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Transcription regulation in cestodes has been little studied. Here, we characterize the Taenia solium TATA-binding protein (TBP) gene. We found binding sites for transcription factors such as NF1, YY1, and AP-1 in the proximal promoter. We also identified two TATA-like elements in the promoter; however, neither could bind TBP. Additionally, we mapped the transcription start site (A+1) within an initiator and identified a putative downstream promoter element (DPE) located at +27 bp relative to the transcription start site. These two elements are important and functional for gene expression. Moreover, we identified the genes encoding T. solium TBP-Associated Factor 6 (TsTAF6) and 9 (TsTAF9). A Western blot assay revealed that both factors are expressed in the parasite; electrophoretic mobility shift assays and super-shift assays revealed interactions between the DPE probe and TsTAF6-TsTAF9. Finally, we used molecular dynamics simulations to formulate an interaction model among TsTAF6, TsTAF9, and the DPE probe; we stabilized the model with interactions between the histone fold domain pair in TAFs and several pairs of nucleotides in the DPE probe. We discuss novel and interesting features of the TsTAF6-TsTAF9 complex for interaction with DPE on T. solium promoters., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2024 Rodríguez-Lima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Alternative transcription start sites can affect transcript isoform diversity and translation levels. In a recently described form of gene regulation, coordinated transcriptional and translational interference results in transcript isoform-dependent changes in protein expression. Specifically, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, interfering with expression of the gene-proximal promoter. Although transcriptional and chromatin features associated with LUTI expression have been described, the mechanism underlying LUTI-based transcriptional interference is not well understood. Using an unbiased genetic approach followed by functional genomics, we uncovered that the Swi/Snf chromatin remodeling complex is required for co-transcriptional nucleosome remodeling that leads to LUTI-based repression. We identified genes with tandem promoters that rely on Swi/Snf function for transcriptional interference during protein folding stress, including LUTI-regulated genes. This study provides clear evidence for Swi/Snf playing a direct role in gene repression via a cis transcriptional interference mechanism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In vertebrates, most protein-coding genes have a peak of GC-content near their 5' transcriptional start site (TSS). This feature promotes both the efficient nuclear export and translation of mRNAs. Despite the importance of GC-content for RNA metabolism, its general features, origin, and maintenance remain mysterious. We investigate the evolutionary forces shaping GC-content at the transcriptional start site (TSS) of genes through both comparative genomic analysis of nucleotide substitution rates between different species and by examining human de novo mutations., Results: Our data suggests that GC-peaks at TSSs were present in the last common ancestor of amniotes, and likely that of vertebrates. We observe that in apes and rodents, where recombination is directed away from TSSs by PRDM9, GC-content at the 5' end of protein-coding gene is currently undergoing mutational decay. In canids, which lack PRDM9 and perform recombination at TSSs, GC-content at the 5' end of protein-coding is increasing. We show that these patterns extend into the 5' end of the open reading frame, thus impacting synonymous codon position choices., Conclusions: Our results indicate that the dynamics of this GC-peak in amniotes is largely shaped by historic patterns of recombination. Since decay of GC-content towards the mutation rate equilibrium is the default state for non-functional DNA, the observed decrease in GC-content at TSSs in apes and rodents indicates that the GC-peak is not being maintained by selection on most protein-coding genes in those species., (© 2024. The Author(s).)

A universal characteristic of eukaryotic transcription is that the promoter recruits RNA polymerase II (RNAPII) to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery selects the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation systems, we show that rapid depletion of an RNAPII-binding protein complex, Integrator, results in robust PROMPT accumulation throughout the genome. Interestingly, the accumulation of PROMPTs is compensated by the reduction of pre-mRNA transcripts in actively transcribed genes. Consistently, Integrator depletion alters the distribution of polymerase between the sense and antisense directions, which is marked by increased RNAPII-carboxy-terminal domain Tyr1 phosphorylation at PROMPT regions and a reduced Ser2 phosphorylation level at transcription start sites. Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPT production. Furthermore, our data indicate that the presence of U1 binding sites on nascent transcripts could counteract the cleavage activity of Integrator. In this process, the absence of robust U1 signal at most PROMPTs allows Integrator to suppress the antisense transcription and shift the transcriptional balance in favor of the sense direction., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Drought stress (DS) is one of the major constraints limiting yield in crop plants including rice. Gene regulation under DS is largely governed by accessibility of the transcription factors (TFs) to their cognate cis-regulatory elements (CREs). In this study, we used DNase I hypersensitive assays followed by sequencing to identify the accessible chromatin regions under DS in a drought-sensitive (IR64) and a drought-tolerant (N22) rice cultivar. Our results indicated that DNase I hypersensitive sites (DHSs) were highly enriched at transcription start sites (TSSs) and numerous DHSs were detected in the promoter regions. DHSs were concurrent with epigenetic marks and the genes harboring DHSs in their TSS and promoter regions were highly expressed. In addition, DS induced changes in DHSs (∆DHSs) in TSS and promoter regions were positively correlated with upregulation of several genes involved in drought/abiotic stress response, those encoding TFs and located within drought-associated quantitative trait loci, much preferentially in the drought-tolerant cultivar. The CREs representing the binding sites of TFs involved in DS response were detected within the ∆DHSs, suggesting differential accessibility of TFs to their cognate sites under DS in different rice cultivars, which may be further deployed for enhancing drought tolerance in rice., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Mammalian RNA polymerase II preinitiation complexes assemble adjacent to a nucleosome whose proximal edge (NPE) is typically 40 to 50 bp downstream of the transcription start site. At active promoters, that +1 nucleosome is universally modified by trimethylation on lysine 4 of histone H3 (H3K4me3). The Pol II preinitiation complex only extends 35 bp beyond the transcription start site, but nucleosomal templates with an NPE at +51 are nearly inactive in vitro with promoters that lack a TATA element and thus depend on TFIID for promoter recognition. Significantly, this inhibition is relieved when the +1 nucleosome contains H3K4me3, which can interact with TFIID subunits. Here, we show that H3K4me3 templates with both TATA and TATA-less promoters are active with +35 NPEs when transcription is driven by TFIID. Templates with +20 NPE are also active but at reduced levels compared to +35 and +51 NPEs, consistent with a general inhibition of promoter function when the proximal nucleosome encroaches on the preinitiation complex. Remarkably, dinucleosome templates support transcription when H3K4me3 is only present in the distal nucleosome, suggesting that TFIID-H3K4me3 interaction does not require modification of the +1 nucleosome. Transcription reactions performed with an alternative protocol retaining most nuclear factors results primarily in early termination, with a minority of complexes successfully traversing the first nucleosome. In such reactions, the +1 nucleosome does not substantially affect the level of termination even with an NPE of +20, indicating that a nucleosome barrier is not a major driver of early termination by Pol II., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The sperm epigenome is thought to affect the developmental programming of the resulting embryo, influencing health and disease in later life. Age-related methylation changes in the sperm of old fathers may mediate the increased risks for reproductive and offspring medical problems. The impact of paternal age on sperm methylation has been extensively studied in humans and, to a lesser extent, in rodents and cattle. Here, we performed a comparative analysis of paternal age effects on protein-coding genes in the human and marmoset sperm methylomes. The marmoset has gained growing importance as a non-human primate model of aging and age-related diseases. Using reduced representation bisulfite sequencing, we identified age-related differentially methylated transcription start site (ageTSS) regions in 204 marmoset and 27 human genes. The direction of methylation changes was the opposite, increasing with age in marmosets and decreasing in humans. None of the identified ageTSS was differentially methylated in both species. Although the average methylation levels of all TSS regions were highly correlated between marmosets and humans, with the majority of TSS being hypomethylated in sperm, more than 300 protein-coding genes were endowed with species-specifically (hypo)methylated TSS. Several genes of the glycosphingolipid (GSL) biosynthesis pathway, which plays a role in embryonic stem cell differentiation and regulation of development, were hypomethylated (<5%) in human and fully methylated (>95%) in marmoset sperm. The expression levels and patterns of defined sets of GSL genes differed considerably between human and marmoset pre-implantation embryo stages and blastocyst tissues, respectively., (© 2024 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

