Transcription Initiation Research Articles

Abstract 373: Transcriptional and post-transcriptional positive regulatory loops between HIF-1a and the RNA-Binding protein hnRNPA18

Abstract Hypoxia, the scarcity of oxygen, is a hallmark of solid tumors to which they adapt by activating the hypoxia-inducible transcription factor 1 (HIF-1). HIF activates the transcription of genes bearing hypoxia responsive elements (RE) in their promoter regions including genes related to angiogenesis, glucose metabolism, cell proliferation, survival, invasion, and metastasis. The HIF-1 pathway is thus an attractive target to prevent cancer aggressiveness and improve the effectiveness of cancer therapy. HIF-1α activity is known to be mainly regulated through post-translational modification by prolyl hydroxylase domain (PHD) enzymes, but accumulating evidence indicate that it is also regulated by other mechanisms such as transcriptional initiation, translational initiation, protein-protein interaction, post-translational modifications and post-transcriptionally, primarily through the action of trans-acting factors (noncoding RNAs and RNA-binding proteins) that interact with the HIF-1α mRNA to regulate its decay and translational rates. We recently identified the RNA binding protein hnRNP A18 as a regulator of HIF-1α translation under hypoxic conditions. hnRNP A18 is a nuclear stress responsive protein that translocates to the cytosol in response to cellular stress including hypoxia to bind to a recognition motif in the 3’UTR of its targeted transcripts including HIF-1α to stabilize the transcripts and increase their translation. Using a bicistronic reporter plasmid containing a HIF-1α internal ribosome entry site (IRES) we now show that hnRNP A18 can also regulate HIF-1α through its IRES in the 5’UTR. In fact, cells expressing hnRNP A18 significantly increase translation of a reporter CAT protein under the control of HIF-1α IRES in the presence of the hypoxia mimetic agent CoCl2. On the other hand, deletion of hnRNP A18 prevents the translation of the CAT reporter protein even in the presence of CoCl2. Moreover, we also identified several HIF-1α REs in hnRNP A18 promoter. Two of the predicted HIF-1α REs, located at -57 and -226 upstream of the hnRNP A18 start codon were validated as bonafide HIF-1α binding sites by Electromobility Shift Assay with recombinant HIF-1α protein and by Chromatin Immunoprecipitation assay in human pancreatic cancer cells Panc 01. These data thus indicate that a positive regulatory loop between hnRNP A18 and HIF1-1α exist to amplify HIF-1α expression under cellular stress. hnRNP A18 can upregulate HIF1α protein expression by stabilizing its transcript at the 3’UTR and by binding to its IRES. On the other hand, HIF-1α can upregulate hnRNP A18 by binding to its proximal promoter. Targeting hnRNP A18 could thus provide a new mechanism to regulate HIF-1α expression and sensitize cancer cells to therapies. Citation Format: Eduardo Solano-Gonzalez, France Carrier. Transcriptional and post-transcriptional positive regulatory loops between HIF-1a and the RNA-Binding protein hnRNPA18 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 373.

