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Aspergillus vadensis CBS 113365, a close relative of A. niger, has been suggested as a more favourable alternative for recombinant protein production as it does not acidify the culture medium and produces very low levels of extracellular proteases. The aim of this study was to investigate the underlying cause of the non-amylolytic and non-proteolytic phenotype of A. vadensis CBS 113365. Our results demonstrate that the non-functionality of the amylolytic transcription factor AmyR in A. vadensis CBS 113365 is primarily attributed to the lack of functionality of its gene's promoter sequence. In contrast, a different mechanism is likely causing the lack of PrtT activity, which is the main transcriptional regulator of protease production. The findings presented here not only expand our understanding of the genetic basis behind the distinct characteristics of A. vadensis CBS 113365, but also underscore its potential as a favourable alternative for recombinant protein production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Long noncoding RNAs (lncRNAs) are implicated in a number of regulatory functions in eukaryotic genomes. In humans, KCNQ1OT1 is a 91 kb imprinted lncRNA that inhibits multiple surrounding genes in cis. Among them, CDKN1C is closely related to KCNQ1OT1 and is involved in multiple epigenetic disorders. Here, we found that pigs also had a relatively conserved paternal allele expressing KCNQ1OT1 and had a shorter 5' end (∼27 kb) compared to human KCNQ1OT1. Knockdown of KCNQ1OT1 using antisense oligonucleotides (ASO) showed that upregulation of CDKN1C expression in pigs. However, porcine KCNQ1OT1 did not affect the DNA methylation status of the CpG islands in the promoters of KCNQ1OT1 and CDKN1C. Inhibition of DNA methyltransferase using Decitabine treatment resulted in a significant increase in both KCNQ1OT1 and CDKN1C expression, suggesting that the regulation between KCNQ1OT1 and CDKN1C may not be dependent on RNA interference. Further use of chromosome conformation capture and reverse transcription-associated trap detection in the region where CDKN1C was located revealed that KCNQ1OT1 bound to the CDKN1C promoter and affected chromosome folding. Phenotypically, inhibition of KCNQ1OT1 at the cumulus-oocyte complex promoted cumulus cell transformation, and to upregulated the expression of ALPL at the early stage of osteogenic differentiation of porcine bone marrow mesenchymal stem cells. Our results confirm that the expression of KCNQ1OT1 imprinting in pigs as well as porcine KCNQ1OT1 regulates the expression of CDKN1C through direct promoter binding and chromatin folding alteration. And this regulatory mechanism played an important role in cell differentiation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DELLA proteins are conserved master growth regulators that play a central role in controlling plant development in response to internal and environmental cues. DELLAs function as transcription regulators, which are recruited to target promoters by binding to transcription factors (TFs) and histone H2A via their GRAS domain. Recent studies showed that DELLA stability is regulated post-translationally via two mechanisms, phytohormone gibberellin-induced polyubiquitination for its rapid degradation, and Small Ubiquitin-like Modifier (SUMO)-conjugation to increase its accumulation. Moreover, DELLA activity is dynamically modulated by two distinct glycosylations: DELLA-TF interactions are enhanced by O-fucosylation, but inhibited by O-linked N-acetylglucosamine (O-GlcNAc) modification. However, the role of DELLA phosphorylation remains unclear as previous studies showing conflicting results ranging from findings that suggest phosphorylation promotes or reduces DELLA degradation to others indicating it has no effect on its stability. Here, we identify phosphorylation sites in REPRESSOR OF ga1-3 (RGA, an AtDELLA) purified from Arabidopsis by mass spectrometry analysis, and show that phosphorylation of two RGA peptides in the PolyS and PolyS/T regions enhances RGA activity by promoting H2A binding and RGA association with target promoters. Notably, phosphorylation does not affect RGA-TF interactions or RGA stability. Our study has uncovered a molecular mechanism of phosphorylation-induced DELLA activity., (© 2024. The Author(s).)

Background: Alternative splicing is a pivotal mechanism of post-transcriptional modification that contributes to the transcriptome plasticity and proteome diversity in metazoan cells. Although many splicing regulations around the exon/intron regions are known, the relationship between promoter-bound transcription factors and the downstream alternative splicing largely remains unexplored., Results: In this study, we present computational approaches to unravel the regulatory relationship between promoter-bound transcription factor binding sites (TFBSs) and the splicing patterns. We curated a fine dataset that includes DNase I hypersensitive site sequencing and transcriptomes across fifteen human tissues from ENCODE. Specifically, we proposed different representations of TF binding context and splicing patterns to examine the associations between the promoter and downstream splicing events. While machine learning models demonstrated potential in predicting splicing patterns based on TFBS occupancies, the limitations in the generalization of predicting the splicing forms of singleton genes across diverse tissues was observed with carefully examination using different cross-validation methods. We further investigated the association between alterations in individual TFBS at promoters and shifts in exon splicing efficiency. Our results demonstrate that the convolutional neural network (CNN) models, trained on TF binding changes in the promoters, can predict the changes in splicing patterns. Furthermore, a systemic in silico substitutions analysis on the CNN models highlighted several potential splicing regulators. Notably, using empirical validation using K562 CTCFL shRNA knock-down data, we showed the significant role of CTCFL in splicing regulation., Conclusion: In conclusion, our finding highlights the potential role of promoter-bound TFBSs in influencing the regulation of downstream splicing patterns and provides insights for discovering alternative splicing regulations., (© 2024. The Author(s).)

Enhlink is a computational tool for scATAC-seq data analysis, facilitating precise interrogation of enhancer function at the single-cell level. It employs an ensemble approach incorporating technical and biological covariates to infer condition-specific regulatory DNA linkages. Enhlink can integrate multi-omic data for enhanced specificity, when available. Evaluation with simulated and real data, including multi-omic datasets from the mouse striatum and novel promoter capture Hi-C data, demonstrate that Enhlink outperfoms alternative methods. Coupled with eQTL analysis, it identified a putative super-enhancer in striatal neurons. Overall, Enhlink offers accuracy, power, and potential for revealing novel biological insights in gene regulation., (© 2024. The Author(s).)