The three-dimensional (3D) organization of chromatin within the nucleus is crucial for gene regulation. However, the 3D architectural features that coordinate the activation of an entire chromosome remain largely unknown. We introduce an omics method, RNA-associated chromatin DNA-DNA interactions, that integrates RNA polymerase II (RNAPII)-mediated regulome with stochastic optical reconstruction microscopy to investigate the landscape of noncoding RNA roX2 -associated chromatin topology for gene equalization to achieve dosage compensation. Our findings reveal that roX2 anchors to the target gene transcription end sites (TESs) and spreads in a distinctive boot-shaped configuration, promoting a more open chromatin state for hyperactivation. Furthermore, roX2 arches TES to transcription start sites to enhance transcriptional loops, potentially facilitating RNAPII convoying and connecting proximal promoter-promoter transcriptional hubs for synergistic gene regulation. These TESs cluster as roX2 compartments, surrounded by inactive domains for coactivation of multiple genes within the roX2 territory. In addition, roX2 structures gradually form and scaffold for stepwise coactivation in dosage compensation.

Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analyzed TSS-seq data to define genuine TSS clusters in 2 species of pathogenic Cryptococcus. We identified 2 types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci, the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pausing of RNA polymerase II (Pol II) at transcription start sites (TSSs) primes target genes for productive elongation. Coincidentally, DNA double-strand breaks (DSBs) enrich at highly transcribed and Pol II-paused genes, although their interplay remains undefined. Using androgen receptor (AR) signaling as a model, we have uncovered AR-interacting protein 4 (ARIP4) helicase as a driver of androgen-dependent transcription induction. Chromatin immunoprecipitation sequencing analysis revealed that ARIP4 preferentially co-occupies TSSs with paused Pol II. Moreover, we found that ARIP4 complexes with topoisomerase II beta and mediates transient DSB formation upon hormone stimulation. Accordingly, ARIP4 deficiency compromised release of paused Pol II and resulted in R-loop accumulation at a panel of highly transcribed AR target genes. Last, we showed that ARIP4 binds and unwinds R-loops in vitro and that its expression positively correlates with prostate cancer progression. We propose that androgen stimulation triggers ARIP4-mediated unwinding of R-loops at TSSs, enforcing Pol II pause release to effectively drive an androgen-dependent expression program.

Understanding the regulatory mechanisms of gene expression is a crucial objective in genomics. Although the DNA sequence near the transcription start site (TSS) offers valuable insights, recent methods suggest that analyzing only the surrounding DNA may not suffice to accurately predict gene expression levels. We developed GENet (Gene Expression Network from Histone and Transcription Factor Integration), a novel approach that integrates essential regulatory signals from transcription factors and histone modifications into a graph-based model. GENet extends beyond simple DNA sequence analysis by incorporating additional layers of genetic control, which are vital for determining gene expression. Our method markedly enhances the prediction of mRNA levels compared to previous models that depend solely on DNA sequence data. The results underscore the significance of including comprehensive regulatory information in gene expression studies. GENet emerges as a promising tool for researchers, with potential applications extending from fundamental biological research to the development of medical therapies.

