Transcription Initiation Site Research Articles

Molecular aspects of regulation of expression of homeologic glutamate dehydrogenase genes in wheat leaves under hypoxia

An increase in GDH activity has been established when plants are exposed to hypoxic conditions in the first 6 hours, which ensures the formation of an adaptive response of cellular metabolism to the lack of oxygen in the cell. A difference in the transcriptional activity of homeologous GDH-1 genes in wheat leaves under stress conditions was shown. An increase in the mRNA content of the GDH-1(5A) gene in wheat leaves when plants are exposed to hypoxic conditions is observed in the first hours of the experiment, which correlates with a change in the catalytic activity of glutamate dehydrogenase. Regulation of this gene is carried out by the transcription factor HIF, the specific landing site of which is found in the transcription initiation site of the promoter of this gene.

Genome-wide quantification of polycistronic transcription in Leishmania major.

Leishmania major is a human-pathogenic, obligate parasite and the etiological agent of the most prevalent, cutaneous form of leishmaniasis, which is an important neglected, tropical disease with ~1.2 million new infections per year. Leishmania, and the whole order Trypanosomatida, are early eukaryotes with highly diverged gene expression and regulation pathways, setting them apart from their mammalian hosts and from most other eukaryotes. Using precision run-on sequence analysis, we performed a genome-wide mapping and density analysis of RNA polymerases in isolated nuclei of the protozoan parasite Leishmania major. We map transcription initiation sites at divergent strand switch regions and head-tail regions within the chromosomes and correlate them with known sites of chromatin modifications. We confirm continuous, polycistronic RNA synthesis in all RNA polymerase II-dependent gene arrays but find small varying RNA polymerase activities in polycistronic transcription units (PTUs), excluding gene-specific transcription regulation, but not PTU-specific variations. Lastly, we find evidence for transcriptional pausing of all three RNA polymerase classes, hinting at a possible mechanism of transcriptional regulation.IMPORTANCELeishmania spp. are pathogens of humans and animals and cause one of the most important neglected tropical diseases. Regulation of gene expression in Leishmania but also in the related Trypanosoma is radically different from all eukaryotic model organisms, dispensing with regulated, gene-specific transcription, and relying instead on highly regulated translation. Our work sheds light on the initiation, elongation, and termination of transcription, maps unidirectional, polycistronic transcription units, provides evidence for transcriptional pausing at or near starting points of RNA synthesis, and quantifies the varying transcription rates of the polycistronic transcription units. Our results will further the understanding of these important pathogens and should provide a valuable resource for researchers in the field of eukaryotic microbiology.

KAS-ATAC reveals the genome-wide single-stranded accessible chromatin landscape of the human genome.

Gene regulation in most eukaryotes involves two fundamental physical processes -- alterations in the packaging of the genome by nucleosomes, with active cis-regulatory elements (CREs) generally characterized by an open-chromatin configuration, and the activation of transcription. Mapping these physical properties and biochemical activities genome-wide -- through profiling chromatin accessibility and active transcription -- are key tools used to understand the logic and mechanisms of transcription and its regulation. However, the relationship between these two states has until now not been accessible to simultaneous measurement. To address this, we developed KAS-ATAC, a combination of the KAS-seq (Kethoxal-Assisted SsDNA sequencing and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) methods for mapping single-stranded DNA (and thus active transcription) and chromatin accessibility, respectively, enabling the genome-wide identification of DNA fragments that are simultaneously accessible and contain ssDNA. We use KAS-ATAC to evaluate levels of active transcription over different classes of regulatory elements in the human genome, to estimate the absolute levels of transcribed accessible DNA over CREs, to map the nucleosomal configurations associated with RNA polymerase activities, and to assess transcription factor association with transcribed DNA through transcription factor binding site (TFBS) footprinting. We observe lower levels of transcription over distal enhancers compared to promoters and distinct nucleosomal configurations around transcription initiation sites associated with active transcription. Most TFs associate equally with transcribed and nontranscribed DNA but a few factors specifically do not exhibit footprints over ssDNA-containing fragments. We anticipate KAS-ATAC to continue to derive useful insights into chromatin organization and transcriptional regulation in other contexts in the future.

