Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms

The eukaryotic ribosomal genes are multi-copy genes, transcribed from the rDNA, and approximately one third of them is actively transcribed in differentiated cells. A number of lncRNAs have been identified from the intergenic spacer between the rRNA genes, among those the spacer RNA and PAPAS that are involved silencing of rRNA gene copies by altering the chromatin configuration. Here, we have identified lncRNAs that are transcribed from the human rDNA loci and modulate the loci; IGS38 positively regulates rRNA gene transcription by associating to the 47S rRNA gene promoter and modulating the rRNA promoter accessibility while IGS32as associates with heterochromatin. IGS38 binds to the 47S gene promoter through the RNA pol I factors TAF1C and RRN3 as well as the Williams Syndrome Transcription Factor (WSTF), a component of the B-WICH chromatin remodelling complex. The increased accessibility of the promoter stabilises the architectural protein Upstream Binding Factor (UBF) at the rRNA promoter, thereby facilitating RNA pol I promoter escape. Furthermore, IGS38 knock down displays and increased dsRNA abundance in the cytoplasm with a weak induction of the dsRNA sensor OAS2, typically induced by interferon and viral dsRNA. Overall, the both IGS38 and IGS32as are chromatin associated lncRNAs involved in rDNA chromatin changes, and IGS38 is stimulating, together with WSTF, rRNA gene transcription in human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/722362v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@14d4159org.highwire.dtl.DTLVardef@fd773forg.highwire.dtl.DTLVardef@a0030dorg.highwire.dtl.DTLVardef@1285301_HPS_FORMAT_FIGEXP M_FIG C_FIG IGS stabilises 47S rRNA transcription, disruption of IGS38 expression leads to the release of dsRNA in the cytoplasm and a weak immune activation of OAS2. Created by biorender (https://biorender.com/shortURL)

The prevalent transcriptional repressor NrdR binds to highly conserved prokaryotic sequences in the promoter regions of operons encoding the essential enzyme ribonucleotide reductase. The NrdR binding sites consist of two partially palindromic 16 bp sequences (NrdR boxes) separated by a 15-16 bp linker sequence. We have assessed the requirement of both boxes for binding, the propensity of different NrdRs to bind to heterologous binding sites, and that the linker sequence is only limited to length and not sequence conservation. As we have observed several deviations from the conserved sequences of the NrdR boxes, we here test the conservation requirements of individual basepairs in the NrdR boxes using a synthetic DNA fragment (Synt DNA) to which the NrdR proteins from the actinomycete Streptomyces coelicolor and the gammaproteobacterium Escherichia coli bind equally well as to their homologous binding sites. By introducing isolated mutations to Synt DNA and testing the binding capacity of NrdR from S. coelicolor and E. coli we expand our understanding of what criteria are needed to build a functional binding site for the NrdR repressor.

DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1{Delta} cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

OBJECTIVEThis study aimed to explore the role and underlying mechanism of microRNA-128 (miR-128) in regulating vascular remodeling in spontaneously hypertensive rats (SHRs), focusing on its targeting of peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}) and modulation of the Toll-like receptor 4/nuclear factor-{kappa}B (TLR4/NF-{kappa}B) inflammatory pathway. METHODSAll experimental procedures were approved by the Animal Care and Use Committee of Fujian Medical University. In vivo, ten-week-old male SHRs were randomly assigned to three groups: renal denervation (RDN, n=6), sacubitril/valsartan (Sac/Val, n=6), and Sham (n=6). Age-matched Wistar-Kyoto (WKY) rats served as normotensive controls (n=6).Eight weeks after intervention, mesenteric arteries were harvested for histological, functional, and molecular analyses. Serum miR-128 levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of key proteins in the vascular wall were assessed via immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting (WB). Bioinformatics analysis and RNA sequencing (RNA-seq) were employed to identify core genes and signaling pathways associated with hypertension-induced pathological inflammation. RESULTSIn vivo, in the SHR sham-operated group, elevated blood pressure, severe vascular remodeling, and impaired vasodilatory function were observed, accompanied by downregulated miR-128 expression and upregulated TLR4/NF-{kappa}B signaling activity (all p < 0.0001).RDN postoperative, miR-128 expression was significantly restored, which in turn inhibited the TLR4/NF-{kappa}B pathway, reduced the production of pro-inflammatory cytokines (including IL-1{beta}, IL-6, and TNF-), and ameliorated vascular dilation dysfunction in SHRs (all p < 0.0001). Mechanistically, miR-128 negatively regulated the TLR4/NF-{kappa}B signaling pathway while upregulating the expression of PPAR-{gamma} (p < 0.05). CONCLUSIONRDN not only exerts a hypotensive effect but also improves hypertensive vascular remodeling. miR-128 inhibits excessive inflammation in vascular smooth muscle cells and alleviates vascular remodeling in SHRs via the PPAR-{gamma}/TLR4/NF-{kappa}B axis. These findings identify miR-128 as a potential therapeutic target for RDN in the treatment of hypertension, providing a novel regulatory strategy for the precision management of cardiovascular diseases.