The long noncoding RNA TERRA is transcribed from telomeres in virtually all eukaryotes with linear chromosomes. In humans, TERRA transcription is driven in part by promoters comprising CpG dinucleotide-rich repeats of 29 bp repeats, believed to be present in half of the subtelomeres. Thus far, TERRA expression has been analyzed mainly using molecular biology-based approaches that only generate partial and somehow biased results. Here, we present a novel experimental pipeline to study human TERRA based on long-read sequencing (TERRA ONTseq). By applying TERRA ONTseq to different cell lines, we show that the vast majority of human telomeres produce TERRA and that the cellular levels of TERRA transcripts vary according to their chromosomes of origin. Using TERRA ONTseq, we also identified regions containing TERRA transcription start sites (TSSs) in more than half of human subtelomeres. TERRA TSS regions are generally found immediately downstream from 29 bp repeat-related sequences, which appear to be more widespread than previously estimated. Finally, we isolated a novel TERRA promoter from the highly expressed subtelomere of the long arm of Chromosome 7. With the development of TERRA ONTseq, we provide a refined picture of human TERRA biogenesis and expression and we equip the scientific community with an invaluable tool for future studies., (© 2024 Rodrigues et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Our study employs pooled CRISPR screens, integrating 2D and 3D culture models, to identify miRNAs critical in Breast Cancer (BC) tumoursphere formation. These screens combine with RNA-seq experiments allowing identification of miRNA signatures and targets essential for tumoursphere growth. miR-4787-3p exhibits significant up-regulation in BC, particularly in basal-like BCs, suggesting its association with aggressive disease. Surprisingly, despite its location within the 5'UTR of a protein coding gene, which defines DROSHA-independent transcription start site (TSS)-miRNAs, we find it dependant on both DROSHA and DICER1 for maturation. Inhibition of miR-4787-3p hinders tumoursphere formation, highlighting its potential as a therapeutic target in BC. Our study proposes elevated miR-4787-3p expression as a potential prognostic biomarker for adverse outcomes in BC. We find that protein-coding genes positively selected in the CRISPR screens are enriched of miR-4787-3p targets. Of these targets, we select ARHGAP17, FOXO3A, and PDCD4 as known tumour suppressors in cancer and experimentally validate the interaction of miR-4787-3p with their 3'UTRs. Our work illuminates the molecular mechanisms underpinning miR-4787-3p's oncogenic role in BC. These findings advocate for clinical investigations targeting miR-4787-3p and underscore its prognostic significance, offering promising avenues for tailored therapeutic interventions and prognostic assessments in BC., (© 2024. Crown.)

Abnormal transcription initiation from alternative first exon has been reported to promote tumorigenesis. However, the prevalence and impact of gene expression regulation mediated by alternative tandem transcription initiation were mostly unknown in cancer. Here, we developed a robust computational method to analyze alternative tandem transcription start site (TSS) usage from standard RNA sequencing data. Applying this method to pan-cancer RNA sequencing datasets, we observed widespread dysregulation of tandem TSS usage in tumors, many of which were independent of changes in overall expression level or alternative first exon usage. We showed that the dynamics of tandem TSS usage was associated with epigenomic modulation. We found that significant 5' untranslated region shortening of gene TIMM13 contributed to increased protein production, and up-regulation of TIMM13 by CRISPR-mediated transcriptional activation promoted proliferation and migration of lung cancer cells. Our findings suggest that dysregulated tandem TSS usage represents an addtional layer of cancer-associated transcriptome alterations.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5' single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4 + T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type-specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type-resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.

The B169L protein (pB169L) of African swine fever virus (ASFV) is a structural protein with an unidentified function during the virus replication. The sequences of the B169L gene and the downstream B438L gene are separated by short intergenic regions. However, the regulatory mode of the gene transcription remains unknown. Here, we identified two distinct promoter regions and two transcription start sites (TSSs) located upstream of the open reading frame (ORF) of B438L . Using the promoter reporter system, we demonstrated that the cis activity of the ORF proximal promoter exhibited significantly higher levels compared with that of the distal promoter located in the B169L gene. Furthermore, transfection with the plasmids with two different promoters for B438L could initiate the transcription and expression of the B438L gene in HEK293T cells, and the cis activity of the ORF proximal promoter also displayed higher activities compared with the distal promoter. Interestingly, the B438L distal promoter also initiated the transcription of the alternatively spliced B169L mRNA ( B169L mRNA2) encoding a truncated pB169L (tpB169L) (amino acids 92-169), and the gene transcription efficiency was increased upon mutation of the initiation codon located upstream of the alternatively spliced B169L gene. Taken together, we demonstrated that the distal promoter of B438L gene initiates the transcription of both the B438L mRNA and B169L mRNA2. Comprehensive analysis of the transcriptional regulatory mode of the B438L gene is beneficial for the understanding of the association of B438L protein and pB169L and the construction of the gene-deleted ASFV.

Lung cancer is a major global health concern with a low survival rate, often due to late-stage diagnosis. Liquid biopsy offers a non-invasive approach to cancer detection and monitoring, utilizing various features of circulating cell-free DNA (cfDNA). In this study, we established two models based on cfDNA coverage patterns at the transcription start sites (TSSs) from 6X whole-genome sequencing: an Early Cancer Screening Model and an EGFR mutation status prediction model. The Early Cancer Screening Model showed encouraging prediction ability, especially for early-stage lung cancer. The EGFR mutation status prediction model exhibited high accuracy in distinguishing between EGFR -positive and wild-type cases. Additionally, cfDNA coverage patterns at TSSs also reflect gene expression patterns at the pathway level in lung cancer patients. These findings demonstrate the potential applications of cfDNA coverage patterns at TSSs in early cancer screening and in cancer subtyping.

bb0616 of Borrelia burgdorferi , the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616 . By analyzing a bb0616 -deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616 . Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi , potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions., Competing Interests: The authors declare no conflict of interest.

Chromatin conformation capture technologies permit the study of chromatin spatial organization on a genome-wide scale at a variety of resolutions. Despite the increasing precision and resolution of high-throughput chromatin conformation capture (Hi-C) methods, it remains challenging to conclusively link transcriptional activity to spatial organizational phenomena. We have developed a clique-based approach for analyzing Hi-C data that helps identify chromosomal hotspots that feature considerable enrichment of chromatin annotations for transcriptional start sites and, building on previously published work, show that these chromosomal hotspots are not only significantly enriched in RNA polymerase II binding sites as identified by the ENCODE project, but also identify a noticeable increase in FANTOM5 and GTEx transcription within our identified cliques across a variety of tissue types. From the obtained data, we surmise that our cliques are a suitable method for identifying transcription factories in Hi-C data, and outline further extensions to the method that may make it useful for locating regions of increased transcriptional activity in datasets where in-depth expression or polymerase data may not be available.