Abstract 1702: Loss of KEAP1 promotes R-loop formation independent of NRF2

Abstract This study investigates the mechanism of R-loop formation as a consequence of KEAP1 depletion. R-loops, described as three-stranded nucleic acid structures consisting of a DNA:RNA hybrid and a non-template single-stranded DNA, are assemblies often attributed with pathological consequences like genomic instability and replication stress. However, they have significant physiological roles, inter alia, regulation of transcription initiation, splicing and termination. Loss in R-loop resolution factors (like RNaseH nucleases, senataxin) can result in “unscheduled” or “pathological” R-loops, which can compromise genome stability. R-loops are commonly associated with an active transcription bubble, with nascent RNA trailing the RNA polymerase re-annealing to the template DNA strand. Increased transcriptional stress can lead to accumulation of R-loops. In a 2016 eLife study by Stork et al., estrogen induction resulted in concomitantly increased transcription and R-loop formation in the same subset of genes. Similar to estrogen induction, activation of the NRF2/KEAP1 pathway upon oxidative stress causes NRF2 to be released from KEAP1 and enter the nucleus to turn on transcription of its target genes. Kelch-like ECH associated protein 1, encoded by the KEAP1 gene, is a canonical binding partner of NRF2. Under basal conditions, KEAP1 binds NRF2 and targets it for degradation. Mutations in KEAP1 and epigenetic modifications have been reported in lung and breast cancer phenotypes. In lieu of chemical manipulation, NRF2 can be activated by knocking down KEAP1 via RNAi. We therefore asked whether transcription activation downstream of the NRF2/KEAP1 pathway can lead to accumulation of R-loops.We have observed the consequences of depleting KEAP1 (for NRF2 activation) with RNAi on global transcription using ethynyl uridine (EU) incorporation with click-iT chemistry and flow cytometry, on R-loop levels using S9.6 antibody immunocytochemistry and on transcriptional profile changes with RNA-seq. We observe that loss of KEAP1 leads to an increased accumulation of R-loops. Surprisingly, this is not accompanied by an expected increase in transcription as measured quantitatively with EU flow cytometry. This phenomenon was found to consistently occur in various cell lines across cancer types (breast, lung). Interestingly, depleting NRF2 cannot mitigate the effects of KEAP1 loss, suggesting that KEAP1 loss-associated R-loop formation is independent of NRF2 activation. Analysis of RNA-seq data shows that TOP2 is downregulated in KEAP1 deficient cells. As TOP2 has been previously known to regulate R-loop formation, we predict that KEAP1 loss can sensitize cells to topoisomerase inhibitors.In conclusion, interaction of KEAP1 with other players in its interactome outside of NRF2 may regulate R-loop formation and therefore R-loop-induced genome instability. Citation Format: Pramiti Mukhopadhyay, Nicklas Bassani, Alexander J. Bishop. Loss of KEAP1 promotes R-loop formation independent of NRF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1702.

Abstract 1413: BRCA1 loss is synthetic lethal with double stranded RNA: A genome wide CRISPR screen to identify mechanisms of RNASEL independent death

Abstract Double-stranded RNAs (dsRNAs) are potent immunostimulatory nucleic acids of viral origin but also physiologically produced by mammalian cells. Recent studies demonstrated that elevating dsRNA levels, either exogenously through dsRNA mimetics like Polyinosinic-polycytidylic acid (pI:C) or endogenously through epigenetic modulation of retroviral elements, can elicit anticancer effects. In response to dsRNA, cells elicit a dual response consisting in Interferon (IFN) production and translational shutdown associated, in some cells, with cell death. These two responses are linked in a feed-forward loop in which induced cytokines themselves enhance dsRNA sensing by the death-inducing pathway, complicating our understanding of the cell-intrinsic mechanisms at play. The 2,5-oligoadenylate synthetase (OAS)-RNASEL system is widely thought to be the main responsible for dsRNA-induced cell death, but it remains unclear how its activity leads to cell death.To elucidate these mechanisms, we decided to minimize the impact of interferon modulation by studying dsRNA-induced death in interferon-saturated conditions. RNASEL-KO A549 cells were, as expected, refractory to IFN+pI:C induced cell death; however, inhibiting protein synthesis or RNA transcription with Cycloheximide (CHX) or Actinomycin D (ActD) abolished the need for RNASEL causing RNASEL-KO cells to undergo apoptosis, although CHX/ActD alone had no effect. In addition, HT-29 cells revealed no clear dependence on RNASEL for dsRNA-induced cell death. Transient ablation through siRNA showed that this novel death-inducing pathway is independent of other canonical dsRNA sensors such as RIG-I, MDA5 and PKR. We then performed a genome-wide CRISPR screen for factors involved in dsRNA-induced death in our interferon-saturated conditions in HT29 cells. Pathway enrichment analysis of screen hits revealed that RNA surveillance, RNA Polymerase II Transcription Initiation, Mitochondrial translation and respiratory chain and DNA Repair genes have a protective role against dsRNA increase. In particular, we validated the synthetic lethality of IFN+pI:C with BRCA1 by CRISPR. Ongoing research is aimed at validating this interaction in vivo models.Our work shed light on potential new mechanisms involved in dsRNA dependent cell death, revealing a connection with DNA damage and BRCA1. If confirmed, these findings may pave the way for the use of dsRNA mimetics, possibly in conjunction with immune checkpoint inhibitors, in the treatment of BRCA1-mutated tumors. Citation Format: Deborah Trastulli, Gianluca Vozza, Veronica Pinamonti, Luca Mazzarella. BRCA1 loss is synthetic lethal with double stranded RNA: A genome wide CRISPR screen to identify mechanisms of RNASEL independent death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1413.

