenotype is observed only after deletion of all their Su(H) binding motifs by CRISPR/Cas9. [ABSTRACT FROM AUTHOR]

Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants. Key message: The combination of enhancers, Cas12a, and sequential transformation strategies enables efficient and precise heritable gene targeting in Arabidopsis. [ABSTRACT FROM AUTHOR]

Asthma is a widespread airway disorder where GATA3-dependent Type-2 helper T (Th2) cells and group 2 innate lymphoid cells (ILC2s) play vital roles. Asthma-associated single nucleotide polymorphisms (SNPs) are enriched in a region located 926-970 kb downstream from GATA3 in the 10p14 (hG900). However, it is unknown how hG900 affects the pathogenesis of allergic airway inflammation. To investigate the roles of the asthma-associated GATA3 enhancer region in experimental allergic airway inflammation, we first examined the correlation between GATA3 expression and the activation of the hG900 region was analyzed by flow cytometry and ChIP-qPCR. We found that The activation of enhancers in the hG900 region was strongly correlated to the levels of GATA3 in human peripheral T cell subsets. We next generated mice lacking the mG900 region (mG900KO mice) were generated by the CRISPR-Cas9 system, and the development and function of helper T cells and ILCs in mG900KO mice were analyzed in steady-state conditions and allergic airway inflammation induced by papain or house dust mite (HDM). The deletion of the mG900 did not affect the development of lymphocytes in steady-state conditions or allergic airway inflammation induced by papain. However, mG900KO mice exhibited reduced allergic inflammation and Th2 differentiation in the HDM-induced allergic airway inflammation. The analysis of the chromatin conformation around Gata3 by circular chromosome conformation capture coupled to high-throughput sequencing (4C-seq) revealed that the mG900 region interacted with the transcription start site of Gata3 with an influencing chromatin conformation in Th2 cells. These findings indicate that the mG900 region plays a pivotal role in Th2 differentiation and thus enhances allergic airway inflammation. [ABSTRACT FROM AUTHOR]

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole-genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation. [ABSTRACT FROM AUTHOR]

The abnormality of the p53 tumor suppressor is crucial in lung cancer development, because p53 regulates target gene promoters to combat cancer. Recent studies have shown extensive p53 binding to enhancer elements. However, whether p53 exerts a tumor suppressor role by shaping the enhancer landscape remains poorly understood. In the current study, we employed several functional genomics approaches to assess the enhancer activity at p53 binding sites throughout the genome based on our established TP53 knockout (KO) human bronchial epithelial cells (BEAS-2B). A total of 943 active regular enhancers and 370 super-enhancers (SEs) disappeared upon the deletion of p53, indicating that p53 modulates the activity of hundreds of enhancer elements. We found that one p53-dependent SE, located on chromosome 9 and designated as KLF4-SE, regulated the expression of the Krüppel-like factor 4 (KLF4) gene. Furthermore, the deletion of p53 significantly decreased the KLF4-SE enhancer activity and the KLF4 expression, but increased colony formation ability in the nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced cell transformation model. Subsequently, in TP53 KO cells, the overexpression of KLF4 partially reversed the increased clonogenic capacity caused by p53 deficiency. Consistently, KLF4 expression also decreased in lung cancer tissues and cell lines. It appeared that overexpression of KLF4 significantly suppressed the proliferation and migration of lung cancer cells. Collectively, our results suggest that the regulation of enhancer formation and activity by p53 is an integral component of the p53 tumor suppressor function. Therefore, our findings offer some novel insights into the regulation mechanism of p53 in lung oncogenesis and introduce a new strategy for screening therapeutic targets. [ABSTRACT FROM AUTHOR]