The lysine 4 of histone H3 (H3K4) can be methylated or acetylated into four states: H3K4me1, H3K4me2, H3K4me3, or H3K4ac. Unlike H3K4 methylation, the genome-wide distribution and functional roles of H3K4ac remain unclear. To understand the relationship of acetylation with methylation at H3K4 and to explore the roles of H3K4ac in the context of chromatin, we analyzed H3K4ac across the human genome and compared it with H3K4 methylation in K562 cells. H3K4ac was positively correlated with H3K4me1/2/3 in reciprocal analysis. A decrease in H3K4ac through the mutation of the histone acetyltransferase p300 reduced H3K4me1 and H3K4me3 at the H3K4ac peaks. H3K4ac was also impaired by H3K4me depletion in the histone methyltransferase MLL3/4-mutated cells. H3K4ac peaks were enriched at enhancers in addition to the transcription start sites (TSSs) of genes. H3K4ac of TSSs and enhancers was positively correlated with mRNA and eRNA transcription. A decrease in H3K4ac reduced H3K4me3 and H3K4me1 in TSSs and enhancers, respectively, and inhibited the eviction of histone H3 from them. The mRNA transcription of highly transcribed genes was affected by the reduced H3K4ac. Interestingly, H3K4ac played a redundant role with regard to H3K27ac in eRNA transcription. These results indicate that H3K4ac serves as a marker of both active TSSs and enhancers and plays a role in histone eviction and RNA transcription by leading to H3K4me1/3., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A 5'-leader, known initially as the 5'-untranslated region, contains multiple isoforms due to alternative splicing (aS) and alternative transcription start site (aTSS). Therefore, a representative 5'-leader is demanded to examine the embedded RNA regulatory elements in controlling translation efficiency. Here, we develop a ranking algorithm and a deep-learning model to annotate representative 5'-leaders for five plant species. We rank the intra-sample and inter-sample frequency of aS-mediated transcript isoforms using the Kruskal-Wallis test-based algorithm and identify the representative aS-5'-leader. To further assign a representative 5'-end, we train the deep-learning model 5'leaderP to learn aTSS-mediated 5'-end distribution patterns from cap-analysis gene expression data. The model accurately predicts the 5'-end, confirmed experimentally in Arabidopsis and rice. The representative 5'-leader-contained gene models and 5'leaderP can be accessed at RNAirport (http://www.rnairport.com/leader5P/). The Stage 1 annotation of 5'-leader records 5'-leader diversity and will pave the way to Ribo-Seq open-reading frame annotation, identical to the project recently initiated by human GENCODE., Competing Interests: Conflict of interest The authors do not have any conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5'-adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured with 5'-monophosphates and 3'-misincorporated nucleotides. The 3'-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a "one precursor, one siRNA" model for the biogenesis of 24-nt siRNAs in Arabidopsis.

UnlabelledIn order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans-activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation.ImportanceThe HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5' ends and evaluation of ICP4 response. These findings provide further insight into the virus' strategy to tightly control expression of lytic cycle genes (especially the neurovirulence factor, ICP34.5) and suggest a mechanism (via ICP4) for the transition from latency to reactivated productive infection.

Bromodomain and extra terminal domain (BET) proteins are important epigenetic regulators facilitating the transcription of genes in chromatin areas linked to acetylated histones. JQ1, a BET protein inhibitor, has antiproliferative activity against many cancers, mainly through inhibition of c-MYC and upregulation of p21. In this research, we investigated the use of JQ1 for human osteosarcoma (OS) treatment. JQ1 significantly inhibited the proliferation and survival of OS cells inducing G1 cell cycle arrest, premature senescence, but little effect on apoptosis. Interestingly, c-MYC protein levels in JQ1-treated cells remained unchanged, whereas the upregulation of p21 protein was still observable. Although effective in vitro, JQ1 alone failed to reduce the size of the MNNG/HOS xenografts in immunocompromised mice. To overcome the resistance of OS cells to JQ1 treatment, we combined JQ1 with rapamycin, an mammalian target of rapamycin (mTOR) inhibitor. JQ1 and rapamycin synergistically inhibited the growth and survival of OS cells in vitro and in vivo. We also identified that RUNX2 is a direct target of bromodomain-containing protein 4 (BRD4) inhibition by JQ1 in OS cells. Chromatin immunoprecipitation (ChIP) showed that enrichment of BRD4 protein around RUNX2 transcription start sites diminished with JQ1 treatment in MNNG/HOS cells. Overexpression of RUNX2 protected JQ1-sensitive OS cells from the effect of JQ1, and siRNA-mediated inhibition of RUNX2 sensitized the same cells to JQ1. In conclusion, our findings suggest that JQ1, in combination with rapamycin, is an effective chemotherapeutic option for OS treatment. We also show that inhibition of RUNX2 expression by JQ1 partly explains the antiproliferative activity of JQ1 in OS cells.

Methylation of cytosines (5(me)C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5(me)C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo.

Psychrophilic methanogenic Archaea contribute significantly to global methane emissions, but archaeal cold adaptation mechanisms remain poorly understood. Hinted by that mRNA architecture determined secondary structure respond to cold more promptly than proteins, differential RNA-seq was used in this work to examine the genome-wide transcription start sites (TSSs) of the psychrophilic methanogen Methanolobus psychrophilus R15 and its response to cold. Unlike most prokaryotic mRNAs with short 5' untranslated regions (5' UTR, median lengths of 20-40 nt), 51% mRNAs of this methanogen have large 5' UTR (>50 nt). For 24% of the mRNAs, the 5' UTR is >150 nt. This implies that post-transcriptional regulation may be significance in the psychrophile. Remarkably, 219 (14%) genes possessed multiple gene TSSs (gTSSs), and 84 genes exhibited temperature-regulated gTSS selection to express alternative 5' UTR. Primer extension studies confirmed the temperature-dependent TSS selection and a stem-loop masking of ribosome binding sites was predicted from the longer 5' UTRs, suggesting alternative 5' UTRs-mediated translation regulation in the cold adaptation as well. In addition, 195 small RNAs (sRNAs) were detected, and Northern blots confirmed that many sRNAs were induced by cold. Thus, this study revealed an integrated transcriptional and post-transcriptional regulation for cold adaptation in a psychrophilic methanogen.