The hox operon in Synechocystis sp. PCC 6803, encoding bidirectional hydrogenase responsible for H 2 production, is transcriptionally upregulated under microoxic conditions. Although several regulators for hox transcription have been identified, their dynamics and higher-order DNA structure of hox region in microoxic conditions remain elusive. We focused on key regulators for the hox operon: cyAbrB2, a conserved regulator in cyanobacteria, and SigE, an alternative sigma factor. Chromatin immunoprecipitation sequencing revealed that cyAbrB2 binds to the hox promoter region under aerobic conditions, with its binding being flattened in microoxic conditions. Concurrently, SigE exhibited increased localization to the hox promoter under microoxic conditions. Genome-wide analysis revealed that cyAbrB2 binds broadly to AT-rich genome regions and represses gene expression. Moreover, we demonstrated the physical interactions of the hox promoter region with its distal genomic loci. Both the transition to microoxic conditions and the absence of cyAbrB2 influenced the chromosomal interaction. From these results, we propose that cyAbrB2 is a cyanobacterial nucleoid-associated protein (NAP), modulating chromosomal conformation, which blocks RNA polymerase from the hox promoter in aerobic conditions. We further infer that cyAbrB2, with altered localization pattern upon microoxic conditions, modifies chromosomal conformation in microoxic conditions, which allows SigE-containing RNA polymerase to access the hox promoter. The coordinated actions of this NAP and the alternative sigma factor are crucial for the proper hox expression in microoxic conditions. Our results highlight the impact of cyanobacterial chromosome conformation and NAPs on transcription, which have been insufficiently investigated., Competing Interests: RK, TO No competing interests declared, (© 2024, Kariyazono and Osanai.)