Background: Recognition of enhancer–promoter Interactions (EPIs) is crucial for human development. EPIs in the genome play a key role in regulating transcription. However, experimental approaches for classifying EPIs are too expensive in terms of effort, time, and resources. Therefore, more and more studies are being done on developing computational techniques, particularly using deep learning and other machine learning techniques, to address such problems. Unfortunately, the majority of current computational methods are based on convolutional neural networks, recurrent neural networks, or a combination of them, which don't take into consideration contextual details and the long-range interactions between the enhancer and promoter sequences. A new transformer-based model called EPI-Trans is presented in this study to overcome the aforementioned limitations. The multi-head attention mechanism in the transformer model automatically learns features that represent the long interrelationships between enhancer and promoter sequences. Furthermore, a generic model is created with transferability that can be utilized as a pre-trained model for various cell lines. Moreover, the parameters of the generic model are fine-tuned using a particular cell line dataset to improve performance. Results: Based on the results obtained from six benchmark cell lines, the average AUROC for the specific, generic, and best models is 94.2%, 95%, and 95.7%, while the average AUPR is 80.5%, 66.1%, and 79.6% respectively. Conclusions: This study proposed a transformer-based deep learning model for EPI prediction. The comparative results on certain cell lines show that EPI-Trans outperforms other cutting-edge techniques and can provide superior performance on the challenge of recognizing EPI. [ABSTRACT FROM AUTHOR]

Background: Enhancers are important gene regulatory elements that promote the expression of critical genes in development and disease. Aberrant enhancer can modulate cancer risk and activate oncogenes that lead to the occurrence of various cancers. However, the underlying mechanism of most enhancers in cancer remains unclear. Here, we aim to explore the function and mechanism of a crucial enhancer in melanoma. Methods: Multi-omics data were applied to identify an enhancer (enh17) involved in melanoma progression. To evaluate the function of enh17, CRISPR/Cas9 technology were applied to knockout enh17 in melanoma cell line A375. RNA-seq, ChIP-seq and Hi-C data analysis integrated with luciferase reporter assay were performed to identify the potential target gene of enh17. Functional experiments were conducted to further validate the function of the target gene ETV4. Multi-omics data integrated with CUT&Tag sequencing were performed to validate the binding profile of the inferred transcription factor STAT3. Results: An enhancer, named enh17 here, was found to be aberrantly activated and involved in melanoma progression. CRISPR/Cas9-mediated deletion of enh17 inhibited cell proliferation, migration, and tumor growth of melanoma both in vitro and in vivo. Mechanistically, we identified ETV4 as a target gene regulated by enh17, and functional experiments further support ETV4 as a target gene that is involved in cancer-associated phenotypes. In addition, STAT3 acts as a transcription factor binding with enh17 to regulate the transcription of ETV4. Conclusions: Our findings revealed that enh17 plays an oncogenic role and promotes tumor progression in melanoma, and its transcriptional regulatory mechanisms were fully elucidated, which may open a promising window for melanoma prevention and treatment. [ABSTRACT FROM AUTHOR]

Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a “viral super-enhancer” that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses. IMPORTANCE In this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a “viral super-enhancer” that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition. [ABSTRACT FROM AUTHOR]

Genes are often regulated by multiple enhancers. It is poorly understood how the individual enhancer activities are combined to control promoter activity. Anecdotal evidence has shown that enhancers can combine sub-additively, additively, synergistically, or redundantly. However, it is not clear which of these modes are more frequent in mammalian genomes. Here, we systematically tested how pairs of enhancers activate promoters using a three-way combinatorial reporter assay in mouse embryonic stem cells. By assaying about 69,000 enhancer-enhancer-promoter combinations we found that enhancer pairs generally combine near-additively. This behaviour was conserved across seven developmental promoters tested. Surprisingly, these promoters scale the enhancer signals in a non-linear manner that depends on promoter strength. A housekeeping promoter showed an overall different response to enhancer pairs, and a smaller dynamic range. Thus, our data indicate that enhancers mostly act additively, but promoters transform their collective effect non-linearly.