Divergent transcription, in which reverse-oriented transcripts occur upstream of eukaryotic promoters in regions devoid of annotated genes, has been suggested to be a general property of active promoters. Here we show that the human basal RNA polymerase II transcriptional machinery and core promoter are inherently unidirectional and that reverse-oriented transcripts originate from their own cognate reverse-directed core promoters. In vitro transcription analysis and mapping of nascent transcripts in HeLa cells revealed that sequences at reverse start sites are similar to those of their forward counterparts. The use of DNase I accessibility to define proximal promoter borders revealed that about half of promoters are unidirectional and that unidirectional promoters are depleted at their upstream edges of reverse core promoter sequences and their associated chromatin features. Divergent transcription is thus not an inherent property of the transcription process but rather the consequence of the presence of both forward- and reverse-directed core promoters.

Alternative polyadenylation (APA) has been implicated in a variety of developmental and disease processes. A particularly dramatic form of APA occurs in the developing nervous system of flies and mammals, whereby various developmental genes undergo coordinate 3' UTR extension. In Drosophila, the RNA-binding protein ELAV inhibits RNA processing at proximal polyadenylation sites, thereby fostering the formation of exceptionally long 3' UTRs. Here, we present evidence that paused Pol II promotes recruitment of ELAV to extended genes. Replacing promoters of extended genes with heterologous promoters blocks normal 3' extension in the nervous system, while extension-associated promoters can induce 3' extension in ectopic tissues expressing ELAV. Computational analyses suggest that promoter regions of extended genes tend to contain paused Pol II and associated cis-regulatory elements such as GAGA. ChIP-seq assays identify ELAV in the promoter regions of extended genes. Our study provides evidence for a regulatory link between promoter-proximal pausing and APA.

BackgroundAt the beginning of the transcription process, the RNA polymerase (RNAP) core enzyme requires a σ-factor to recognize the genomic location at which the process initiates. Although the crucial role of σ-factors has long been appreciated and characterized for many individual promoters, we do not yet have a genome-scale assessment of their function.ResultsUsing multiple genome-scale measurements, we elucidated the network of σ-factor and promoter interactions in Escherichia coli. The reconstructed network includes 4,724 σ-factor-specific promoters corresponding to transcription units (TUs), representing an increase of more than 300% over what has been previously reported. The reconstructed network was used to investigate competition between alternative σ-factors (the σ70 and σ38 regulons), confirming the competition model of σ substitution and negative regulation by alternative σ-factors. Comparison with σ-factor binding in Klebsiella pneumoniae showed that transcriptional regulation of conserved genes in closely related species is unexpectedly divergent.ConclusionsThe reconstructed network reveals the regulatory complexity of the promoter architecture in prokaryotic genomes, and opens a path to the direct determination of the systems biology of their transcriptional regulatory networks.

Transcription start sites (TSSs) lying inside annotated genes, on the same or opposite strand, have been observed in diverse bacteria, but the function of these unexpected transcripts is unclear. Here, we use the metal-reducing bacterium Shewanella oneidensis MR-1 and its relatives to study the evolutionary conservation of unexpected TSSs. Using high-resolution tiling microarrays and 5'-end RNA sequencing, we identified 2,531 TSSs in S. oneidensis MR-1, of which 18% were located inside coding sequences (CDSs). Comparative transcriptome analysis with seven additional Shewanella species revealed that the majority (76%) of the TSSs within the upstream regions of annotated genes (gTSSs) were conserved. Thirty percent of the TSSs that were inside genes and on the sense strand (iTSSs) were also conserved. Sequence analysis around these iTSSs showed conserved promoter motifs, suggesting that many iTSS are under purifying selection. Furthermore, conserved iTSSs are enriched for regulatory motifs, suggesting that they are regulated, and they tend to eliminate polar effects, which confirms that they are functional. In contrast, the transcription of antisense TSSs located inside CDSs (aTSSs) was significantly less likely to be conserved (22%). However, aTSSs whose transcription was conserved often have conserved promoter motifs and drive the expression of nearby genes. Overall, our findings demonstrate that some internal TSSs are conserved and drive protein expression despite their unusual locations, but the majority are not conserved and may reflect noisy initiation of transcription rather than a biological function. Importance: The first step of gene expression is the initiation of transcription from promoters, which have been traditionally thought to be located upstream of genes. Recently, studies showed that in diverse bacteria, promoters are often located inside genes. It has not been clear if these unexpected promoters are important to the organism or if they result from transcriptional noise. Here, we identify and examine promoters in eight related bacterial species. Promoters that lie within genes on the sense strand are often conserved as locations and in their sequences. Furthermore, these promoters often affect the bacterium's growth. Thus, many of these unexpected promoters are likely functional. Fewer promoters that lie within genes on the antisense strand are conserved, but the conserved ones seem to drive the expression of nearby genes.