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.

Abstract 1692: MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal type of pancreatic neoplasm. Unfortunately, most patients are diagnosed with unresectable or metastatic disease, making current treatments insufficient. As a result, the 5-year survival rate for PDAC is below 10%.Pancreatic neoplasms are primarily initiated by the KRAS mutation, accounting for 95% of cases. However, the progression of PDAC to a highly aggressive and therapy-resistant cancer is accelerated by chronic inflammation. The role of mutant KRAS signaling and NF-κB inflammatory signaling in promoting PDAC progression has been extensively studied. Yet, the transcriptional mechanism of their cooperation remains unclear. Additionally, the interaction between PDAC tumor cells and signals in the tumor microenvironment (TME) is not yet fully understood. Our study demonstrates that the simultaneous activation of KRAS/MAPK and NF-κB by epidermal growth factor (EGF) and TNFα, respectively, increases cell migration as shown through live-cell imaging analysis. An integrated analysis of epigenetic and transcriptomic data (ChIP-seq and RNA-seq) was performed after dual stimulation of both pathways. This revealed a high occupancy of the active mark, H3K27ac at transcription start sites of a subset of genes involved in cell migration and subtype switch. Through gain and loss-of-function studies, FOSL1 and RELA were identified as the key transcription factors. The epigenome mapping of FOSL1 and RELA after the stimulation of both pathways revealed their occupancy at RELA-dominant and FOSL1/RELA co-occupied regions. Inhibitor treatments and loss-of-function studies revealed that RELA binding controls RNA polymerase recruitment for transcription initiation and enhances cell migration. Further analysis of RNA-seq data on PDAC cell lines and cells from patient-derived xenografts highlighted that active inflammatory signaling contributes to a subtype switch from the classical to the more migratory basal subtype. Our study, combined with single-cell RNA-seq analysis of published data, shows that macrophages in the tumor microenvironment supply TNFα that activates NF-κB signaling, leading to changes in cell migration. Our study has provided a comprehensive understanding of the mechanism by which mutant KRAS and inflammatory signaling contribute to the progression of PDAC. These findings could offer a new perspective in the development of mechanism-based therapeutic approaches that can improve the efficacy of chemotherapeutic agents and make tumors more amenable to surgical resection. Citation Format: Joana E. Aggrey-Fynn, Meghana Manjunath, Steven A. Johnsen. MAPK and NF-kappaB signaling converge on the epigenome to transcriptionally activate genes involved in pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1692.

Viral reprogramming of host transcription initiation.

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.

ERG and c-MYC regulate a critical gene network in BCR::ABL1-driven B cell acute lymphoblastic leukemia.

Philadelphia chromosome-positive B cell acute lymphoblastic leukemia (B-ALL), characterized by the BCR::ABL1 fusion gene, remains a poor prognosis cancer needing new therapeutic approaches. Transcriptomic profiling identified up-regulation of oncogenic transcription factors ERG and c-MYC in BCR::ABL1 B-ALL with ERG and c-MYC required for BCR::ABL1 B-ALL in murine and human models. Profiling of ERG- and c-MYC-dependent gene expression and analysis of ChIP-seq data established ERG and c-MYC coordinate a regulatory network in BCR::ABL1 B-ALL that controls expression of genes involved in several biological processes. Prominent was control of ribosome biogenesis, including expression of RNA polymerase I (POL I) subunits, the importance of which was validated by inhibition of BCR::ABL1 cells by POL I inhibitors, including CX-5461, that prevents promoter recruitment and transcription initiation by POL I. Our results reveal an essential ERG- and c-MYC-dependent transcriptional network involved in regulation of metabolic and ribosome biogenesis pathways in BCR::ABL1 B-ALL, from which previously unidentified vulnerabilities and therapeutic targets may emerge.