Background/aim: Matrix metalloproteinase-2 (MMP-2) has been implicated in the pathogenesis of breast cancer (BC). However, there is limited research on the role of MMP-2 genotypes in BC risk. This study aimed to investigate the associations between two MMP-2 promoter polymorphisms, rs243865 and rs2285053, and BC risk., Materials and Methods: MMP-2 genotypes were analyzed using PCR-based RFLP methodology in a cohort comprising 1,232 BC cases and 1,232 controls., Results: Genotypic frequencies of MMP-2 rs243865 and rs2285053 in controls were consistent with Hardy-Weinberg equilibrium (p=0.3702 and 0.2036, respectively). There were no significant differences in the distribution of rs243865 and rs2285053 genotypes between BC cases and controls (p for trend=0.1602 and 0.2170, respectively). Variant genotypes at rs243865 and rs2285053 appeared to confer a protective effect, although not statistically significant (all p>0.05). Similarly, the variant T allele at rs243865 and rs2285053 showed a non-significant trend towards decreased BC risk (OR=0.84 and 0.89, 95%CI=0.69-1.02 and 0.78-1.02, p=0.0811 and 0.1043, respectively). There was no interaction observed between MMP-2 rs243865 or rs2285053 genotypes and age. Stratified analysis did not reveal significant associations between MMP-2 rs243865 or rs2285053 genotypes and triple-negative breast cancer (TNBC) (p=0.6458 and 0.8745, respectively). Among both TNBC and non-TNBC cases, none of the variant genotypes at rs243865 or rs2285053 showed significant associations with TNBC (all p>0.05)., Conclusion: MMP-2 rs243865 and rs2285053 genotypes appear to have a minimal impact on individual susceptibility to BC or TNBC., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Armadillo repeat-containing proteins (ARMCs) are a large family found throughout eukaryotes, which play prominent roles in cell adhesion, signaling and cytoskeletal regulation. The ARMC6 protein is highly conserved in primates, including humans, but to date does not have a clear function beyond initial hints of a link to cancer and telomerase activity. We report here in vitro experiments showing ARMC6 binding to DNA promoter sequences from several cancer-related genes (e.g., EGFR, VEGF and c-MYC), and also to the telomeric RNA repeat (TERRA). ARMC6 binding activity appears to recognize G-quadruplex motifs, which are being increasingly implicated as structure-based protein binding sites in chromosome maintenance and repair. In vivo investigation of ARMC6 function revealed that when this protein is overexpressed in human cell lines, there is different expression of genes connected with oncogenic pathways and those implicated in downstream non-canonical telomerase pathways (e.g., VEGF, hTERT, c-MYC, ESM1, MMP3). ARMC6 is already known to interact with human shelterin protein TRF2 and telomerase. The protein binds G-quadruplex structures and does so preferentially to RNA over DNA. As such, this protein may be an example of how a non-canonical nucleic acid structural motif allows mediation between gene regulation and telomeric chromatin rearrangement pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We constructed a new Aspergillus expression vector (pSENSU2512nid) under the control of the enolase promoter with 12 tandem repeats of cis-acting elements (region III) and the heat shock protein 12 (Hsp12) 5' untranslated region (UTR). Bilirubin oxidase (EC: 1.3.3.5) from Myrothecium verrucaria, which catalyzes the oxidation of bilirubin to biliverdin, was overexpressed in Aspergillus oryzae and A. niger. The productivity was estimated to be approximately 1.2 g/L in the culture broth, which was approximately 6-fold higher than that of recombinant bilirubin oxidase (BOD) expressed in Pichia pastoris (Komagataella phaffii). BOD was purified using hydrophobic interaction chromatography, followed by ion exchange chromatography. The specific activity of the purified BOD against 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate was 57.6 U/mg and 66.4 U/mg for A. oryzae and A. niger, respectively. l-Ascorbic acid (4 mM) addition and storage under deoxygenated conditions for 3-7 d increased the specific activity of these Aspergillus-expressed BODs approximately 2.3-fold (154.1 U/mg). The BOD specific activity was enhanced by incubation at higher temperature (30-50 °C). Further characterization of the enzyme catalytic efficiency revealed that the K m value remained unchanged, whereas the k cat value improved 3-fold. In conclusion, this high-level of BOD expression meets the requirements for industrial-level production. Additionally, we identified an effective method to enhance the low specific activity during expression, making it advantageous for industrial applications., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A certain degree of chromatin openness is necessary for the activity of transcription-regulating regions within the genome, facilitating accessibility to RNA polymerases and subsequent synthesis of regulatory element RNAs (regRNAs) from these regions. The rapidly increasing number of studies underscores the significance of regRNAs across diverse cellular processes and diseases, challenging the paradigm that these transcripts are non-functional transcriptional noise. This review explores the multifaceted roles of regRNAs in human cells, encompassing rather well-studied entities such as promoter RNAs and enhancer RNAs (eRNAs), while also providing insights into overshadowed silencer RNAs and insulator RNAs. Furthermore, we assess notable examples of shorter regRNAs, like miRNAs, snRNAs, and snoRNAs, playing important roles. Expanding our discourse, we deliberate on the potential usage of regRNAs as biomarkers and novel targets for cancer and other human diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stasevich E. M. reports financial support was provided by Russian Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The second messenger guanosine 3',5'-bis(diphosphate)/guanosine tetraphosphate (ppGpp) and guanosine 3'-diphosphate 5'-triphosphate/guanosine pentaphosphate (pppGpp) ((p)ppGpp) has been shown to be crucial for the survival of mycobacteria under hostile conditions. Unexpectedly, deletion of primary (p)ppGpp synthetase-Rel did not completely diminish (p)ppGpp levels leading to the discovery of novel bifunctional enzyme-RelZ, which displayed guanosine 5'-monophosphate,3'-diphosphate (pGpp), ppGpp, and pppGpp ((pp)pGpp) synthesis and RNAseHII activity. What conditions does it express itself under, and does it work in concert with Rel? The regulation of its transcription and whether the Rel enzyme plays a role in such regulation remain unclear. In this article, we have studied relZ promoter and compared its activity with rel promoter in different growth conditions. We observed that the promoter activity of relZ was constitutive; it is weaker than rel promoter, lies within 200 bp upstream of translation-start site, and it increased under carbon starvation. Furthermore, the promoter activity of relZ was compromised in the rel-knockout strain in the stationary phase. Our study unveils the dynamic regulation of relZ promoter activity by SigA and SigB sigma factors in different growth phases in mycobacteria. Importantly, elucidating the regulatory network of RelZ would enable the development of the targeted interventions for treating mycobacterial infections., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Dunaliella salina is an innovative expression system due to its distinct advantages such as high salt tolerance, low susceptibility to contamination, and the absence of the cell wall. While nuclear transformation has been extensively studied, research on D. salina chloroplast transformation remains in the preliminary stages. In this study, we established an efficient chloroplast expression system for D. salina using Golden Gate assembly. We developed a D. salina toolkit comprising essential components such as chloroplast-specific promoters, terminators, homologous fragments, and various vectors. We confirmed its functionality by expressing the EGFP protein. Moreover, we detailed the methodology of the entire construction process. This expression system enables the specific targeting of foreign genes through simple homologous recombination, resulting in stable expression in chloroplasts. The toolkit achieved a relatively high transformation efficiency within a shorter experimental cycle. Consequently, the construction and utilization of this toolkit have the potential to enhance the efficiency of transgenic engineering in D. salina and advance the development of microalgal biofactories., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The study characterized the transcriptionally regulatory mechanism and functions of three zinc (Zn) transporters (znt4, znt5 and znt10) in Zn 2+ metabolism in yellow catfish (Pelteobagrus fulvidraco), commonly freshwater fish in China and other countries. We cloned the sequences of znt4 promoter, spanning from -1217 bp to +80 bp relative to TSS (1297 bp); znt5, spanning from -1783 bp to +49 bp relative to TSS (1832 bp) and znt10, spanning from -1923 bp to +190 bp relative to TSS (2113 bp). In addition, after conducting the experiments of sequential deletion of promoter region and mutation of potential binding site, we found that the Nrf2 binding site (-607/-621 bp) and Klf4 binding site (-5/-14 bp) were required on znt4 promoter, the Mtf-1 binding site (-1674/-1687 bp) and Atf4 binding site (-444/-456 bp) were required on znt5 promoter and the Atf4 binding site (-905/-918 bp) was required on znt10 promoter. Then, according to EMSA and ChIP, we found that Zn 2+ incubation increased DNA affinity of Atf4 to znt5 or znt10 promoter, but decreased DNA affinity of Nrf2 to znt4 promoter, Klf4 to znt4 promoter and Mtf-1 to znt5 promoter. Using fluorescent microscopy, it was revealed that Znt4 and Znt10 were located in the lysosome and Golgi, and Znt5 was located in the Golgi. Finally, we found that znt4 knockdown reduced the zinc content of lysosome and Golgi in the control and zinc-treated group; znt5 knockdown reduced the zinc content of Golgi in the control and zinc-treated group and znt10 knockdown reduced the zinc content of Golgi in the zinc-treated group. High dietary zinc supplement up-regulated Znt4 and Znt5 protein expression. Above all, for the first time, we revealed that Klf4 and Nrf2 transcriptionally regulated the activities of znt4 promoter; Mtf-1 and Atf4 transcriptionally regulated the activities of znt5 promoter and Atf4 transcriptionally regulated the activities of znt10 promoter, which provided innovative regulatory mechanism of zinc transporting in yellow catfish. Our study also elucidated their subcellular location, and regulatory role of zinc homeostasis in yellow catfish., Competing Interests: Declaration of competing interest The authors declared that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Suaeda australis is one of typical halophyte owing to high levels of salt tolerance. In addition, the bZIP gene family assumes pivotal functions in response to salt stress. However, there are little reports available regarding the bZIP gene family in S. australis., Results: In this study, we successfully screened 44 bZIP genes within S. australis genome. Subsequently, we conducted an extensive analysis, encompassing investigations into chromosome location, gene structure, phylogenetic relationship, promoter region, conserved motif, and gene expression profile. The 44 bZIP genes were categorized into 12 distinct groups, exhibiting an uneven distribution among the 9 chromosomes of S. australis chromosomes, but one member (Sau23745) was mapped on unanchored scaffolds. Examination of cis-regulatory elements revealed that bZIP promoters were closely related to anaerobic induction, transcription start, and light responsiveness. Comparative transcriptome analysis between ST1 and ST2 samples identified 2,434 DEGs, which were significantly enriched in some primary biological pathways related to salt response-regulating signaling based on GO and KEGG enrichment analysis. Expression patterns analyses clearly discovered the role of several differently expressed SabZIPs, including Sau08107, Sau08911, Sau11415, Sau16575, and Sau19276, which showed higher expression levels in higher salt concentration than low concentration and a response to salt stress. These expression patterns were corroborated through RT-qPCR analysis. The six differentially expressed SabZIP genes, all localized in the nucleus, exhibited positive regulation involved in the salt stress response. SabZIP14, SabZIP26, and SabZIP36 proteins could bind to the promoter region of downstream salt stress-related genes and activate their expressions., Conclusions: Our findings offer valuable insights into the evolutionary trajectory of the bZIP gene family in S. australis and shed light on their roles in responding to salt stress. In addition to fundamental genomic information, these results would serve as a foundational framework for future investigations into the regulation of salt stress responses in S. australis., (© 2024. The Author(s).)