In wheat, weak seed dormancy (SD) is related to an increased tendency for pre-harvest sprouting (PHS), which reduces yield and quality. However, the molecular mechanism underlying SD remains elusive. Here, we identified a wheat R2R3-MYB transcription factor (TaMYB83-7B) related to SD. Expression analysis showed that TaMYB83-7B was highly expressed in wheat seeds, and was more highly expressed in strong-dormancy varieties than in weak-dormancy varieties. Sequence and association analysis indicated that T/C mutations at -907 bp and -1133 bp in the TaMYB83-7B promoter were significantly associated with wheat SD, with C at both sites related to strong dormancy. Dual-luciferase reporter assays demonstrated that the transcriptional activity of the TaMYB83-7B promoter was significantly higher in strong-dormancy varieties than in weak-dormancy varieties. Further analyses indicated that TaMYB83-7B functions as a transcriptional inhibitor. Germination experiments revealed that overexpression of TaMYB83-7B significantly enhanced SD, while its loss-of-function reduced SD. Finally, TaMYB83-7B was found to regulate SD by influencing the balance between abscisic acid (ABA) and gibberellin (GA) in wheat seeds. Overall, the results of this study enhance our understanding of the complex regulatory mechanism underlying SD, and provide gene targets and molecular markers for the genetic improvement of PHS resistance in wheat.

WD40 domains share a widespread {beta}-propeller fold, and often act as versatile scaffold proteins. Despite their central role in organizing dynamic cellular complexes, the molecular and structural mechanisms of many WD40 proteins remain poorly understood. Among them, DCAF7, an ubiquitously expressed and essential gene in human, also encodes a highly conserved WD40 protein in eukaryotic organisms. It is known to interact with multiple and functionnally diverse partners to coordinates cellular activity of several protein kinases as well as transcriptional regulators, thereby modulating key cellular processes such as cell growth, differentiation, and transcriptional regulation. However, the precise mode of action of DCAF7 is unknown and its important divergence in sequence from better characterize WD40 prevent information transfer by similarity. Structural interactomic can reveal how protein-protein interactions (PPIs) occur within an organism and are essential for understanding biological functions and developing new therapeutic strategies. Using SLiMAn2, AlphaFold2/3 and PSSMsearch, we identified a conserved -helical short linear motif (SLiM) in several well known DCAF7 partners that binds to the top surface of its {beta}-propeller. This motif was subsequently used to generate a regular expression, to identify potential new direct binders across the DCAF7 meta-interactome and the human proteome. Domain-domain interactions were also predicted for some other partners. Finally, modeling of oligomeric complexes with such new hits reveals the structural basis of DCAF7 scaffolding, with links to neurodevelopmental disorders such as autism.