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.

ATX1 and HUB1/2 promote recruitment of the transcription elongation factor VIP2 to modulate the floral transition in Arabidopsis.

Histone 2B ubiquitination (H2Bub) and trimethylation of H3 at lysine 4 (H3K4me3) are associated with transcription activation. However, the function of these modifications in transcription in plants remains largely unknown. Here, we report that coordination of H2Bub and H3K4me3 deposition with the binding of the RNA polymerase-associated factor VERNALIZATION INDEPENDENCE2 (VIP2) to FLOWERING LOCUS C (FLC) modulates flowering time in Arabidopsis. We found that RING domain protein HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 (we refer as HUB1/2), which are responsible for H2Bub, interact with ARABIDOPSIS TRITHORAX1 (ATX1), which is required for H3K4me3 deposition, to promote the transcription of FLC and repress the flowering time. The atx1-2 hub1-10 hub2-2 triple mutant in FRIGIDIA (FRI) background displayed early flowering like FRI hub1-10 hub2-2 and overexpression of ATX1 failed to rescue the early flowering phenotype of hub1-10 hub2-2. Mutations in HUB1 and HUB2 reduced the ATX1 enrichment at FLC, indicating that HUB1 and HUB2 are required for ATX1 recruitment and H3K4me3 deposition at FLC. We also found that the VIP2 directly binds to HUB1, HUB2, and ATX1 and that loss of VIP2 in FRI hub1-10 hub2-2 and FRI atx1-2 plants resulted in early flowering like that observed in FRI vip2-10. Loss of function of HUB2 and ATX1 impaired VIP2 enrichment at FLC, and reduced the transcription initiation and elongation of FLC. In addition, mutations in VIP2 reduced HUB1 and ATX1 enrichment and H2Bub and H3K4me3 levels at FLC. Together, our findings revealed that HUB1/2, ATX1, and VIP2 coordinately modulate H2Bub and H3K4me3 deposition, FLC transcription, and flowering time.

Reverse transcription of plasma-derived HIV-1 RNA generates multiple artifacts through tRNA(Lys-3)-priming.

In vitro reverse transcription of full-length HIV-1 RNA extracted from the blood plasma of people living with HIV-1 remains challenging. Here, we describe the initiation of reverse transcription of plasma-derived viral RNA in the absence of an exogenous primer. Real-time PCR and Sanger sequencing were applied to identify the source and to monitor the outcome of this reaction. Results demonstrated that during purification of viral RNA from plasma, tRNA(Lys-3) is co-extracted in a complex with the viral RNA. In the presence of a reverse transcription enzyme, this tRNA(Lys-3) can induce reverse transcription, a reaction that is not confined to transcription of the 5' end of the viral RNA. A range of cDNA products is generated, most of them indicative for the occurrence of in vitro strand transfer events that involve translocation of cDNA from the 5' end to random positions on the viral RNA. This process results in the formation of cDNAs with large internal deletions. However, near full-length cDNA and cDNA with sequence patterns resembling multiple spliced HIV-1 RNA were also detected. Despite its potential to introduce significant bias in the interpretation of results across various applications, tRNA(Lys-3)-driven reverse transcription has been overlooked thus far. A more in-depth study of this tRNA-driven in vitro reaction may provide new insight into the complex process of in vivo HIV-1 replication.IMPORTANCEThe use of silica-based extraction methods for purifying HIV-1 RNA from viral particles is a common practice, but it involves co-extraction of human tRNA(Lys-3) due to the strong interactions between these molecules. This co-extraction becomes particularly significant when the extracted RNA is used in reverse transcription reactions, as the tRNA(Lys-3) then serves as a primer. Reverse transcription from tRNA(Lys-3) is not confined to cDNA synthesis of the 5' end of the viral RNA but extends across various regions of the viral genome through in vitro strand transfer events. Co-extraction of tRNA(Lys-3) has been overlooked thus far, despite its potential to introduce bias in downstream, reverse transcription-related applications. The observed events in the tRNA(Lys-3)-induced in vitro reverse transcription resemble in vivo replication processes. Therefore, these reactions may offer a unique model to better understand the replication dynamics of HIV-1.