Core promoter identification and transcriptional regulation of porcine ACSL3 gene.

Intramuscular fat (IMF) content is an important factor that affects the edible and processing quality of pork. Studying the transcriptional regulation mechanisms of genes affecting intramuscular fat deposition can provide theoretical support for genetic improvement in pigs. Long-chain fatty acyl-CoA synthase 3 (ACSL3), as a key enzyme in the process of lipid synthesis in mammals. However, no information about the core promoter of the ACSL3 gene and its transcriptional regulation has been reported so far. In this experiment, we successfully cloned 3112 bp of the porcine ACSL3 gene promoter region. In order to find out the core promoter of the ACSL3 gene. The results indicated that the core promoter region of the ACSL3 gene is located from -111 bp to -59 bp upstream of the transcription initiation site (TSS). To identify the interaction between SP1 and the ACSL3 gene promoter, we mutated the predicted binding sites of ACSL3 gene promoter. The results showed that the activity of the promoter was decreased by site-specific mutagenesis of the SP1 transcription factor binding site, while overexpression of SP1 increased the expression of the ACSL3 gene. In summary, our study identified a core promoter region of the porcine ACSL3 gene, and the SP1 binding site is responsible for the promoter activity.

Поліморфізм +1245G/T гена COL1A1 у дітей із дефіцитом гормону росту

Among a significant number of existing polymorphisms of the COL1A1 gene, the most studied is the polymorphism in the transcription initiation site SpI (+1245 G/T, rs1800012), and the heterozygous polymorphism of the COL1A1 gene determines the degree of bone mineral density reduction and, accordingly, linear bone growth. The +1245G/T polymorphism of the COL1A1 gene in children with growth hormone (GH) deficiency was investigated by polymerase chain reaction. A genetic study of 28 children (21 boys, 7 girls) with GH deficiency was performed. The age of the children was 10.86 ± 3.15 years, and the growth retardation was -2.34 (±0.85) SDS. At the time of the study, the children were in a state of euthyroidism. They had a polymorphism of the COL1A1 gene, namely +1245 G/T (rs1800012). In the group of patients with GH deficiency, the proportion of the T/G genotype was 1.4 times higher than in the controls. The presence of homozygous TT and GG genotypes can be protective against GH deficiency (OR = 0.38, 95%CI 0.08-1.79; P = 0.22 and OR = 0.99, 95%CI 0.40-2.18; P = 0.88, respectively). The ratio of allele frequencies in children with GH deficiency (pT = 0.268, qC = 0,732) was significantly different from 1:1, indicating a bias in the study group, possibly due to small sample size. The allele frequencies in patients with GH deficiency were practically indistinguishable from the control group, and the distribution of genotypes corresponded to the Hardy-Weinberg equilibrium. The main allele in the control group was G (pT = 0.693), as well as in the group with GH deficiency (pT = 0.732). Thus, in children with GH deficiency, carrying the G allele of the polymorphic locus +1245G/T (rs1800012) of the COL1A1 gene prevails, which may be a prerequisite for the development of this pathology.

An Antisense Long Non-Coding RNA, LncRsn, Is Involved in Sexual Reproduction and Full Virulence in Fusarium graminearum.

Fusarium head blight (FHB), primarily caused by Fusarium graminearum, is a devastating crop disease that leads to significant declines in wheat yield and quality worldwide. Long non-coding RNAs (lncRNAs) are found to play significant functions in various biological processes, but their regulatory functions in the sexual reproduction and pathogenicity of F. graminearum have not been studied extensively. This study identified an antisense lncRNA, named lncRsn, located in the transcription initiation site region between the 5'-flanking gene FgSna and the 3'-flanking gene FgPta. A deletion mutant of lncRsn (ΔlncRsn) was constructed through homologous recombination. ΔlncRsn exhibited huge reductions in pathogen and sexual reproduction. Additionally, the deletion of lncRsn disrupted the biosynthesis of deoxynivalenol (DON) and impaired the formation of infection structures. RT-qPCR analysis reveals that lncRsn may negatively regulate the transcription of the target gene FgSna. This study found that lncRsn plays an important role in sexual and asexual reproduction, pathogenicity, virulence, osmotic stress, and cell wall integrity (CWI) in F. graminearum. Further characterization of pathogenesis-related genes and the reaction between lncRsn and protein-coding genes will aid in developing novel approaches for controlling F. graminearum diseases.