After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli (E. coli) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 (σ 70 ) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α-C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ 70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ-independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation., (© 2024. The Author(s).)

The human silencing hub (HUSH) complex binds to transcripts of LINE-1 retrotransposons (L1s) and other genomic repeats, recruiting MORC2 and other effectors to remodel chromatin. How HUSH and MORC2 operate alongside DNA methylation, a central epigenetic regulator of repeat transcription, remains largely unknown. Here we interrogate this relationship in human neural progenitor cells (hNPCs), a somatic model of brain development that tolerates removal of DNA methyltransferase DNMT1. Upon loss of MORC2 or HUSH subunit TASOR in hNPCs, L1s remain silenced by robust promoter methylation. However, genome demethylation and activation of evolutionarily-young L1s attracts MORC2 binding, and simultaneous depletion of DNMT1 and MORC2 causes massive accumulation of L1 transcripts. We identify the same mechanistic hierarchy at pericentromeric α-satellites and clustered protocadherin genes, repetitive elements important for chromosome structure and neurodevelopment respectively. Our data delineate the epigenetic control of repeats in somatic cells, with implications for understanding the vital functions of HUSH-MORC2 in hypomethylated contexts throughout human development., (© 2024. The Author(s).)

During tumor development, promoter CpG islands that are normally silenced by Polycomb repressive complexes (PRCs) become DNA-hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) [DNMT(s)] catalyze CpG methylation at PRC-regulated regions remains unclear. Here, we report a cryo-electron microscopy structure of the DNMT3A long isoform (DNMT3A1) amino-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine-119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 amino terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Further, aberrant redistribution of DNMT3A1 to Polycomb target genes recapitulates the cancer-associated DNA hypermethylation signature and inhibits their transcriptional activation during cell differentiation. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for mediating promoter CpG island DNA hypermethylation, a major molecular hallmark of cancer.

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.)

Enhancing thermogenic brown adipose tissue (BAT) function is a promising therapeutic strategy for metabolic disease. However, predominantly thermoneutral modern human living conditions deactivate BAT. We demonstrate that selective adipocyte deficiency of the oxygen-sensor HIF-prolyl hydroxylase (PHD2) gene overcomes BAT dormancy at thermoneutrality. Adipocyte-PHD2-deficient mice maintain higher energy expenditure having greater BAT thermogenic capacity. In human and murine adipocytes, a PHD inhibitor increases Ucp1 levels. In murine brown adipocytes, antagonising the major PHD2 target, hypoxia-inducible factor-(HIF)-2a abolishes Ucp1 that cannot be rescued by PHD inhibition. Mechanistically, PHD2 deficiency leads to HIF2 stabilisation and binding of HIF2 to the Ucp1 promoter, thus enhancing its expression in brown adipocytes. Serum proteomics analysis of 5457 participants in the deeply phenotyped Age, Gene and Environment Study reveal that serum PHD2 associates with increased risk of metabolic disease. Here we show that adipose-PHD2-inhibition is a therapeutic strategy for metabolic disease and identify serum PHD2 as a disease biomarker., (© 2024. The Author(s).)

Obesity poses a public health threat, reaching epidemic proportions. Our hypothesis suggests that some of this epidemic stems from its transmission across generations via paternal epigenetic mechanisms. To investigate this possibility, we focused on examining the paternal transmission of CpG methylation. First-generation male Wistar rats were fed either a high-fat diet (HF) or chow and were mated with females fed chow. We collected sperm from these males. The resulting offspring were raised on a chow diet until day 35, after which they underwent a dietary challenge. Diet-induced obese (DIO) male rats passed on the obesogenic trait to both male and female offspring. We observed significant hypermethylation of the Pomc promoter in the sperm of HF-treated males and in the hypothalamic arcuate nucleus (Arc) of their offspring at weaning. However, these differences in Arc methylation decreased later in life. This hypermethylation is correlated with increased expression of DNMT3B. Further investigating genes in the Arc that might be involved in obesogenic transgenerational transmission, using reduced representation bisulfite sequencing (RRBS) we identified 77 differentially methylated regions (DMRs), highlighting pathways associated with neuronal development. These findings support paternal CpG methylation as a mechanism for transmitting obesogenic traits across generations., (© 2024. The Author(s).)