Flexible gold standards for transcription factor regulatory interactions in Escherichia coli K-12: architecture of evidence types.

Post-genomic implementations have expanded the experimental strategies to identify elements involved in the regulation of transcription initiation. Here, we present for the first time a detailed analysis of the sources of knowledge supporting the collection of transcriptional regulatory interactions (RIs) of Escherichia coli K-12. An RI groups the transcription factor, its effect (positive or negative) and the regulated target, a promoter, a gene or transcription unit. We improved the evidence codes so that specific methods are incorporated and classified into independent groups. On this basis we updated the computation of confidence levels, weak, strong, or confirmed, for the collection of RIs. These updates enabled us to map the RI set to the current collection of HT TF-binding datasets from ChIP-seq, ChIP-exo, gSELEX and DAP-seq in RegulonDB, enriching in this way the evidence of close to one-quarter (1329) of RIs from the current total 5446 RIs. Based on the new computational capabilities of our improved annotation of evidence sources, we can now analyze the internal architecture of evidence, their categories (experimental, classical, HT, computational), and confidence levels. This is how we know that the joint contribution of HT and computational methods increase the overall fraction of reliable RIs (the sum of confirmed and strong evidence) from 49% to 71%. Thus, the current collection has 3912 reliable RIs, with 2718 or 70% of them with classical evidence which can be used to benchmark novel HT methods. Users can selectively exclude the method they want to benchmark, or keep for instance only the confirmed interactions. The recovery of regulatory sites in RegulonDB by the different HT methods ranges between 33% by ChIP-exo to 76% by ChIP-seq although as discussed, many potential confounding factors limit their interpretation. The collection of improvements reported here provides a solid foundation to incorporate new methods and data, and to further integrate the diverse sources of knowledge of the different components of the transcriptional regulatory network. There is no other genomic database that offers this comprehensive high-quality architecture of knowledge supporting a corpus of transcriptional regulatory interactions.

Achieving an optimal pregnancy outcome through the combined utilization of micro-TESE and ICSI in cryptorchidism associated with a non-canonical splicing variant in RXFP2.

To identify the genetic cause of a cryptorchidism patient carrying a non-canonical splicing variant highlighted by SPCards platform in RXFP2 and to provide a comprehensive overview of RXFP2 variants with cryptorchidism correlation. We identified a homozygous non-canonical splicing variant by whole-exome sequencing and Sanger sequencing in a case with cryptorchidism and non-obstructive azoospermia (NOA). As the pathogenicity of this non-canonical splicing variant remained unclear, we initially utilized the SPCards platform to predict its pathogenicity. Subsequently, we employed a minigene splicing assay to further evaluate the influence of the identified splicing variant. Microdissection testicular sperm extraction (micro-TESE) combined with intracytoplasmic sperm injection (ICSI) was performed. PubMed and Human Genome Variant Database (HGMD) were queried to search for RXFP2 variants. We identified a homozygous non-canonical splicing variant (NM_130806: c.1376-12A > G) in RXFP2, and confirmed this variant caused aberrant splicing of exons 15 and 16 of the RXFP2 gene: 11 bases were added in front of exon 16, leading to an abnormal transcript initiation and a frameshift. Fortunately, the patient successfully obtained his biological offspring through micro-TESE combined with ICSI. Four cryptorchidism-associated variants in RXFP2 from 90 patients with cryptorchidism were identified through a literature search in PubMed and HGMD, with different inheritance patterns. This is the first cryptorchidism case carrying a novel causative non-canonical splicing RXFP2 variant. The combined approach of micro-TESE and ICSI contributed to an optimal pregnancy outcome. Our literature review demonstrated that RXFP2 variants caused cryptorchidism in a recessive inheritance pattern, rather than a dominant pattern.

Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces.

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the -35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β' zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.

Engineering RNA polymerase to construct biotechnological host strains of cyanobacteria.