Full-Length Transcriptome Profiling of the Complete Mitochondrial Genome of Sericothrips houjii (Thysanoptera: Thripidae: Sericothripinae) Featuring Extensive Gene Rearrangement and Duplicated Control Regions.

The mitochondrial genome (mitogenome) of Thysanoptera has extensive gene rearrangement, and some species have repeatable control regions. To investigate the characteristics of the gene expression, transcription and post-transcriptional processes in such extensively gene-rearranged mitogenomes, we sequenced the mitogenome and mitochondrial transcriptome of Sericothrips houjii to analyze. The mitogenome was 14,965 bp in length and included two CRs contains 140 bp repeats between COIII-trnN (CR1) and trnT-trnP (CR2). Unlike the putative ancestral arrangement of insects, S. houjii exhibited only six conserved gene blocks encompassing 14 genes (trnL2-COII, trnD-trnK, ND2-trnW, ATP8-ATP6, ND5-trnH-ND4-ND4L and trnV-lrRNA). A quantitative transcription map showed the gene with the highest relative expression in the mitogenome was ND4-ND4L. Based on analyses of polycistronic transcripts, non-coding RNAs (ncRNAs) and antisense transcripts, we proposed a transcriptional model of this mitogenome. Both CRs contained the transcription initiation sites (TISs) and transcription termination sites (TTSs) of both strands, and an additional TIS for the majority strand (J-strand) was found within antisense lrRNA. The post-transcriptional cleavage processes followed the "tRNA punctuation" model. After the cleavage of transfer RNAs (tRNAs), COI and ND3 matured as bicistronic mRNA COI/ND3 due to the translocation of intervening tRNAs, and the 3' untranslated region (UTR) remained in the mRNAs for COII, COIII, CYTB and ND5. Additionally, isoform RNAs of ND2, srRNA and lrRNA were identified. In summary, the relative mitochondrial gene expression levels, transcriptional model and post-transcriptional cleavage process of S. houjii are notably different from those insects with typical mitochondrial gene arrangements. In addition, the phylogenetic tree of Thripidae including S. houjii was reconstructed. Our study provides insights into the phylogenetic status of Sericothripinae and the transcriptional and post-transcriptional regulation processes of extensively gene-rearranged insect mitogenomes.

FTMT-dependent mitophagy is crucial for ferroptosis resistance in cardiac fibroblast

Metabolic responses to cellular stress are pivotal in cell ferroptosis, with mitophagy serving as a crucial mechanism in both metabolic processes and ferroptosis. This study aims to elucidate the effects of high glucose on cardiomyocytes (CMs) and cardiac fibroblasts (CFs) regarding ferroptosis and to uncover the underlying mechanisms involved. We examined alterations in glycolysis, mitochondrial oxidative phosphorylation (OXPHOS), and mitophagy, which are essential for metabolic adaptations and ferroptosis. High glucose exposure induced ferroptosis specifically in CMs, while CFs exhibited resistance to ferroptosis, increased glycolytic activity, and no change in OXPHOS. Moreover, high glucose treatment enhanced mitophagy and upregulated mitochondrial ferritin (FTMT). Notably, the combination of FTMT and the autophagy-related protein nuclear receptor coactivator 4 (NCOA4) increased under high glucose conditions. Silencing FTMT significantly impeded mitophagy and eliminated ferroptosis resistance in CFs cultured under high glucose conditions. The transcription factor forkhead box A1 (FOXA1) was upregulated in CFs upon high glucose exposure, playing a crucial role in the increased expression of FTMT. Within the 5′-flanking sequence of the FTMT mRNA, approximately −500 nt from the transcription initiation site, three putative FOXA1 binding sites were identified. High glucose augmented the binding affinity between FOXA1 and these sequences, thereby promoting FTMT transcription. In summary, high glucose upregulated FOXA1 expression and stimulated FTMT promoter activity in CFs, thereby promoting FTMT-dependent mitophagy and conferring ferroptosis resistance in CFs.