Retinoic acid (RA) has diverse biological effects. The liver stores vitamin A, generates RA, and expresses receptors for RA. The current study examines the hepatic binding profile of two RA receptor isoforms, RARA (RARα) and RARB (RARβ), in response to RA treatment in mouse livers. Our data uncovered 35,521, and 14,968 genomic bindings for RARA and RARB, respectively. Each expressed unique and common bindings, implying their redundant and specific roles. RARB has higher RA responsiveness than RARB. RA treatment generated 18,821 novel RARB bindings but only 14,798 of RARA bindings, compared with the control group. RAR frequently bound the consensus hormone response element [HRE; (A/G)G(G/T)TCA], which often contained the motifs assigned to SP1, GABPA, and FOXA2, suggesting potential interactions between those transcriptional factors. Functional annotation coupled with principle component analysis revealed that the function of RAR target genes were motif dependent. Taken together, the cistrome of RARA and RARB revealed their extensive biological roles in the mouse liver. RAR target genes are enriched in various biological processes. The hepatic RAR genome-wide binding data can help us understand the global molecular mechanisms underlying RAR and RA-mediated gene and pathway regulation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

UnlabelledThe genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5' RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance.ImportanceSulfate-reducing bacteria contribute to global nutrient cycles and are a nuisance for the petroleum industry. Despite their environmental and industrial significance, the genomes of sulfate-reducing bacteria remain poorly characterized. Here, we describe a genetic approach to fill gaps in our knowledge of sulfate-reducing bacteria. We generated a large collection of archived, transposon mutants in Desulfovibrio alaskensis G20 and used the phenotypes of these mutant strains to infer the function of genes involved in gene regulation, methionine biosynthesis, and choline utilization. Our findings and mutant resources will enable systematic investigations into gene function, energy generation, stress response, and metabolism for this important group of bacteria.

Rare copy number variants (CNVs) disrupting ASTN2 or both ASTN2 and TRIM32 have been reported at 9q33.1 by genome-wide studies in a few individuals with neurodevelopmental disorders (NDDs). The vertebrate-specific astrotactins, ASTN2 and its paralog ASTN1, have key roles in glial-guided neuronal migration during brain development. To determine the prevalence of astrotactin mutations and delineate their associated phenotypic spectrum, we screened ASTN2/TRIM32 and ASTN1 (1q25.2) for exonic CNVs in clinical microarray data from 89 985 individuals across 10 sites, including 64 114 NDD subjects. In this clinical dataset, we identified 46 deletions and 12 duplications affecting ASTN2. Deletions of ASTN1 were much rarer. Deletions near the 3' terminus of ASTN2, which would disrupt all transcript isoforms (a subset of these deletions also included TRIM32), were significantly enriched in the NDD subjects (P = 0.002) compared with 44 085 population-based controls. Frequent phenotypes observed in individuals with such deletions include autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), speech delay, anxiety and obsessive compulsive disorder (OCD). The 3'-terminal ASTN2 deletions were significantly enriched compared with controls in males with NDDs, but not in females. Upon quantifying ASTN2 human brain RNA, we observed shorter isoforms expressed from an alternative transcription start site of recent evolutionary origin near the 3' end. Spatiotemporal expression profiling in the human brain revealed consistently high ASTN1 expression while ASTN2 expression peaked in the early embryonic neocortex and postnatal cerebellar cortex. Our findings shed new light on the role of the astrotactins in psychopathology and their interplay in human neurodevelopment.

Fluctuations in nutrient availability profoundly impact gene expression. Previous work revealed postrecruitment regulation of RNA polymerase II (Pol II) during starvation and recovery in Caenorhabditis elegans, suggesting that promoter-proximal pausing promotes rapid response to feeding. To test this hypothesis, we measured Pol II elongation genome wide by two complementary approaches and analyzed elongation in conjunction with Pol II binding and expression. We confirmed bona fide pausing during starvation and also discovered Pol II docking. Pausing occurs at active stress-response genes that become downregulated in response to feeding. In contrast, "docked" Pol II accumulates without initiating upstream of inactive growth genes that become rapidly upregulated upon feeding. Beyond differences in function and expression, these two sets of genes have different core promoter motifs, suggesting alternative transcriptional machinery. Our work suggests that growth and stress genes are both regulated postrecruitment during starvation but at initiation and elongation, respectively, coordinating gene expression with nutrient availability.

Exome sequence analysis of affected individuals from two families with autosomal-dominant inheritance of coloboma identified two different cosegregating heterozygous nonsense mutations (c.370C>T [p.Arg124*] and c. 1066G>T [p.Glu356*]) in YAP1. The phenotypes of the affected families differed in that one included no extraocular features and the other manifested with highly variable multisystem involvement, including hearing loss, intellectual disability, hematuria, and orofacial clefting. A combined LOD score of 4.2 was obtained for the association between YAP1 loss-of-function mutations and the phenotype in these families. YAP1 encodes an effector of the HIPPO-pathway-induced growth response, and whole-mount in situ hybridization in mouse embryos has shown that Yap1 is strongly expressed in the eye, brain, and fusing facial processes. RT-PCR showed that an alternative transcription start site (TSS) in intron 1 of YAP1 and Yap1 is widely used in human and mouse development, respectively. Transcripts from the alternative TSS are predicted to initiate at codon Met179 relative to the canonical transcript (RefSeq NM_001130145). In these alternative transcripts, the c.370C>T mutation in family 1305 is within the 5' UTR and cannot result in nonsense-mediated decay (NMD). The c. 1066G>T mutation in family 132 should result in NMD in transcripts from either TSS. Amelioration of the phenotype by the alternative transcripts provides a plausible explanation for the phenotypic differences between the families.

Genome-wide maps of DNase I hypersensitive sites (DHSs) reveal that most human promoters contain perpetually active cis-regulatory elements between -150 bp and +50 bp (-150/+50 bp) relative to the transcription start site (TSS). Transcription factors (TFs) recruit cofactors (chromatin remodelers, histone/protein-modifying enzymes, and scaffold proteins) to these elements in order to organize the local chromatin structure and coordinate the balance of post-translational modifications nearby, contributing to the overall regulation of transcription. However, the rules of TF-mediated cofactor recruitment to the -150/+50 bp promoter regions remain poorly understood. Here, we provide evidence for a general model in which a series of cis-regulatory elements (here termed 'cardinal' motifs) prefer acting individually, rather than in fixed combinations, within the -150/+50 bp regions to recruit TFs that dictate cofactor signatures distinctive of specific promoter subsets. Subsequently, human promoters can be subclassified based on the presence of cardinal elements and their associated cofactor signatures. In this study, furthermore, we have focused on promoters containing the nuclear respiratory factor 1 (NRF1) motif as the cardinal cis-regulatory element and have identified the pervasive association of NRF1 with the cofactor lysine-specific demethylase 1 (LSD1/KDM1A). This signature might be distinctive of promoters regulating nuclear-encoded mitochondrial and other particular genes in at least some cells. Together, we propose that decoding a signature-based, expanded model of control at proximal promoter regions should lead to a better understanding of coordinated regulation of gene transcription.