Enhancers are key epigenetic regulatory elements that orchestrate spatiotemporal gene expression and are critical in mammalian development, gene regulation, and disease. Histone modifications such as H3K4me1 (a canonical enhancer mark) and H3K27ac (which distinguishes active enhancers) remain poorly characterized during early mammalian embryogenesis. Using low-input CUT&RUN (Cleavage Under Targets and Release Using Nuclease) with input as low as 50 cells, this study profiles genome-wide H3K4me1 and H3K27ac patterns in mouse oocytes and pre-implantation embryos. Both marks are enriched in distal regions and exhibit distinct sequence preferences and reprogramming dynamics in pre-implantation embryos. H3K27ac is reprogrammed at the 2-cell stage and marks active enhancers, while H3K4me1 is remodeled at the 4-cell stage and co-localizes with H3K27ac, overlapping with accessible chromatin regions. Interestingly, the co-localization of H3K4me1 and H3K27ac is also detected in promoter regions, where they exhibit a mutually exclusive pattern with H3K4me3. Three enhancer types-active (H3K4me1/H3K27ac), primed (H3K4me1), and poised (H3K4me1/H3K27me3)-are dynamically remodeled during maternal-to-zygotic transition (MZT), with active enhancers increasing significantly after zygotic genome activation. Furthermore, genome-wide super-enhancers are identified and mainly enriched in promoters. The differences in gene expression at different stages may be related to the specific motifs enriched by super-enhancers.

Cells employ a bevy of transcriptional and post-translational stress responses to tolerate the burden of misfolded proteins induced by stress. In particular, the heat shock response facilitates the upregulation of molecular chaperones and protein remodeling factors that mediate proteostasis in response to accumulated misfolded proteins in the nucleus and cytosol. However, in response to stress neurons struggle to induce a canonical heat shock response, highlighting our poor understanding of how neurons maintain proteostasis. Specifically, the ability of post-mitotic respiring cells to regulate the heat shock response in comparison to their rapidly dividing, predominantly glycolytic counterparts has been under-studied. In this study, we employ yeast models that are easily manipulated to generate energy via glycolysis or mitochondrial respiration by changing the carbon source in the media. Using this model, we demonstrate that Hsf1 activity, the heat shock response and proteostasis are impaired in respiring cells. Interestingly, our data show that reduced Hsf1 activity regulates viability of respiring cells, with respiring cells poorly tolerating constitutively activated Hsf1. Finally, we describe alternative post-translational programming of the molecular chaperones Hsp70 and Hsp104 that plausibly enables respiring cells to mediate proteostasis despite a dampened heat shock response. Our findings offer new insights into possible proteostatic strategies employed by cells in different metabolic conditions.

Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 hours post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression. HighlightsPrdm3 and Prdm16 associate with nucleosomes for regulation of gene expression Gene targets are altered in prdm3 and prdm16 mutant zebrafish Reduced binding is observed in respective mutants

Existing databases of interphase chromosome conformations typically store three-dimensional coordinates of genomic segments. However, since interphase chromatin is highly dynamic, such databases are dominated by transient configurations and unstructured regions, whose positions vary continuously between cells and over time, unlike folded proteins such as globin, which adopt similar structures in every cell. These drawbacks motivated the inception of a database based on strion (a portmanteau of a string capturing structure and function). A strion concisely describes the structure and activity of all transcription units in one cell, by retaining only functionally relevant positional information. Sets of strions describing structures in different cells sampled at different times are compiled into a super-strion. Then, 46 super-strions summarise the range of structure and activity of a human cell type, including information on all transcription units, how often each co-fires and co-clusters with others in transcription factories/hubs, enhancer interactomes and small-world expression networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/724942v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@13a1263org.highwire.dtl.DTLVardef@18d2c78org.highwire.dtl.DTLVardef@162865corg.highwire.dtl.DTLVardef@1631d65_HPS_FORMAT_FIGEXP M_FIG C_FIG

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

Histone lactylation is a recently identified histone post-translational modification (PTM) that links energy metabolism to chromatin regulation. Although histone lactylation has been implicated in transcriptional activation, its function in meiotic chromatin remains unclear. Previously, we identified enrichment of multiple histone lactylation marks within the meiotic karyosome, a highly condensed and transcriptionally repressive chromatin structure formed in Drosophila oocytes. Here, through an RNAi-based screen, we identified the CBP family protein dCBP as a regulator of histone lactylation in the karyosome. Germline-specific knockdown of dCBP preferentially reduced histone lactylation, including H4K8 lactylation, and caused premature disruption of the synaptonemal complex, abnormal egg chamber development with excess nurse cells, reduced egg production, and decreased embryonic viability. Corresponding histone acetylation marks were comparatively less affected than histone lactylation by dCBP knockdown. Together, our findings provide evidence that dCBP-mediated histone lactylation contributes to meiotic chromosome maintenance and suggest a potential link between energy metabolism and meiotic chromatin regulation.