Bacterial and bacteriophage RNA polymerases (RNAPs) have divergently evolved and share the RNA hairpin-dependent intrinsic termination of transcription. Here, we examined phage T7, T3 and SP6 RNAP terminations utilizing the single-molecule fluorescence assays we had developed for bacterial terminations. We discovered the phage termination mode or outcome is virtually single with decomposing termination. Therein, RNAP is displaced forward along DNA and departs both RNA and DNA for one-step decomposition, three-dimensional diffusion and reinitiation at any promoter. This phage displacement-mediated decomposing termination is much slower than readthrough and appears homologous with the bacterial one. However, the phage sole mode of termination contrasts with the bacterial dual mode, where both decomposing and recycling terminations occur compatibly at any single hairpin- or Rho-dependent terminator. In the bacterial recycling termination, RNA is sheared from RNA·DNA hybrid, and RNAP remains bound to DNA for one-dimensional diffusion, which enables facilitated recycling for reinitiation at the nearest promoter located downstream or upstream in the sense or antisense orientation. Aligning with proximity of most terminators to adjacent promoters in bacterial genomes, the shearing-mediated recycling termination could be bacterial adaptation for the facilitated reinitiations repeated at a promoter for accelerated expression and coupled at adjoining promoters for coordinated regulation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The MHYT domain, identified over two decades ago for its potential to detect diatomic gases like CO, O2 or NO, has awaited experimental validation as a protein sensory domain. Here, we characterize the MHYT domain-containing transcriptional regulator CoxC, which governs the expression of the cox genes responsible for aerobic CO oxidation in the carboxidotrophic bacterium Afipia carboxidovorans OM5. The C-terminal LytTR-type DNA-binding domain of CoxC binds to an operator region consisting of three direct repeats sequences overlapping the -35 box at the target PcoxB promoter, which is consistent with the role of CoxC as a specific transcriptional repressor of the cox genes. Notably, the N-terminal transmembrane MHYT domain endows CoxC with the ability to sense CO as an effector molecule, as demonstrated by the relief of CoxC-mediated repression and binding to the PcoxB promoter upon CO exposure. Furthermore, copper serves as the essential divalent cation for the interaction of CO with CoxC, thereby confirming previous hypothesis regarding the role of copper in the gas-sensing mechanism of MHYT domains. CoxC represents the prototype of a novel subfamily of single-component LytTR transcriptional regulators, characterized by the fusion of a DNA-binding domain with a membrane-bound MHYT sensor domain., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Intrinsically disordered regions (IDRs) guide transcription factors (TFs) to their genomic binding sites, raising the question of how structure-lacking regions encode for complex binding patterns. We investigated this using the TF Gln3, revealing sets of IDR-embedded determinants that direct Gln3 binding to respective groups of functionally related promoters, and enable tuning binding preferences between environmental conditions, phospho-mimicking mutations, and orthologs. Through targeted mutations, we defined the role of short linear motifs (SLiMs) and co-binding TFs (Hap2) in stabilizing Gln3 at respiration-chain promoters, while providing evidence that Gln3 binding at nitrogen-associated promoters is encoded by the IDR amino-acid composition, independent of SLiMs or co-binding TFs. Therefore, despite their apparent simplicity, TF IDRs can direct and regulate complex genomic binding patterns through a combination of SLiM-mediated and composition-encoded interactions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Cis-regulatory elements (CREs) are crucial for regulating gene expression, and G-quadruplexes (G4s), as prototypal non-canonical DNA structures, may play a role in this regulation. However, the relationship between G4s and CREs, especially with non-promoter-like functional elements, requires further systematic investigation. We aimed to investigate the associations between G4s and human cCREs (candidate CREs) inferred from the Encyclopedia of DNA Elements (ENCODE) data., Results: We found that G4s are prominently enriched in most types of cCREs, especially those with promoter-like signatures (PLS). The co-occurrence of CTCF signals with H3K4me3 or H3K27ac signals strengthens the association between cCREs and G4s. Genetic variants in G4s, particularly within their G-runs, exhibit higher regulatory potential and deleterious effects compared to cCREs. The G-runs within G4s near transcriptional start sites (TSSs) are more evolutionarily constrained compared to G-runs in cCREs, while those far from the TSS are relatively less conserved. The presence of G4s is often linked to a more favorable local chromatin environment for the activation and execution of regulatory function of cCREs, potentially attributable to the formation of G4 secondary structures. Finally, we discovered that G4-associated cCREs exhibit widespread activation in a variety of cancers., Conclusions: Our study suggests that G4s are integral components of human cis-regulatory elements, extending beyond their potential role in promoters. The G4 primary sequences are associated with the localization of CREs, while the G4 structures are linked to the activation of these elements. Therefore, we propose defining G4s as pivotal regulatory elements in the human genome., (© 2024. The Author(s).)

Enhanced drug resistance poses a significant challenge in treating ovarian cancer (OC). Phenylethyl isothiocyanate (PEITC) is involved in drug resistance in OC, but the mechanism remains unclear. In this study, we investigated the molecular regulatory mechanism of carboplatin sensitivity in OC associated with PEITC, MAF BZIP Transcription Factor F (MAFF), and Zinc finger proteins (ZNF) 711. The carboplatin sensitivity was significantly increased in OC cells after PEITC treatment. Knockdown of MAFF significantly enhanced the carboplatin sensitivity of OC cells, promoted apoptosis, inhibited colony-forming efficiency in vitro, and suppressed tumor growth in vivo. The binding site of MAFF to the ZNF711 promoter was predicted, and the knockdown of MAFF significantly increased the ZNF711 expression. Results of the dual luciferase assay and ChIP-PCR confirmed the binding of MAFF to the ZNF711 promoter. Immunofluorescence and CoIP results demonstrated the colocalization and the binding of MAFF and its interacting protein, BZIP Transcription Factor ATF-like 3 (BATF3). Similarly, we confirmed the binding of BATF3 to the ZNF711 promoter. Knockdown of BATF3 alone and simultaneous knockdown of BATF3 and MAFF showed similar regulatory effects on ZNF711 transcription and apoptosis. These suggested that the binding of MAFF to BATF3 inhibited ZNF711 transcription and reduced carboplatin sensitivity in OC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The regulation of leaf senescence and disease resistance plays a crucial role in determining rice grain yield and quality, which are important to meet the ever-increasing food demands of the world. Here, we identified an atypical Dof transcriptional factor OsDes1 that contributes to the stay-green phenotype, grain yield, and disease resistance in rice. The expression level of OsDes1 is positively associated with stay-green in natural variations of japonica rice, suggesting that OsDes1 would be alternatively used in breeding programs. Mechanistically, OsDes1 targets the promoter of the Rieske FeS protein gene OsPetC to activate its expression and interacts with OsPetC to protect against its degradation, thus promoting stay-green and ultimately improving the grain yield. OsDes1 also binds to the promoter region of defense-related genes, such as OsPR1b , and activates their expression, leading to enhanced disease resistance. These findings offer a potential strategy for breeding rice to enhance grain yield and disease resistance.

MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II (RNAPII). While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYC's effects on transcription. Here, we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organisation at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localisation to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localise to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA., Competing Interests: RV, EL, SH, MM, DF, CS, DP, IR, LU, CA, PG, RB, GB No competing interests declared, ME founder and shareholder of Tucana Biosciences, (© 2024, Vidal et al.)