Application of cyanobacteria for bioproduction, bioremediation and biotransformation is being increasingly explored. Photoautotrophs are carbon-negative by default, offering a direct pathway to reducing emissions in production systems. More robust and versatile host strains are needed for constructing production strains that would function as efficient and carbon-neutral cyanofactories. We have tested if the engineering of sigma factors, regulatory units of the bacterial RNA polymerase, could be used to generate better host strains of the model cyanobacterium Synechocystis sp. PCC 6803. Overexpressing the stress-responsive sigB gene under the strong psbA2 promoter (SigB-oe) led to improved tolerance against heat, oxidative stress and toxic end-products. By targeting transcription initiation in the SigB-oe strain, we could simultaneously activate a wide spectrum of cellular protective mechanisms, including carotenoids, the HspA heat shock protein, and highly activated non-photochemical quenching. Yellow fluorescent protein was used to test the capacity of the SigB-oe strain to produce heterologous proteins. In standard conditions, the SigB-oe strain reached a similar production as the control strain, but when cultures were challenged with oxidative stress, the production capacity of SigB-oe surpassed the control strain. We also tested the production of growth-rate-controlled host strains via manipulation of RNA polymerase, but post-transcriptional regulation prevented excessive overexpression of the primary sigma factor SigA, and overproduction of the growth-restricting SigC factor was lethal. Thus, more research is needed before cyanobacteria growth can be manipulated by engineering RNA polymerase.

Identification of Salt-Sensitive and Salt-Tolerant Genes through Weighted Gene Co-Expression Networks across Multiple Datasets: A Centralization and Differential Correlation Analysis.

Salt stress is a significant challenge that severely hampers rice growth, resulting in decreased yield and productivity. Over the years, researchers have identified biomarkers associated with salt stress to enhance rice tolerance. However, the understanding of the mechanism underlying salt tolerance in rice remains incomplete due to the involvement of multiple genes. Given the vast amount of genomics and transcriptomics data available today, it is crucial to integrate diverse datasets to identify key genes that play essential roles during salt stress in rice. In this study, we propose an integration of multiple datasets to identify potential key transcription factors. This involves utilizing network analysis based on weighted co-expression networks, focusing on gene-centric measurement and differential co-expression relationships among genes. Consequently, our analysis reveals 86 genes located in markers from previous meta-QTL analysis. Moreover, six transcription factors, namely LOC_Os03g45410 (OsTBP2), LOC_Os07g42400 (OsGATA23), LOC_Os01g13030 (OsIAA3), LOC_Os05g34050 (OsbZIP39), LOC_Os09g29930 (OsBIM1), and LOC_Os10g10990 (transcription initiation factor IIF), exhibited significantly altered co-expression relationships between salt-sensitive and salt-tolerant rice networks. These identified genes hold potential as crucial references for further investigation into the functions of salt stress response in rice plants and could be utilized in the development of salt-resistant rice cultivars. Overall, our findings shed light on the complex genetic regulation underlying salt tolerance in rice and contribute to the broader understanding of rice's response to salt stress.

Conceptual breakthroughs of the long noncoding RNA functional system and its endogenous regulatory role in the cancerous regime.

Long noncoding RNAs (lncRNAs) derived from noncoding regions in the human genome were once regarded as junks with no biological significance, but recent studies have shown that these molecules are highly functional, prompting an explosion of studies on their biology. However, these recent efforts have only begun to recognize the biological significance of a small fraction (< 1%) of the lncRNAs. The basic concept of these lncRNA functions remains controversial. This controversy arises primarily from conventional biased observations based on limited datasets. Fortunately, emerging big data provides a promising path to circumvent conventional bias to understand an unbiased big picture of lncRNA biology and advance the fundamental principles of lncRNA biology. This review focuses on big data studies that break through the critical concepts of the lncRNA functional system and its endogenous regulatory roles in all cancers. lncRNAs have unique functional systems distinct from proteins, such as transcriptional initiation and regulation, and they abundantly interact with mitochondria and consume less energy. lncRNAs, rather than proteins as traditionally thought, function as the most critical endogenous regulators of all cancers. lncRNAs regulate the cancer regulatory regime by governing the endogenous regulatory network of all cancers. This is accomplished by dominating the regulatory network module and serving as a key hub and top inducer. These critical conceptual breakthroughs lay a blueprint for a comprehensive functional picture of the human genome. They also lay a blueprint for combating human diseases that are regulated by lncRNAs.