Bdelloid rotifers are known to survive desiccation and high doses of ionizing radiation. This extreme resistance is notably due to their capacity to cope with numerous DNA double-strand breaks (DSBs). Genes encoding key components of the non-homologous end joining (NHEJ) DNA repair pathway are strongly upregulated in the bdelloid rotifer Adineta vaga following exposure to ionizing radiation. Considering the notably high doses tolerated by these organisms, their capacity to efficiently restore genome integrity is particularly striking. Although NHEJ is generally regarded as less accurate than homologous recombination (HR), the absence of major genomic rearrangements in the descendants of irradiated rotifers suggests that DNA repair occurs with high fidelity. Terwagne et al. recently reported a delayed repair in germline nuclei, occurring during oocyte development when homologous chromosomes pair, thereby enabling template-based repair through HR. In this study, we established an in situ hybridization approach on A. vaga cryosections to investigate the spatial and temporal expression of key actors involved in NHEJ, HR, and Base excision repair (BER) pathways in somatic and germline tissues. We show that NHEJ (KU80) and BER-related genes (PARPs) as well as A. vaga Ligase E (putatively involved in DNA repair) are expressed early after radiation exposure in the somatic syncytium. In contrast, HR-related genes (Rad51: two paralogs, Rad54), as well as PCNA (involved in DNA replication, NER, BER, HR) are expressed later in maturing oocytes, indicating the activation of a delayed homologous recombination repair pathway in germline nuclei. Nurse cells, which express genes associated with both HR and NHEJ pathways, may rely on both mechanisms for their own DNA repair while also supplying mRNAs to the maturing oocyte. Our results provide new evidence for a differential regulation of DNA DSB repair pathways between soma and germline in bdelloids, with NHEJ predominating in somatic tissues and HR in the germline of A. vaga. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722046v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3b1f3borg.highwire.dtl.DTLVardef@17f5eb5org.highwire.dtl.DTLVardef@122ef14org.highwire.dtl.DTLVardef@7e4413_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure:C_FLOATNO Summary of in situ hybridization results: genes coding for actors of NHEJ are expressed in the somatic nuclei and in the nurse nuclei of Adineta vaga individuals 2.5 hours post X-rays radiation, while genes coding for HR actors and PCNA (involved in multiple pathways including DNA replication and DNA repair: NER, BER, MR, HR) are expressed in the nurse nuclei 2.5 hours post radiation, and later in the maturing oocyte during oogenesis and in the laid eggs. Genes coding for actors highly expressed post-radiation, involved in the BER pathway appear to be only expressed in the somatic syncytium 2.5 hours post radiation, as well as the gene coding for the Ligase E, likely involved in DNA repair. C_FIG

TAR DNA-binding protein 43 (TDP-43) is a multifunctional DNA/RNA-binding protein implicated in transcriptional and post-transcriptional regulation. Dysregulation of TDP-43 is closely correlated with human diseases such as cancer and neurodegenerative diseases. Although its roles in RNA metabolism are well characterized, its function in transcriptional regulation remains largely underexplored. DNA G-quadruplexes (dG4s) are non-canonical nucleic acid structures enriched at gene promoters and regulatory elements, where they facilitate chromatin looping and gene transcription. Here, we investigated the transcriptional regulatory role of TDP-43 by integrating multi-omics datasets, including Hi-C, dG4 ChIP-seq, TDP-43 ChIP-seq, RNA-seq and ATAC-seq from K562 and HepG2 cells. Our analyses demonstrate TDP-43 binding and dG4s formation are highly colocalized at chromatin loop anchors, particularly at promoter and enhancer regions. TDP-43 occupancy at these anchors correlates with increased dG4 stability, chromatin loop interaction frequency, elevated chromatin accessibility, and upregulated gene expression. Morover, TDP-43 knockdown in HepG2 cells revealed a significant reduction in dG4 formation and loop interaction strength, accompanied by widespread transcriptional dysregulation. Collectively, our findings highlight a novel regulatory role of TDP-43 in facilitating long-range chromatin interactions and transcriptional activation through binding to and stabilizing dG4 structures, providing a mechanistic basis for gene dysregulation driven by TDP-43 dysfunction in diseases.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Zinc is an essential trace element involved in numerous biological processes, including cellular signalling, development, and reproduction. Zinc homeostasis is regulated by zinc transporters, yet the physiological roles of many transporters remain poorly understood in vivo. Here, we investigated the function of the zinc transporter ZIP9 (SLC39A9) using a zebrafish (Danio rerio) knockout model. Elemental imaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) revealed altered zinc distribution in zip9-deficient larvae. Synchrotron-based X-ray fluorescence (XRF) imaging further showed reduced zinc levels in the brain region of mutant zebrafish. Consistent with these observations, loss of zip9 was associated with altered expression of key neuroendocrine genes within the hypothalamic-pituitary-gonadal (HPG) axis. Zip9 mutant females exhibited disrupted ovarian follicle development, reduced spawning rates, and decreased egg production. In addition, embryos derived from zip9 mutant parents displayed reduced size, impaired early development, and decreased survival. Together, these findings identify ZIP9 as a regulator of zinc distribution in vivo and suggest that ZIP9-mediated zinc signalling contributes to reproductive regulation in zebrafish.