DNA-binding one zinc finger (DOF) transcription factors are crucial plant-specific regulators involved in growth, development, signal transduction, and abiotic stress response generation. However, the genome-wide identification and characterization of AcDOF genes and their regulatory elements in kiwifruit ( Actinidia chinensis ) has not been thoroughly investigated. In this study, we screened the kiwifruit genome database and identified 42 AcDOF genes ( AcDOF1 to AcDOF42 ). Phylogenetic analysis facilitated the categorization of these genes into five subfamilies ( DOF -a, DOF -b, DOF -c, DOF -d, and DOF -e). We further analyzed the motifs, conserved domains, gene structures, and collinearity of the AcDOF gene family. Gene ontology (GO) enrichment analysis indicated significant enrichment in the "flower development" term and the "response to abiotic stress" category. Promoter prediction analysis revealed numerous cis-regulatory elements related to responses to light, hormones, and low-temperature and drought stress in AcDOF promoters. RNA-seq expression profiles demonstrated the tissue-specific expression of AcDOF genes. Quantitative real-time PCR results showed that six selected genes ( AcDOF04 , AcDOF09 , AcDOF11 , AcDOF13 , AcDOF21 , and AcDOF22 ) were differentially induced by abscisic acid (ABA), methyl jasmonate (MeJA), and cold, salt, and drought stresses, with AcDOF22 specifically expressed at high levels in drought-tolerant cultivars. Further experiments indicated that transient AcDOF22 overexpression in kiwifruit leaf disks reduced water loss and chlorophyll degradation. Additionally, AcDOF22 was localized to the nucleus and exhibited transcriptional activation, enhancing drought resistance by activating the downstream drought marker gene AcDREB2A . These findings lay the foundation for elucidating the molecular mechanisms of drought resistance in kiwifruit and offer new insights into drought-resistant breeding.

Carnitine-acylcarnitine translocase (CACT) is a nuclear-encoded mitochondrial carrier that catalyzes the transfer of long-chain fatty acids across the inner mitochondrial membrane for β-oxidation. In this study, we conducted a structural and functional characterization of the CACT promoter to investigate the molecular mechanism underlying the transcriptional regulation of the CACT gene by n-3 PUFA, EPA and DHA. In hepatic BRL3A cells, EPA and DHA stimulate CACT mRNA and protein expression. Deletion promoter analysis using a luciferase reporter gene assay identified a n-3 PUFA response region extending from -202 to -29 bp. This region did not contain a response element for PPARα, a well-known PUFA-responsive nuclear receptor. Instead, bioinformatic analysis revealed two highly conserved GABP responsive elements within this region. Overexpression of GABPα and GABPβ subunits, but not PPARα, increased CACT promoter activity, more remarkably upon treatment with EPA and DHA. ChIP assays showed that n3-PUFA enhanced the binding of GABPα to the -202/-29 bp sequence. Furthermore, both EPA and DHA induced nuclear accumulation of GABPα. In conclusion, our findings indicate that the upregulation of CACT by n3-PUFA in hepatic cells is independent from PPARα and could be mediated by GABP activation.

CRISPR/Cas-based transcriptional activators can be enhanced by intrinsically disordered regions (IDRs). However, the underlying mechanisms are still debatable. Here, we examine 12 well-known IDRs by fusing them to the dCas9-VP64 activator, of which only seven can augment activation, albeit independently of their phase separation capabilities. Moreover, modular domains (MDs), another class of multivalent molecules, though ineffective in enhancing dCas9-VP64 activity on their own, show substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and fusing dCas9-VP64 with different IDRs/MDs, we uncover that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs yields synergistic effects, which can be further amplified via enhancing chromatin interactions. Overall, our study develops a versatile platform for efficient gene activation and sheds important insights into CRIPSR-based transcriptional activators enhanced with multivalent molecules., (© 2024. The Author(s).)

Background: Methyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation., Results: This study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively., Conclusion: This is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives., (© 2024. The Author(s).)

Background: The dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes., Methods: Based on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses., Results: The use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35-75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies., Conclusions: A DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established., (© 2024. The Author(s).)

Long non-coding RNAs (lncRNAs) play key roles in cellular pathways and disease progression, yet their molecular mechanisms remain largely understudied. The lncRNA lncTCF7 has been shown to promote tumor progression by recruiting the SWI/SNF complex to the TCF7 promoter, activating its expression and the WNT signaling pathway. However, how lncTCF7 recruits SWI/SNF remains to be determined, and lncTCF7-specific binding partners are unknown. Using RNA-pulldown and quantitative mass spectrometry, we identified a novel interacting protein partner for lncTCF7, SND1, a multifunctional RNA binding protein that can also function as a transcription co-activator. Knockdown analysis of lncTCF7 and SND1 reveals that they are both required for the expression of TCF7. Chromatin immunoprecipitation assays suggest that both SND1 and lncTCF7 are required for recruiting the SWI/SNF chromatin remodeling complex, and these functions, in tandem, activate the expression of TCF7. Finally, using structural probing and RNA-pulldown of lncTCF7 and its subdomains, we highlight the potential binding region for SND1 in the 3'-end of lncTCF7. Overall, this study highlights the critical roles lncRNAs play in regulating gene expression and provides new insights into the complex network of interactions that underlie this process., (© 2024. The Author(s).)

In the past decade, the exploration of genetic resources in rice has significantly enhanced the efficacy of rice breeding. However, the exploration of genetic resources is hindered by the identification of candidate genes. To expedite the identification of candidate genes, this study examined tapetum programmed cell death-related genes OsiWAK1, OsPDT1, EAT1, TDR, and TIP2 to assess the efficacy of the Dual-Luciferase (Dual-LUC) assay in rapidly determining gene relationships. The study found that, in the Dual-LUC assay, OsiWAK1 and its various recombinant proteins exhibit comparable activation abilities on the EAT1 promoter, potentially indicating a false positive. However, the Dual-LUC assay can reveal that OsiWAK1 impacts both the function of its upstream regulatory factor OsPDT1 and the TDR/TIP2 transcription complex. By rapidly studying the relationship between diverse candidate genes and regulatory genes in a well-known trait via the Dual-LUC assay, this study provides a novel approach to expedite the determination of candidate genes such as genome-wide association study., (© 2024. The Author(s).)

The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA., (© 2024. The Author(s).)