Abstract Or106: DNA Double-stranded Breaks, a Hallmark of Aging, Defined at the Nucleotide Resolution, Are Increased and Associated with Transcription in the Cardiac Myocyte Genome in LMNA-Cardiomyopathy

Background: An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction. Hypothesis and Aims: We posit that DSBs would be more prevalent at the genomic sites associated with gene expression. As Lamin-A (LMNA) regulates active transcription through Lamin associated domains (LADS) we predict for DSBs to be more common in non-LAD regions. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes. Methods: We identified the genome-wide DSBs in ~ 1 million cardiac myocytes per heart in 3 wild-type and 3 myocyte-specific LMNA-deficient ( Myh6-Cre:Lmna F/F ) mice by END-Sequencing, the LADs were defined in cardiac myocytes via LMNA CUT&RUN assay (N=5). Results: The prevalence of DSBs was 0.8% and 2.2% in the wild-type and Myh6-Cre:Lmna F/F myocytes, respectively. The END-Seq signals for 8 DSBs were enriched in the wild-type and 6,764 in Myh6-Cre:Lmna F/F myocytes, respectively (q<0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the LADs, we defined the LADs in cardiac myocytes and found an average of 818 LADs per myocyte. LADs that were shared by at least three mice (N= 2,572) were considered constitutive LADs (cLADs), which comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N=3,975) were ~ 16-fold lower than those at the non-LAD regions (N=~17,778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of LAD regions, defined as the juxtaposed genomic regions in the Myh6-Cre:Lmna F/F that corresponded to the LAD regions in the wild-type myocytes. Conclusions: To our knowledge, this is the first identification of DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various disease states and aging.

Position-dependent function of human sequence-specific transcription factors

Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites1–3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF’s distinct functional profiles—many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.

Cap-snatching mechanism-mediated unveiling of the transcriptional initiation sites of two distinct begomoviruses

Significant economic losses are inflicted by plant viruses, which pose a risk to sustainable agriculture. The proliferation of novel viral diseases is predominantly attributable to factors such as climate change, international trade, and the rapid evolutionary capabilities of viruses. Begomoviruses are a major group of plant-infecting viruses that pose an imminent threat to global agriculture by causing devastating viral diseases in many crop species. The transcriptional start sites (TSSs) of many plant viruses are typically found in the intergenic region (IR), which is the non-coding (NC) area between the viral genes. The promoters play a crucial role in initiating the transcription process by aiding in the recruitment of cellular transcription machinery. The TSSs are precise nucleotide sequences where RNA polymerase initiates the transcription process. The primary objective of this study was to determine the total number of TSSs for two devastating begomoviruses, family: Geminiviridae, Cotton leaf curl Multan virus (CLCuMuV) and Ageratum yellow vein mosaic virus (AYVMV), using the cap-snatching method in conjunction with one heterologous plant virus. These two begomoviruses, along with their infectious clones, were intentionally infected with selected heterologous plant virus in N. benthamiana plants. The identification of the 5′ ends of heterologous viral mRNA was accomplished by employing high-throughput sequencing to assess the capped RNA leaders (CRLs). The determination of the 5′ termini of suspected begomoviral mRNAs was achieved by aligning the collected CRLs of heterologous virus with the genome of each begomovirus, taking into account only those that were a perfect match with the begomoviral genome. In this study, the TSSs of both begomoviruses were identified via complementary approach. The utilization of high-throughput sequencing for both begomoviruses has facilitated the acquisition of millions of sequences. Comprehending the TSSs and promoter components of begomoviruses is crucial for understanding their ability to cause disease, their interactions with host organisms, and for developing effective methods to manage and control the diseases, they inflict on economically significant crop plants.