The β-catenin-dependent canonical Wnt signaling is pivotal in organ development, tissue homeostasis, and cancer. Here, we identified an upstream enhancer of Ctnnb1 – the coding gene for β-catenin, named ieCtnnb1 (intestinal enhancer of Ctnnb1), which is crucial for intestinal homeostasis. ieCtnnb1 is predominantly active in the base of small intestinal crypts and throughout the epithelia of large intestine. Knockout of ieCtnnb1 led to a reduction in Ctnnb1 transcription, compromising the canonical Wnt signaling in intestinal crypts. Single-cell sequencing revealed that ieCtnnb1 knockout altered epithelial compositions and potentially compromised functions of small intestinal crypts. While deletion of ieCtnnb1 hampered epithelial turnovers in physiologic conditions, it prevented occurrence and progression of Wnt/β-catenin-driven colorectal cancers. Human ieCTNNB1 drove reporter gene expression in a pattern highly similar to mouse ieCtnnb1. ieCTNNB1 contains a single-nucleotide polymorphism associated with CTNNB1 expression levels in human gastrointestinal epithelia. The enhancer activity of ieCTNNB1 in colorectal cancer tissues was stronger than that in adjacent normal tissues. HNF4α and phosphorylated CREB1 were identified as key trans-factors binding to ieCTNNB1 and regulating CTNNB1 transcription. Together, these findings unveil an enhancer-dependent mechanism controlling the dosage of Wnt signaling and homeostasis in intestinal epithelia.

Abstract Background Cervical cancer is a human papillomavirus (HPV)-related disease. HPV type 16 (HPV16), which is the predominant cause of cervical cancer, can encode miRNAs (HPV16-miRNAs). However, the role of HPV16-miRNAs in the pathogenesis of cervical cancer remains unclear. Methods Human cervical cancer cell lines SiHa (HPV16-positive) and C33A (HPV-negative), and cervical cancer tissues were collected to investigate the expression levels of two HPV16-miRNAs (HPV16-miR-H1 and HPV16-miR-H6). The overexpression and knockdown of HPV16-miR-H1 and HPV16-miR-H6 were performed using the lentiviral vector system and miRNA inhibitors, respectively. RNA-sequencing (RNA-seq) analysis and H3K27ac chromatin immunoprecipitation and sequencing (CHIP-seq) experiments were utilized to explore the roles of HPV16-miR-H1 and HPV16-miR-H6 facilitated by enhancers. CCK8, EdU, transwell, and wound healing assays were performed to verify the effects of HPV16-miR-H1 and HPV16-miR-H6 on cell proliferation and migration. Results HPV16-miR-H1 and HPV16-miR-H6 were highly expressed in both SiHa cells and tissue samples from HPV16-positive cervical cancer patients. RNA-seq analysis showed that HPV16-miR-H1 and HPV16-miR-H6 induced the upregulation of numerous tumor progression-associated genes. H3K27ac CHIP-seq experiments further revealed that HPV16-miR-H1 and HPV16-miR-H6 modulated the expression of critical genes by regulating their enhancer activity. The functional study demonstrated that HPV16-miR-H1 and HPV16-miR-H6 increased the migratory capacity of SiHa cells. Conclusions Our data shed light on the role of HPV16-encoded miRNAs in cervical cancer, particularly emphasizing their involvement in the miRNA-enhancer-target gene system. This novel regulatory mechanism of HPV16-miRNAs provides new insights and approaches for the development of therapeutic strategies by targeting HPV16-positive cervical cancer.

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy.

The Grey allele in horses is causing premature hair greying and susceptibility to melanoma. The causal mutation is a 4.6 kb tandem duplication in intron 6 of the Syntaxin 17 gene. A recent study demonstrated that the most common allele at the Grey locus (G3) involves three tandem copies of this sequence, whilst a more rare allele (G2) has two tandem copies and the wild-type allele (G1) only one copy. The G3 allele is causing fast greying and high incidence of skin melanoma, whereas the G2 allele is causing slow greying and no obvious increase in melanoma incidence. Further somatic copy number expansion has been documented in melanoma tissue from Grey horses. Functional studies showed that this intronic sequence acts as a weak melanocyte-specific enhancer that becomes substantially stronger by the copy number expansion. The Grey mutation is associated with upregulated expression of both Syntaxin 17 and the neighbouring NR4A3 gene in Grey horse melanomas. It is still an open question which of these genes is most important for the phenotypic effects or if causality is due to the combined effect of upregulation of both genes. Interestingly, RNAseq data in the Human Protein Atlas give support for a possible role of NR4A3 because it is particularly upregulated in human skin cancer, and it belongs to a cluster of genes associated with skin cancer and melanin biosynthesis. The Grey mutation and its association with melanoma provide a possibility to study the path to tumour development in numerous Grey horses carrying exactly the same predisposing mutation.