Long noncoding RNAs (lncRNAs) are regulatory RNAs. Saccharomyces cerevisiae strains transcribe hundreds of lncRNAs. LncRNAs can regulate the expression of adjacent genes (cis-regulation) or distant genes from lncRNAs (trans-regulation). Here, we analyzed the potential global cis and trans-regulation of lncRNAs of yeast subjected to ethanol stress. For potential cis regulation, for BMA641-A and S288C strains, we observed that most lncRNA-neighbor gene pairs increased the expression at a certain point followed by a decrease, and vice versa. Based on the transcriptome profile and triple helix prediction between lncRNAs and promoters of coding genes, we observed nine different ways of potential trans regulation that work in a strain-specific manner. Our data provide an initial landscape of potential cis and trans regulation in yeast, which seems to be strain-specific., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

DEAD-box RNA helicases, a prominent subfamily within the RNA helicase superfamily 2 (SF2), play crucial roles in the growth, development, and abiotic stress responses of plants. This study identifies 146 DEAD-box RNA helicase genes (GhDEADs) and categorizes them into four Clades (Clade A-D) through phylogenetic analysis. Promoter analysis reveals cis-acting elements linked to plant responses to light, methyl jasmonate (MeJA), abscisic acid (ABA), low temperature, and drought. RNA-seq data demonstrate that Clade C GhDEADs exhibit elevated and ubiquitous expression across different tissues, validating their connection to leaf development through real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Notably, over half of GhDEADs display up-regulation in the leaves of virus-induced gene silencing (VIGS) plants of GhVIR-A/D (members of m 6 A methyltransferase complex, which regulate leaf morphogenesis). In conclusion, this study offers a comprehensive insight into GhDEADs, emphasizing their potential involvement in leaf development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (β-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.

Although the role of G-quadruplex (G4) DNA structures has been suggested in chromosomal looping this was not tested directly. Here, to test causal function, an array of G4s, or control sequence that does not form G4s, were inserted within chromatin in cells. In vivo G4 formation of the inserted G4 sequence array, and not the control sequence, was confirmed using G4-selective antibody. Compared to the control insert, we observed a remarkable increase in the number of 3D chromatin looping interactions from the inserted G4 array. This was evident within the immediate topologically associated domain (TAD) and throughout the genome. Locally, recruitment of enhancer histone marks and the transcriptional coactivator p300/Acetylated-p300 increased in the G4-array, but not in the control insertion. Resulting promoter-enhancer interactions and gene activation were clear up to 5 Mb away from the insertion site. Together, these show the causal role of G4s in enhancer function and long-range chromatin interactions. Mechanisms of 3D topology are primarily based on DNA-bound architectural proteins that induce/stabilize long-range interactions. Involvement of the underlying intrinsic DNA sequence/structure in 3D looping shown here therefore throws new light on how long-range chromosomal interactions might be induced or maintained., Competing Interests: SR, SB, SV, AS, CT, RK, SS, SC No competing interests declared, (© 2024, Roy et al.)

CCHC-type zinc finger proteins (CCHC-ZFPs), ubiquitous across plant species, are integral to their growth, development, hormonal regulation, and stress adaptation. Roses ( Rosa sp.), as one of the most significant and extensively cultivated ornamentals, account for more than 30% of the global cut-flower market. Despite its significance, the CCHC gene family in roses ( Rosa sp.) remains unexplored. This investigation identified and categorized 41 CCHC gene members located on seven chromosomes of rose into 14 subfamilies through motif distribution and phylogenetic analyses involving ten additional plant species, including Ginkgo biloba , Ostreococcus lucimarinus , Arabidopsis thaliana , and others. This study revealed that dispersed duplication likely plays a crucial role in the diversification of the CCHC genes, with the Ka/Ks ratio suggesting a history of strong purifying selection. Promoter analysis highlighted a rich presence of cis-acting regulatory elements linked to both abiotic and biotic stress responses. Differential expression analysis under drought conditions grouped the 41 CCHC gene members into five distinct clusters, with those in group 4 exhibiting pronounced regulation in roots and leaves under severe drought. Furthermore, virus-induced gene silencing (VIGS) of the RcCCHC25 member from group 4 compromised drought resilience in rose foliage. This comprehensive analysis lays the groundwork for further investigations into the functional dynamics of the CCHC gene family in rose physiology and stress responses.

Parallel-stranded G-quadruplex structures are found to be common in the human promoter sequences. We tested highly fluorescent 9-methoxyluminarine ligand (9-MeLM) binding interactions with different parallel G-quadruplexes DNA by spectroscopic methods such as fluorescence and circular dichroism (CD) titration as well as UV melting profiles. The results showed that the studied 9-MeLM ligand interacted with the intramolecular parallel G-quadruplexes (G4s) with similar affinity. The binding constants of 9-methoxyluminarine with different parallel G4s were determined. The studies upon oligonucleotides with different flanking sequences on c- MYC G-quadruplex suggest that 9-methoxyluminarine may preferentially interact with 3'end of the c- MYC promoter. The high decrease in 9-MeLM ligand fluorescence upon binding to all tested G4s indicates that 9-methoxyluminarine molecule can be used as a selective fluorescence turn-off probe for parallel G-quadruplexes.

Plant senescence is a highly coordinated process that is intricately regulated by numerous endogenous and environmental signals. The involvement of phytic acid in various cell signaling and plant processes has been recognized, but the specific roles of phytic acid metabolism in Arabidopsis leaf senescence remain unclear. Here, we demonstrate that in Arabidopsis thaliana the multiple inositol phosphate phosphatase (AtMINPP) gene, encoding an enzyme with phytase activity, plays a crucial role in regulating leaf senescence by coordinating the ethylene signal transduction pathway. Through overexpressing AtMINPP ( AtMINPP-OE ), we observed early leaf senescence and reduced chlorophyll contents. Conversely, a loss-of-function heterozygous mutant ( atminpp/+ ) exhibited the opposite phenotype. Correspondingly, the expression of senescence-associated genes (SAGs) was significantly upregulated in AtMINPP-OE but markedly decreased in atminpp/+ . Yeast one-hybrid and chromatin immunoprecipitation assays indicated that the EIN3 transcription factor directly binds to the promoter of AtMINPP. Genetic analysis further revealed that AtMINPP-OE could accelerate the senescence of ein3-1eil1-3 mutants. These findings elucidate the mechanism by which AtMINPP regulates ethylene-induced leaf senescence in Arabidopsis, providing insights into the genetic manipulation of leaf senescence and plant growth.