IPSI(2L)-EDL: a Two-layer Predictor for Identifying Promoters and their Types based on Ensemble Deep Learning

Abstract: Promoters are DNA fragments located near the transcription initiation site, they can be divided into strong promoter type and weak promoter type according to transcriptional activation and expression level. Identifying promoters and their strengths in DNA sequences is essential for understanding gene expression regulation. Therefore, it is crucial to further improve predictive quality of predictors for real-world application requirements. Here, we constructed the latest training dataset based on the RegalonDB website, where all the promoters in this dataset have been experimentally validated, and their sequence similarity is less than 85%. We used one-hot and nucleotide chemical property and density (NCPD) to represent DNA sequence samples. Additionally, we proposed an ensemble deep learning framework containing a multi-head attention module, long short-term memory present, and a convolutional neural network module. : The results showed that iPSI(2L)-EDL outperformed other existing methods for both promoter prediction and identification of strong promoter type and weak promoter type, the AUC and MCC for the iPSI(2L)-EDL in identifying promoter were improved by 2.23% and 2.96% compared to that of PseDNC-DL on independent testing data, respectively, while the AUC and MCC for the iPSI(2L)- EDL were increased by 3.74% and 5.86% in predicting promoter strength type, respectively. The results of ablation experiments indicate that CNN plays a crucial role in recognizing promoters, the importance of different input positions and long-range dependency relationships among features are helpful for recognizing promoters. : Furthermore, to make it easier for most experimental scientists to get the results they need, a userfriendly web server has been established and can be accessed at http://47.94.248.117/IPSW(2L)-EDL.

DNA double-stranded breaks, a hallmark of aging, defined at the nucleotide resolution, are increased and associated with transcription in the cardiac myocytes in LMNA-cardiomyopathy.

An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction. We posit that DSBs would be more prevalent at the genomic sites that are associated with gene expression. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes. We identified the genome-wide DSBs in ∼1 million cardiac myocytes per heart in three wild-type and three myocyte-specific LMNA-deficient (Myh6-Cre:LmnaF/F) mice by END-Sequencing. The prevalence of DSBs was 0.8% and 2.2% in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively. The END-Seq signals were enriched for 8 and 6764 DSBs in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively (q < 0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the lamin-associated domains (LADs), we defined the LADs in cardiac myocytes by a Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay (N = 5). On average there were 818 LADs per myocyte. Constitutive LADs (cLADs), defined as LADs that were shared by at least three genomes (N = 2572), comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N = 3975) were ∼16-fold lower than those at the non-LAD regions (N = ∼17 778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of-LAD regions, defined as the genomic regions in the Myh6-Cre:LmnaF/F that were juxtaposed to the LAD regions in the wild-type myocytes. To our knowledge, this is the first identification of the DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various states and aging.

Association of single nucleotide polymorphisms in ITLN1 gene with ischemic stroke risk in Xi'an population, Shaanxi province.

Ischemic stroke (IS) is the main cause of death and adult disability. However, the pathogenesis of this complicated disease is unknown. The present study aimed to assess the relationship between ITLN1 single nucleotide polymorphisms (SNPs) and the susceptibility to IS in Xi'an population, Shaanxi province. In this study, we designed polymerase chain reaction (PCR) primers located at -3,308 bp upstream of the transcription initiation site within promoter region of the ITLN1 gene. The target fragment was amplified by PCR and identified by agarose gel electrophoresis. Sanger sequencing was then performed in the samples extracted from a cohort comprising 1,272 participants (636 controls and 636 cases), and the obtained sequences were compared with the reference sequences available on the National Center for Biotechnology Information (NCBI) website to detect SNPs in the ITLN1 gene promoter region. Logistic regression analysis was employed to assess the relationship between ITLN1 polymorphisms and IS risk, with adjustments for age and gender. Significant positive results were tested by false-positive report probability (FPRP) and false discovery rate (FDR). The interaction among noteworthy SNPs and their predictive relationship with IS risk were explored using the Multi-Factor Dimensionality Reduction (MDR) software. The results of Sanger sequencing were compared with the reference sequences on the NCBI website, and we found 14 SNPs in ITLN1 gene promoter satisfied Hardy-Weinberg equilibrium (HWE). Logistic regression analysis showed that ITLN1 was associated with a decreased risk of IS (rs6427553: Homozygous C/C: adjusted OR: 0.69, 95% CI [0.48-0.97]; Log-additive: adjusted OR: 0.83, 95% CI [0.70-0.98]; rs7411035: Homozygous G/G: adjusted OR: 0.66, 95% CI [0.47-0.94]; Dominant G/T-G/G: adjusted OR: 0.78, 95% CI [0.62-0.98]; Log-additive: adjusted OR: 0.81, 95% CI [0.69-0.96]; rs4656958: Heterozygous G/A: adjusted OR: 0.74, 95% CI [0.59-0.94]; Homozygous A/A: adjusted OR: 0.51, 95% CI [0.31-0.84]; Dominant G/A-A/A: adjusted OR: 0.71, 95% CI [0.57-0.89]; Recessive A/A: adjusted OR: 0.59, 95% CI [0.36-0.96]; Log-additive: adjusted OR: 0.73, 95% CI [0.61-0.88]), especially in people aged less than 60 years and males. In short, our study revealed a correlation between ITLN1 variants (rs6427553, rs7411035 and rs4656958) and IS risk in Xi'an population, Shaanxi province, laying a foundation for ITLN1 gene as a potential biomarker for predicting susceptibility to IS.