Unsaturated fatty acids in avocado oil can help reduce erythema brought on by prolonged UV-B exposure. The effectivity of sunscreen absorption into the skin will be enhanced by the use of isopropyl myristate (IPM) in nanoemulsion. The purpose of this study was to determine the physical characteristics and sunscreen effectiveness of avocado oil nanoemulsion (AVN) modified with IPM. 1% (FI), 3% (FII), and 5% (FIII) IPM variation were used to make AVN with 5% oil. The AVN were tested for physical characteristics such as organoleptic, pH, viscosity, rheology, particle size and polydispersity index (PI). The products were also tested for sunscreen effectivity by in vitro and Minimum Erythemal Dose (MED) method. The data obtained were analyzed statistically. The results showed that the AVN was pale yellow and clear with transmittance percentage were 96%. The rheogram showed that the products were newtonian. The pH values range were from 6.62 to 6.66; viscosity 1.65-1.84 dPa.s; particle size < 17 nm, zeta potential was in range of -30,54±1,72 to -37,85±3,11 and PI < 0.5 for all formula. In vitro SPF values were 16.43 ± 4.50 (FI), 16.27 ± 4.20 (FII) and 17.88 ± 3.20 (FIII) (p >0.05), and categorized as ultra protection. MED value were 12.28 ± 1.34 (FI); 12.51 ± 1.68 (FII); and 13.22 ± 1.84 (FIII) (p< 0.05) and categorized as maximum protection. Isopropyl myristate increased the sunscreen product's MED value without changing its physical characteristics.

Summary: Several empirical and theoretical studies suggest the presence of multiple enhancers per gene that collectively regulate gene expression, and that common sequence variation impacting on the activities of these enhancers is a major source of inter-individual gene expression variability. However, for the vast majority of genes, enhancers and the underlying regulatory variation remains unknown. Even for the genes with well-characterized enhancers, the nature of the combined effects from multiple enhancers and their variants, when known, on gene expression regulation remains unexplored. Here, we have evaluated the combined effects from five SCN5A enhancers and their regulatory variants that are known to collectively correlate with SCN5A cardiac expression and underlie QT interval association in the general population. Using small deletions centered at the regulatory variants in episomal reporter assays in a mouse cardiomyocyte cell line, we demonstrate that the variants and their flanking sequences play critical role in individual enhancer activities, likely being a transcription factor (TF) binding site. By oligonucleotide-based pulldown assays on predicted TFs, we identify the TFs likely driving allele-specific enhancer activities. Using all 32 possible allelic synthetic constructs in reporter assays, representing the five bi-allelic enhancers, we demonstrate combined additive effects on overall enhancer activities. Using transient enhancer assays in zebrafish embryos we demonstrate that four elements act as enhancers in vivo. Together, these studies uncover the TFs driving the enhancer activities of QT interval associated SCN5A regulatory variants, reveal the additive effects from allelic combinations of these regulatory variants, and prove their potential to act as enhancers in vivo.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascll, Egfr, and Olig2. The increased Ascll expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regu-latory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer is a human papillomavirus (HPV)-related disease. HPV type 16 (HPV16), which is the predominant cause of cervical cancer, can encode miRNAs (HPV16-miRNAs). However, the role of HPV16-miRNAs in the pathogenesis of cervical cancer remains unclear. Methods: Human cervical cancer cell lines SiHa (HPV16-positive) and C33A (HPV-negative), and cervical cancer tissues were collected to investigate the expression levels of two HPV16-miRNAs (HPV16-miR-H1 and HPV16-miR-H6). The overexpression and knockdown of HPV16-miR-H1 and HPV16-miR-H6 were performed using the lentiviral vector system and miRNA inhibitors, respectively. RNA-sequencing (RNA-seq) analysis and H3K27ac chromatin immunoprecipitation and sequencing (CHIP-seq) experiments were utilized to explore the roles of HPV16-miR-H1 and HPV16-miR-H6 facilitated by enhancers. CCK8, EdU, transwell, and wound healing assays were performed to verify the effects of HPV16-miR-H1 and HPV16-miR-H6 on cell proliferation and migration. Results: HPV16-miR-H1 and HPV16-miR-H6 were highly expressed in both SiHa cells and tissue samples from HPV16-positive cervical cancer patients. RNA-seq analysis showed that HPV16-miR-H1 and HPV16-miR-H6 induced the upregulation of numerous tumor progression-associated genes. H3K27ac CHIP-seq experiments further revealed that HPV16-miR-H1 and HPV16-miR-H6 modulated the expression of critical genes by regulating their enhancer activity. The functional study demonstrated that HPV16-miR-H1 and HPV16-miR-H6 increased the migratory capacity of SiHa cells. Conclusions: Our data shed light on the role of HPV16-encoded miRNAs in cervical cancer, particularly emphasizing their involvement in the miRNA-enhancer-target gene system. This novel regulatory mechanism of HPV16-miRNAs provides new insights and approaches for the development of therapeutic strategies by targeting HPV16-positive cervical cancer. [ABSTRACT FROM AUTHOR]