Cytokinins (CKs) are a group of phytohormones that are involved in plant growth, development, and disease resistance. The isopentenyl transferase (IPT) and cytokinin oxidase/dehydrogenase (CKX) families comprise key enzymes controlling CK biosynthesis and degradation. However, an integrated analysis of these two gene families in radish has not yet been explored. In this study, 13 RsIPT and 12 RsCKX genes were identified and characterized, most of which had four copies in Brassica napus and two copies in radish and other diploid Brassica species. Promoter analysis indicated that the genes contained at least one phytohormone or defense and stress responsiveness cis-acting element. RsIPTs and RsCKXs were expanded through segmental duplication. Moreover, strong purifying selection drove the evolution of the two gene families. The expression of the RsIPT and RsCKX genes distinctly showed diversity in different tissues and developmental stages of the root. Expression profiling showed that RsCKX1-1/1-2/1-3 was significantly upregulated in club-resistant materials during primary infection, suggesting their vital function in clubroot resistance. The interaction network of CKX proteins with similar 3D structures also reflected the important role of RsCKX genes in disease resistance. This study provides a foundation for further functional study on the IPT and CKX genes for clubroot resistance improvement in Raphanus .

Background: Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers., Results: In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter., Conclusion: Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts., (© 2024. The Author(s).)

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Friedhoff 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.)

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

Background: Increasing evidence suggests that a substantial proportion of disease-associated mutations occur in enhancers, regions of non-coding DNA essential to gene regulation. Understanding the structures and mechanisms of the regulatory programs this variation affects can shed light on the apparatuses of human diseases., Results: We collect epigenetic and gene expression datasets from seven early time points during neural differentiation. Focusing on this model system, we construct networks of enhancer-promoter interactions, each at an individual stage of neural induction. These networks serve as the base for a rich series of analyses, through which we demonstrate their temporal dynamics and enrichment for various disease-associated variants. We apply the Girvan-Newman clustering algorithm to these networks to reveal biologically relevant substructures of regulation. Additionally, we demonstrate methods to validate predicted enhancer-promoter interactions using transcription factor overexpression and massively parallel reporter assays., Conclusions: Our findings suggest a generalizable framework for exploring gene regulatory programs and their dynamics across developmental processes; this includes a comprehensive approach to studying the effects of disease-associated variation on transcriptional networks. The techniques applied to our networks have been published alongside our findings as a computational tool, E-P-INAnalyzer. Our procedure can be utilized across different cellular contexts and disorders., (© 2024. The Author(s).)

Human adenoviruses (HAdVs) are small DNA viruses that generally cause mild disease. Certain strains, particularly those belonging to species B HAdVs, can cause severe pneumonia and have a relatively high mortality rate. Little is known about the molecular aspects of how these highly pathogenic species affect the infected cell and how they suppress innate immunity. The present study provides molecular insights into how species B adenoviruses suppress the interferon signaling pathway. Our study shows that these viruses, unlike HAdV-C2, are resistant to type I interferon. This resistance likely arises due to the highly efficient suppression of interferon-stimulated gene expression. Unlike in HAdV-C2, HAdV-B7 and B14 sequester STAT2 and RNA polymerase II from interferon-stimulated gene promoters in infected cells. This results in suppressed interferon- stimulated gene activation. In addition, we show that RuvBL1 and RuvBL2, cofactors important for RNA polymerase II recruitment to promoters and interferon-stimulated gene activation, are redirected to the cytoplasm forming high molecular weight complexes that, likely, are unable to associate with chromatin. Proteomic analysis also identified key differences in the way these viruses affect the host cell, providing insights into species B-associated high pathogenicity. Curiously, we observed that at the level of protein expression changes to the infected cell, HAdV-C2 and B7 were more similar than those of the same species, B7 and B14. Collectively, our study represents the first such study of innate immune suppression by the highly pathogenic HAdV-B7 and B14, laying an important foundation for future investigations.IMPORTANCEHuman adenoviruses form a large family of double-stranded DNA viruses known for a variety of usually mild diseases. Certain strains of human adenovirus cause severe pneumonia leading to much higher mortality and morbidity than most other strains. The reasons for this enhanced pathogenicity are unknown. Our study provides a molecular investigation of how these highly pathogenic strains might inactivate the interferon signaling pathway, highlighting the lack of sensitivity of these viruses to type I interferon in general while providing a global picture of how viral changes in cellular proteins drive worse disease outcomes., Competing Interests: The authors declare no conflict of interest.

DNA methylation plays an important role in the development and tissue differentiation of eukaryotes. In this study, bisulfite sequencing (BS-seq) technology was used to analyze the DNA methylation profiles of liver tissues taken from Rongchang pigs at three postnatal feeding stages, including newborn, suckling, and adult. The DNA methylation pattern across the genomes or genic region showed little difference between the three stages. We observed 419 differentially methylated regions (DMRs) in promoters, corresponding to 323 genes between newborn and suckling stages, in addition to 288 DMRs, corresponding to 134 genes, between suckling and adult stages and 351 DMRs, corresponding to 293 genes, between newborn and adult stages. These genes with DMRs were mainly enriched in metabolic, immune-related functional processes. Correlation analysis showed that the methylation level of gene promoters was significantly negatively correlated with gene expression. Further, we found that genes related to nutritional metabolism, e.g., carbohydrate metabolism (FAHD1 and GUSB) or fatty acid metabolism (LPIN1 and ACOX2), lost DNA methylation in their promoter, with mRNA expression increased in newborn pigs compared with those in the suckling stage. A few fatty acid metabolism-related genes (SLC27A5, ACOX2) were hypomethylated and highly expressed in the newborn stage, which might satisfy the nutritional requirements of Rongchang pigs with high neonatal birth rates. In the adult stage, HMGCS2-which is related to fatty acid β-oxidation-was hypomethylated and highly expressed, which explains that the characteristics of high energy utilization in adult Rongchang pigs and their immune-related genes (CD68, STAT2) may be related to the establishment of liver immunity. This study provides a comprehensive analysis of genome-wide DNA methylation patterns in pig liver postnatal development and growth. Our findings will serve as a valuable resource in hepatic metabolic studies and the agricultural food industry.

Xanthomonas citri subsp. citri ( Xcc ), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1 . This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620 , which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1., Competing Interests: XC, HZ, TZ, WR, WW, XF No competing interests declared, (© 2024, Chen et al.)