A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae reveals novel insights into factors that stabilize chromatin structure on newly replicated DNA.

The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.

Interplay between coding and non-coding regulation drives the Arabidopsis seed-to-seedling transition

Translation of seed stored mRNAs is essential to trigger germination. However, when RNAPII re-engages RNA synthesis during the seed-to-seedling transition has remained in question. Combining csRNA-seq, ATAC-seq and smFISH in Arabidopsis thaliana we demonstrate that active transcription initiation is detectable during the entire germination process. Features of non-coding regulation such as dynamic changes in chromatin accessible regions, antisense transcription, as well as bidirectional non-coding promoters are widespread throughout the Arabidopsis genome. We show that sensitivity to exogenous ABSCISIC ACID (ABA) during germination depends on proximal promoter accessibility at ABA-responsive genes. Moreover, we provide genetic validation of the existence of divergent transcription in plants. Our results reveal that active enhancer elements are transcribed producing non-coding enhancer RNAs (eRNAs) as widely documented in metazoans. In sum, this study defining the extent and role of coding and non-coding transcription during key stages of germination expands our understanding of transcriptional mechanisms underlying plant developmental transitions.

ElemeNT 2023: an enhanced tool for detection and curation of core promoter elements.

Prediction and identification of core promoter elements and transcription factor binding sites is essential for understanding the mechanism of transcription initiation and deciphering the biological activity of a specific locus. Thus, there is a need for an up-to-date tool to detect and curate core promoter elements/motifs in any provided nucleotide sequences. Here, we introduce ElemeNT 2023-a new and enhanced version of the Elements Navigation Tool, which provides novel capabilities for assessing evolutionary conservation and for readily evaluating the quality of high-throughput transcription start site (TSS) datasets, leveraging preferential motif positioning. ElemeNT 2023 is accessible both as a fast web-based tool and via command line (no coding skills are required to run the tool). While this tool is focused on core promoter elements, it can also be used for searching any user-defined motif, including sequence-specific DNA binding sites. Furthermore, ElemeNT's CORE database, which contains predicted core promoter elements around annotated TSSs, is now expanded to cover 10 species, ranging from worms to human. In this applications note, we describe the new workflow and demonstrate a case study using ElemeNT 2023 for core promoter composition analysis of diverse species, revealing motif prevalence and highlighting evolutionary insights. We discuss how this tool facilitates the exploration of uncharted transcriptomic data, appraises TSS quality, and aids in designing synthetic promoters for gene expression optimization. Taken together, ElemeNT 2023 empowers researchers with comprehensive tools for meticulous analysis of sequence elements and gene expression strategies. ElemeNT 2023 is freely available at https://www.juven-gershonlab.org/resources/element-v2023/. The source code and command line version of ElemeNT 2023 are available at https://github.com/OritAdato/ElemeNT. No coding skills are required to run the tool.

Identification of Highly Repetitive Enhancers with Long-range Regulation Potential in Barley via STARR-seq.

Enhancers are DNA sequences that can strengthen transcription initiation. However, the global identification of plant enhancers is complicated due to uncertainty in the distance and orientation of enhancers, especially in species with large genomes. In this study, we performed self-transcribing active regulatory region sequencing (STARR-seq) for the first time to identify enhancers across the barley genome. A total of 7323 enhancers were successfully identified, and among 45 randomly selected enhancers, over 75% were effective as validated by a dual-luciferase reporter assay system in the lower epidermis of tobacco leaves. Interestingly, up to 53.5% of the barley enhancers were repetitive sequences, especially transposable elements (TEs), thus reinforcing the vital role of repetitive enhancers in gene expression. Both the common active mark H3K4me3 and repressive mark H3K27me3 were abundant among the barley STARR-seq enhancers. In addition, the functional range of barley STARR-seq enhancers seemed much broader than that of rice or maize and extended to ±100 kb of the gene body, and this finding was consistent with the high expression levels of genes in the genome. This study specifically depicts the unique features of barley enhancers and provides available barley enhancers for further utilization.