High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of MGMT, a gene encoding the repair protein MGMT. [ABSTRACT FROM AUTHOR]

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy. EHF is upregulated by a core transcriptional regulatory circuitry and promotes malignancy of gastroesophageal adenocarcinoma via AJUBA coactivation through the KRAS pathway, offering a novel dual-targeting therapeutic strategy. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Methods: Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An a-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the a-globin domain in erythroid chicken cells. Results: Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An a-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over aD and aA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that a-globin CRE-2 is required for the induction of aD and aA. Analysis of TF binding motifs at a-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Conclusion: Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells. [ABSTRACT FROM AUTHOR]

ASCL1 is a transcription factor that directs neural progenitors towards lineage differentiation. Although many of the molecular mechanisms underlying its action have been described, several of its targets remain unidentified. We identified in the chick genome a putative enhancer (cE1) upstream of the transcription factor Scratch2 (Scrt2) locus with a predicted heterodimerization motif for ASCL1 and POU3F2. In this study, we investigated the role of ASCL1 and this enhancer in regulating the expression of the Scrt2 in the embryonic spinal cord. We confirmed that cE1 region interacted with the Scrt2 promoter. cE1 was sufficient to mediate ASCL1-driven expression in the neural tube through the heterodimerization sites. Moreover, Scrt2 expression was inhibited when we removed cE1 from the genome. These findings strongly indicate that ASCL1 regulates Scrt2 transcription in the neural tube through cE1. [ABSTRACT FROM AUTHOR]

Human endogenous retroviruses (HERVs) are derived from the infection and integration of exogenetic retroviruses. HERVs account for 8% of human genome, and the majority of HERVs are solitary LTRs (solo-LTRs) due to homologous recombination. Multiple findings have showed that solo-LTRs could provide an enormous reservoir of transcriptional regulatory sequences involved in diverse biological processes, especially carcinogenesis and cancer development. The link between solo-LTRs and human diseases still remains poorly understood. This review focuses on the regulatory modules of solo-LTRs, which contribute greatly to the diversification and evolution of human genes. More importantly, although inactivating mutations, insertions and deletions have been identified in solo-LTRs, the inherited regulatory elements of solo-LTRs initiate the expression of chimeric lncRNA transcripts, which have been reported to play crucial roles in human health and disease. These findings provide valuable insights into the evolutionary and functional mechanisms underlying the presence of HERVs in human genome. Taken together, in this review, we will present evidences showing the regulatory and encoding capacity of solo-LTRs as well as the significant impact on various aspects of human biology. [ABSTRACT FROM AUTHOR]