Dissection of a CTCF topological boundary uncovers principles of enhancer-oncogene regulation

Enhancer-gene communication is dependent on topologically associating domains (TADs) and boundaries enforced by the CCCTC-binding factor (CTCF) insulator, but the underlying structures and mechanisms remain controversial. Here, we investigate a boundary that typically insulates fibroblast growth factor (FGF) oncogenes but is disrupted by DNA hypermethylation in gastrointestinal stromal tumors (GISTs). The boundary contains an array of CTCF sites that enforce adjacent TADs, one containing FGF genes and the other containing ANO1 and its putative enhancers, which are specifically active in GIST and its likely cell of origin. We show that coordinate disruption of four CTCF motifs in the boundary fuses the adjacent TADs, allows the ANO1 enhancer to contact FGF3, and causes its robust induction. High-resolution micro-C maps reveal specific contact between transcription initiation sites in the ANO1 enhancer and FGF3 promoter that quantitatively scales with FGF3 induction such that modest changes in contact frequency result in strong changes in expression, consistent with a causal relationship.

HIV-1 RNAs whose transcription initiates from the third deoxyguanosine of GGG tract in the 5' long terminal repeat serve as a dominant genome for efficient provirus DNA formation.

Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.

Classifying promoters by interpreting the hidden information of DNA sequences for disease prediction in clinical laboratories using Gaussian decision boundary estimation

A promoter is a brief stretch of DNA (100–1,000 bp) where RNA polymerase starts to transcribe a gene. A DNA (Deoxyribonucleic Acid) base pair is a fundamental unit of DNA structure and represents the pairing of two complementary nucleotide bases within the DNA double helix. The four DNA nucleotide bases are adenine (A), thymine (T), cytosine (C), and guanine (G). DNA base pairs are the building blocks of the DNA molecule, and their complementary pairing is central to the storage and transmission of genetic information in all living organisms. Normally, a promoter is found at the 5′ end of the transcription initiation site or immediately upstream. Numerous human disorders, particularly diabetes, cancer, and Huntington’s disease, have been shown to have DNA promoter as their root cause. The scientific community has long been interested in learning crucial information about protein-coding genes. Finding the promoters is therefore the first step in finding genes in DNA sequences. The scientific world has always been attracted by the effort to glean crucial knowledge about protein-coding genes. Consequently, identifying promoters has emerged as an intriguing challenge that has caught the interest of numerous researchers in the field of bioinformatics. We proposed Gaussian Decision Boundary Estimation in machine learning models to detect transcription start sites (promoters) in the DNA sequences of a common bacteria, Escherichia coli. The best features are identified through a score-based function to select relevant nucleotides that are directly responsible for promoter recognition, in order maximise the models’ performance. The Gaussian Decision Boundary Estimation based support-vector-machine model is trained with these features and finds the best hyperplane that separates the data into different classes. Throughout this study, promoter regions could be identified with high accuracy 99.9% which is better compared to other state of art algorithms. The comparison of machine learning classification models is another major emphasis of this paper in order to identify the model that most accurately predicts DNA sequence promoters. It provides analysis for further biological research as well as precision medicine.