Embryonic patterning displays remarkable consistency from individual to individual despite frequent environmental perturbations and diverse genetic contexts. Stochastic influences on the cellular environment may cause transcription rates to fluctuate, but these fluctuations rarely lead to developmental defects or disease. Here we characterize a set of recessive alleles of the Toll pathway component tube that destabilize embryonic dorsoventral patterning in Drosophila melanogaster. Females bearing these tube alleles generate embryos of an unusually wide range of dorsalized phenotypes, with the distributions across this range being unique for each allele. We determine that the mutant lines have in common a retrotransposon insertion upstream of the tube transcription start site. Genetic and molecular approaches demonstrate that this insertion dramatically reduces maternal expression of tube, thereby uncovering the inherent variability in gene expression. We further find that additional transposable element insertions near the tube gene synergistically enhance the phenotype caused by the sensitizing upstream insertion. These studies document how phenotypic variability can arise from normally occurring fluctuations around reduced mean expression and illustrate the contribution of transposons, individually and combinatorially, to such a state.

Class switch DNA recombination (CSR) crucially diversifies Ab biologic effector functions. 14-3-3γ specifically binds to the 5'-AGCT-3' repeats in the IgH locus switch (S) regions. By interacting directly with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. In this study, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by LPSs, and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A.

RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ∼60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.

Bartonella quintana is a vector-borne bacterial pathogen that causes fatal disease in humans. During the infectious cycle, B. quintana transitions from the hemin-restricted human bloodstream to the hemin-rich body louse vector. Because extracytoplasmic function (ECF) sigma factors often regulate adaptation to environmental changes, we hypothesized that a previously unstudied B. quintana ECF sigma factor, RpoE, is involved in the transition from the human host to the body louse vector. The genomic context of B. quintana rpoE identified it as a member of the ECF15 family of sigma factors found only in alphaproteobacteria. ECF15 sigma factors are believed to be the master regulators of the general stress response in alphaproteobacteria. In this study, we examined the B. quintana RpoE response to two stressors that are encountered in the body louse vector environment, a decreased temperature and an increased hemin concentration. We determined that the expression of rpoE is significantly upregulated at the body louse (28°C) versus the human host (37°C) temperature. rpoE expression also was upregulated when B. quintana was exposed to high hemin concentrations. In vitro and in vivo analyses demonstrated that RpoE function is regulated by a mechanism involving the anti-sigma factor NepR and the response regulator PhyR. The ΔrpoE ΔnepR mutant strain of B. quintana established that RpoE-mediated transcription is important in mediating the tolerance of B. quintana to high hemin concentrations. We present the first analysis of an ECF15 sigma factor in a vector-borne human pathogen and conclude that RpoE has a role in the adaptation of B. quintana to the hemin-rich arthropod vector environment.

Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II (Pol II) at the transcription start site. The molecular mechanism and function of this assembly have remained elusive due to lack of structural information. Here we have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed pre-initiation complex and the open-promoter complex, and to regulate start--initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, support a DNA translocation model of XPB and explain its essential role in promoter opening.

Transcription factor RBPJ is the central component in Notch signal transduction and directly forms a coactivator complex together with the Notch intracellular domain (NICD). While RBPJ protein levels remain constant in most tissues, dynamic expression of Notch target genes varies depending on the given cell-type and the Notch activity state. To elucidate dynamic RBPJ binding genome-wide, we investigated RBPJ occupancy by ChIP-Seq. Surprisingly, only a small set of the total RBPJ sites show a dynamic binding behavior in response to Notch signaling. Compared to static RBPJ sites, dynamic sites differ in regard to their chromatin state, binding strength and enhancer positioning. Dynamic RBPJ sites are predominantly located distal to transcriptional start sites (TSSs), while most static sites are found in promoter-proximal regions. Importantly, gene responsiveness is preferentially associated with dynamic RBPJ binding sites and this static and dynamic binding behavior is repeatedly observed across different cell types and species. Based on the above findings we used a machine-learning algorithm to predict Notch responsiveness with high confidence in different cellular contexts. Our results strongly support the notion that the combination of binding strength and enhancer positioning are indicative of Notch responsiveness., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Viruses are master remodelers of the host cell environment in support of infection and virus production. For example, viruses typically regulate cell gene expression through modulating canonical cell promoter activity. Here, we show that Epstein Barr virus (EBV) replication causes 'de novo' transcription initiation at 29674 new transcription start sites throughout the cell genome. De novo transcription initiation is facilitated in part by the unique properties of the viral pre-initiation complex (vPIC) that binds a TATT[T/A]AA, TATA box-like sequence and activates transcription with minimal support by additional transcription factors. Other de novo promoters are driven by the viral transcription factors, Zta and Rta and are influenced by directional proximity to existing canonical cell promoters, a configuration that fosters transcription through existing promoters and transcriptional interference. These studies reveal a new way that viruses interact with the host transcriptome to inhibit host gene expression and they shed light on primal features driving eukaryotic promoter function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Insulator-binding proteins (IBPs) play a critical role in genome architecture by forming and maintaining contact domains. While the involvement of several IBPs in organising chromatin architecture in Drosophila has been described, the specific contribution of the Suppressor of Hairy wings (Su(Hw)) insulator-binding protein to genome topology remains unclear., Results: In this study, we provide evidence for the existence of long-range interactions between chromatin bound Su(Hw) and Combgap, which was first characterised as Polycomb response elements binding protein. Loss of Su(Hw) binding to chromatin results in the disappearance of Su(Hw)-Combgap long-range interactions and in a decrease in spatial self-interactions among a subset of Su(Hw)-bound genome sites. Our findings suggest that Su(Hw)-Combgap long-range interactions are associated with active chromatin rather than Polycomb-directed repression. Furthermore, we observe that the majority of transcription start sites that are down-regulated upon loss of Su(Hw) binding to chromatin are located within 2 kb of Combgap peaks and exhibit Su(Hw)-dependent changes in Combgap and transcriptional regulators' binding., Conclusions: This study demonstrates that Su(Hw) insulator binding protein can form long-range interactions with Combgap, Polycomb response elements binding protein, and that these interactions are associated with active chromatin factors rather than with Polycomb dependent repression., (© 2024. The Author(s).)