Surgical castration can effectively avoid boar taint and improve pork quality by removing the synthesis of androstenone in the testis, thereby reducing its deposition in adipose tissue. The expression of genes involved in testis-derived hormone metabolism was altered following surgical castration, but the upstream regulatory factors and underlying mechanism remain unclear. In this study, we systematically profiled chromatin accessibility and transcriptional dynamics in liver tissue of castrated and intact full-sibling Yorkshire pigs. First, we identified 897 differentially expressed genes and 6864 differential accessible regions (DARs) using RNA- and ATAC-seq. By integrating the RNA- and ATAC-seq results, 227 genes were identified, and a significant positive correlation was revealed between differential gene expression and the ATAC-seq signal. We constructed a transcription factor regulatory network after motif analysis of DARs and identified a candidate transcription factor (TF) SP1 that targeted the HSD3B1 gene, which was responsible for the metabolism of androstenone. Subsequently, we annotated DARs by incorporating H3K27ac ChIP-seq data, marking 2234 typical enhancers and 245 super enhancers involved in the regulation of all testis-derived hormones. Among these, four typical enhancers associated with HSD3B1 were identified. Furthermore, an in-depth investigation was conducted on the androstenone-related enhancers, and an androstenone-related mutation was identified in a newfound candidatetypical enhancer (andEN) with dual-luciferase assays. These findings provide further insights into how enhancers function as links between phenotypic and non-coding area variations. The discovery of upstream TF and enhancers of HSD3B1 contributes to understanding the regulatory networks of androstenone metabolism and provides an important foundation for improving pork quality. [ABSTRACT FROM AUTHOR]

Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a) is an active histone mark catalyzed by protein arginine methyltransferase 1 (PRMT1), a major arginine methyltransferase in vertebrates catalyzing asymmetric dimethylation of arginine. H4R3me2a stimulates the activity of lysine acetyltransferases such as CBP/p300, which catalyze the acetylation of H3K27, a mark of active enhancers, super-enhancers, and promoters. There are a few studies on the genomic location of H4R3me2a. In chicken polychromatic erythrocytes, H4R3me2a is found in introns and intergenic regions and binds to the globin locus control region (a super-enhancer) and globin regulatory regions. In this report, we analyzed chromatin immunoprecipitation sequencing data for the genomic location of H4R3me2a in the breast cancer cell line MCF7. As in avian cells, MCF7 H4R3me2a is present in intronic and intergenic regions. Nucleosomes with H4R3me2a and H3K27ac next to nucleosome-free regions are found at super-enhancers, enhancers, and promoter regions of expressed genes. Genes with critical roles in breast cancer cells have broad domains of nucleosomes with H4R3me2a, H3K27ac, and H3K4me3. Our results are consistent with PRMT1-mediated H4R3me2a playing a key role in the chromatin organization of regulatory regions of vertebrate genomes. [ABSTRACT FROM AUTHOR]

High metastatic propensity of osteosarcoma leads to its therapeutic failure and poor prognosis. Although nuclear activation miRNAs (NamiRNAs) are reported to activate gene transcription via targeting enhancer and further promote tumor metastasis, it remains uncertain whether NamiRNAs regulate osteosarcoma metastasis and their exact mechanism. Here, we found that extracellular vesicles of the malignant osteosarcoma cells (143B) remarkably increased the migratory abilities of MNNG cells representing the benign osteosarcoma cells by two folds, which attributed to their high miR‐1246 levels. Specially, miR‐1246 located in nucleus could activate the migration gene expression (such as MMP1) to accelerate MNNG cell migration through elevating the enhancer activities via increasing H3K27ac enrichment. Instead, MMP1 expression was dramatically inhibited after Argonaute 2 (AGO2) knockdown. Notably, in vitro assays demonstrated that AGO2 recognized the hybrids of miR‐1246 and its enhancer DNA via PAZ domains to prevent their degradation from RNase H and these protective roles of AGO2 may favor the gene activation by miR‐1246 in vivo. Collectively, our findings suggest that miR‐1246 could facilitate osteosarcoma metastasis through interacting with enhancer to activate gene expression dependent on AGO2, highlighting the nuclear AGO2 as a guardian for NamiRNA‐targeted gene activation and the potential of miR‐1246 for osteosarcoma metastasis therapy. [ABSTRACT FROM AUTHOR]