P059 GSDMB triggers intestinal inflammation via regulating BHLHA15-mediated hyperactive unfolded protein response

Abstract Background Impaired intestinal epithelial barrier has been considered to be associated with an increasing variety of gastrointestinal diseases, especially inflammatory bowel disease (IBD) encompassing Crohn’s disease (CD) and ulcerative colitis (UC). Herein, we investigated the role of Gasdermin B (GSDMB) in modulating intestinal epithelial barrier integrity and proposed a promising therapeutic strategy. Methods We generated GSDMB (GSDMBfl/fl;Villin-Cre) intestinal epithelial-specific knock in mice to observe the functions of GSDMB in intestinal epithelial barrier. RNA-seq analysis and human and murine intestine-derived organoids were used to determine the mechanism of function of GSDMB. Results To interrogate the intestinal functions of GSDMB in vivo, we generated GSDMB conditional knock in mice (heterozygote: GSDMBfl/-;Villin-Cre; homozygote: GSDMBfl/fl;Villin-Cre) by crossing a mouse line containing the floxed alleles of human GSDMB and a transcriptional stop codon at the transcriptional initiation site (GSDMBfl/fl) with the mouse line expressing Cre-recombinase under control of the Villin promoter (Villin-Cre). GSDMBfl/fl;Villin-Cre mice developed spontaneous enterocolitis and exhibited aberrant intestinal barrier integrity (as shown in the figure). GSDMB prominently provoked cell death of absorptive enterocytes and affected the function of goblet cells and Paneth cells in this setting. Mechanistically, epithelial GSDMB modulated hyperactive unfolded protein response of IECs by up-regulating BHLHA15 to mediate intestinal barrier injury instead of by the invasion of pathogenic microorganisms. Conclusion We have uncovered an important and a previously unrecognized role of GSDMB in intestinal inflammation, which represents a potential therapeutic target for IBD.

Intra-promoter switch of transcription initiation sites in proliferation signaling-dependent RNA metabolism

Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5′ terminal oligopyrimidine (5′TOP) sequence, dramatically increasing the range of genes potentially subjected to 5′TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K–Akt–mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.

A High-Efficiency Capture-Based NGS Approach for Comprehensive Analysis of Mitochondrial Transcriptome.

The transcription of the mitochondrial genome is pivotal for maintenance of mitochondrial functions, and the deregulated mitochondrial transcriptome contributes to various pathological changes. Despite substantial progress having been achieved in uncovering the transcriptional complexity of the nuclear transcriptome, many unknowns and controversies remain for the mitochondrial transcriptome, partially owing to the lack of a highly efficient mitochondrial RNA (mtRNA) sequencing and analysis approach. Here, we first comprehensively evaluated the influence of essential experimental protocols, including strand-specific library construction, two RNA enrichment strategies, and optimal rRNA depletion, on accurately profiling mitochondrial transcriptome in whole-transcriptome sequencing (WTS) data. Based on these insights, we developed a highly efficient approach specifically suitable for targeted sequencing of whole mitochondrial transcriptome, termed capture-based mtRNA seq (CAP), in which strand-specific library construction and optimal rRNA depletion were applied. Compared with WTS, CAP has a great decrease of required data volume without affecting the sensitivity and accuracy of detection. In addition, CAP also characterized the unannotated mt-tRNA transcripts whose expression levels are below the detection limits of conventional WTS. As a proof-of-concept characterization of mtRNAs, the transcription initiation sites and mtRNA cleavage ratio were accurately identified in CAP data. Moreover, CAP had very reliable performance in plasma and single-cell samples, highlighting its wide application. Altogether, the present study has established a highly efficient pipeline for targeted sequencing of mtRNAs, which may pave the way toward functional annotation of mtRNAs and mtRNA-based diagnostic and therapeutic strategies in various diseases.