Local mutation rates in human are highly heterogeneous, with known variability at the scale of megabase-sized chromosomal domains, and, on the other extreme, at the scale of oligonucleotides. The intermediate, kilobase-scale heterogeneity in mutation risk is less well characterized. Here, by analyzing thousands of somatic genomes, we studied mutation risk gradients along gene bodies, representing a genomic scale spanning roughly 1-10 kb, hypothesizing that different mutational mechanisms are differently distributed across gene segments. The main heterogeneity concerns several kilobases at the transcription start site and further downstream into 5' ends of gene bodies; these are commonly hypomutated with several mutational signatures, most prominently the ubiquitous C > T changes at CpG dinucleotides. The width and shape of this mutational coldspot at 5' gene ends is variable across genes, and corresponds to variable interval of lowered DNA methylation depending on gene activity level and regulation. Such hypomutated loci, at 5' gene ends or elsewhere, correspond to DNA hypomethylation that can associate with various landmarks, including intragenic enhancers, Polycomb-marked regions, or chromatin loop anchor points. Tissue-specific DNA hypomethylation begets tissue-specific local hypomutation. Of note, direction of mutation risk is inverted for AID/APOBEC3 cytosine deaminase activity, whose signatures are enriched in hypomethylated regions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

ABSTRACT An alternative transcription start site (ATSS) is a major driving force for increasing the complexity of transcripts in human tissues. As a transcriptional regulatory mechanism, ATSS has biological significance. Many studies have confirmed that ATSS plays an important role in diseases and cell development and differentiation. However, exploration of its dynamic mechanisms remains insufficient. Identifying ATSS change points during cell differentiation is critical for elucidating potential dynamic mechanisms. For relative ATSS usage as percentage data, the existing methods lack sensitivity to detect the change point for ATSS longitudinal data. In addition, some methods have strict requirements for data distribution and cannot be applied to deal with this problem. In this study, the Bayesian change point detection model was first constructed using reparameterization techniques for two parameters of a beta distribution for the percentage data type, and the posterior distributions of parameters and change points were obtained using Markov Chain Monte Carlo (MCMC) sampling. With comprehensive simulation studies, the performance of the Bayesian change point detection model is found to be consistently powerful and robust across most scenarios with different sample sizes and beta distributions. Second, differential ATSS events in the real data, whose change points were identified using our method, were clustered according to their change points. Last, for each change point, pathway and transcription factor motif analyses were performed on its differential ATSS events. The results of our analyses demonstrated the effectiveness of the Bayesian change point detection model and provided biological insights into cell differentiation .

Background: Untranslated regions (UTRs) are important mediators of post-transcriptional regulation. The length of UTRs and the composition of regulatory elements within them are known to vary substantially across genes, but little is known about the reasons for this variation in humans. Here, we set out to determine whether this variation, specifically in 5'UTRs, correlates with gene dosage sensitivity., Results: We investigate 5'UTR length, the number of alternative transcription start sites, the potential for alternative splicing, the number and type of upstream open reading frames (uORFs) and the propensity of 5'UTRs to form secondary structures. We explore how these elements vary by gene tolerance to loss-of-function (LoF; using the LOEUF metric), and in genes where changes in dosage are known to cause disease. We show that LOEUF correlates with 5'UTR length and complexity. Genes that are most intolerant to LoF have longer 5'UTRs, greater TSS diversity, and more upstream regulatory elements than their LoF tolerant counterparts. We show that these differences are evident in disease gene-sets, but not in recessive developmental disorder genes where LoF of a single allele is tolerated., Conclusions: Our results confirm the importance of post-transcriptional regulation through 5'UTRs in tight regulation of mRNA and protein levels, particularly for genes where changes in dosage are deleterious and lead to disease. Finally, to support gene-based investigation we release a web-based browser tool, VuTR, that supports exploration of the composition of individual 5'UTRs and the impact of genetic variation within them., (© 2024. The Author(s).)

Lysine lactylation (Kla) links metabolism and gene regulation and plays a key role in multiple biological processes. However, the regulatory mechanism and functional consequence of Kla remain to be explored. Here, we report that HBO1 functions as a lysine lactyltransferase to regulate transcription. We show that HBO1 catalyzes the addition of Kla in vitro and intracellularly, and E508 is a key site for the lactyltransferase activity of HBO1. Quantitative proteomic analysis further reveals 95 endogenous Kla sites targeted by HBO1, with the majority located on histones. Using site-specific antibodies, we find that HBO1 may preferentially catalyze histone H3K9la and scaffold proteins including JADE1 and BRPF2 can promote the enzymatic activity for histone Kla. Notably, CUT&Tag assays demonstrate that HBO1 is required for histone H3K9la on transcription start sites (TSSs). Besides, the regulated Kla can promote key signaling pathways and tumorigenesis, which is further supported by evaluating the malignant behaviors of HBO1- knockout (KO) tumor cells, as well as the level of histone H3K9la in clinical tissues. Our study reveals HBO1 serves as a lactyltransferase to mediate a histone Kla-dependent gene transcription., (© 2024. The Author(s).)