Human-specific insertions play important roles in human phenotypes and diseases. Here we reported a 446-bp insertion (Insert-446) in intron 11 of the TBC1D8B gene, located on chromosome X, and traced its origin to a portion of intron 6 of the EBF1 gene on chromosome 5. Interestingly, Insert-446 was present in the human Neanderthal and Denisovans genomes, and was fixed in humans after human-chimpanzee divergence. We have demonstrated that Insert-446 acts as an enhancer through binding transcript factors that promotes a higher expression of human TBC1D8B gene as compared with orthologs in macaques. In addition, over-expression TBC1D8B promoted cell proliferation and migration through "a dual finger" catalytic mechanism (Arg538 and Gln573) in the TBC domain in vitro and knockdown of TBC1D8B attenuated tumorigenesis in vivo. Knockout of Insert-446 prevented cell proliferation and migration in cancer and normal cells. Our results reveal that the human-specific Insert-446 promotes cell proliferation and migration by upregulating the expression of TBC1D8B gene. These findings provide a significant insight into the effects of human-specific insertions on evolution. [ABSTRACT FROM AUTHOR]

During the early development of tetrapods, including humans, the embryonic body elongates caudally once the anterior-posterior axis is established. During this process, region-specific vertebral morphogenesis occurs, with the determination of limb positioning along the anterior-posterior axis. We previously reported that Gdf11 functions as an anatomical integration system that determines the positioning of hindlimbs and sacral vertebrae where Gdf11 is expressed. However, the molecular mechanisms underlying induction of Gdf11 expression remain unclear. In this study, we searched for non-coding regions near the Gdf11 locus that were conserved across species to elucidate the regulatory mechanisms of Gdf11 expression. We identified an enhancer of the Gdf11 gene in intron 1 and named it highly conserved region (HCR). In HCR knockout mice, the expression level of endogenous Gdf11 was decreased, and the position of the sacral-hindlimb unit was shifted posteriorly. We also searched for factors upstream of Gdf11 based on the predicted transcription factor binding sites within the HCR. We found that inhibition of FGF signaling increased endogenous Gdf11 expression, suggesting that FGF signaling negatively regulates Gdf11 expression. However, FGF signaling does not regulate HCR activity. Our results suggest that there are species-specific Gdf11 enhancers other than HCR and that FGF signaling regulates Gdf11 expression independent of HCR. [ABSTRACT FROM AUTHOR]

Super-enhancers are a category of active super-enhancers densely occupied by transcription factors and chromatin regulators, controlling the expression of disease-related genes and cellular identity. Recent studies have demonstrated the formation of complex structures by various factors and super-enhancers, particularly in various cancers. However, our current knowledge of super-enhancers, such as their genomic locations, interaction with factors, functions, and distinction from other super-enhancers regions, remains limited. This research aims to employ deep learning techniques to detect and differentiate between super-enhancers and enhancers based on genomic and epigenomic features and compare the accuracy of the results with other machine learning methods In this study, in addition to evaluating algorithms, we trained a set of genomic and epigenomic features using a deep learning algorithm and the Python-based cross-platform software to detect super-enhancers in DNA sequences. We successfully predicted the presence of super-enhancers in the sequences with higher accuracy and precision. [ABSTRACT FROM AUTHOR]