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

BackgroundMetazoan adenosine-to-inosine (A-to-I) mRNA editing temporospatially diversifies the neuronal transcriptome and proteome. The limited read length from next-generation sequencing (NGS) constrains the quantification of the potentially differential editing levels across different splicing isoforms, restricting our understanding of the extent to which RNA editing contributes to molecular diversity and its interplay with splicing. MethodsWe employed reverse transcription nested PCR (RT-nPCR) and developed a novel interfering-Primer PCR (iPrimer PCR) technique to distinguish different transcripts of any gene. We selected multiple essential genes exhibiting RNA editing in coding sequences (CDSs) or untranslated regions (UTRs) for isoform-specific amplification and Sanger sequencing. ResultsNine different Adar isoforms together with pre-mRNA had distinct editing levels at the S>G auto-recoding site, which was predicted to have isoform-specific effects on catalytic activities. Although pre-mRNA editing might exert isoform-dependent promotion/suppression of splicing, closely located editing sites, such as those in neuronal genes qvr and stj, still exhibited high correlation in editing levels due to co-editing. iPrimer strategy further discovered differential recoding levels between the long/short 3UTR isoforms of gene jef. ConclusionsWe provide the first comprehensive solution for isoform-specific PCR amplification of any gene, enabling quantification of RNA editing level of different isoforms. Our results offer insights into how RNA editing interplays with splicing, and highlight its complicated role in expanding molecular diversity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725286v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ebc82org.highwire.dtl.DTLVardef@1ea365dorg.highwire.dtl.DTLVardef@1971aceorg.highwire.dtl.DTLVardef@160d053_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed isoform-specific PCR followed by Sanger sequencing, and achieved the quantification of differential RNA editing levels in different transcripts of a gene.

The nuclear receptor superfamily is comprised of ligand-regulated transcription factors that contain an intrinsically disordered domain at the amino-terminal end, known as the N-terminal domain (NTD). While this poorly conserved domain is known to possess ligand-independent activation function (AF-1), few NTD functions are conserved between nuclear receptors (NRs). Identified roles in other receptors include androgen receptor (AR), estrogen receptor (ER) and mineralocorticoid receptor (MR). Here, we aim to define the function of the NTD of the farnesoid X receptor (FXR), a crucial regulator of lipid and bile acid metabolism. We show that the NTD engages in interdomain contact with other FXR domains. We also observe that the NTD interacts directly with coregulator proteins. Using mutagenesis, mammalian two-hybrid assays and molecular dynamics simulations, we identify and validate a novel SXXLF motif in the NTD which mediates interactions with both coregulators and the ligand binding domain. Mutation of the motif induces large changes in conformational and allosteric coupling in FXR. Our study identifies a new nuclear receptor-interacting motif that modulates the transcriptional activity of FXR. Graphical AbstractFXR-NTD regulates transcriptional activity through interdomain communication with the LBD and is also involved in co-activator recruitment. The SENLF motif is the first defined functional element within the FXR-NTD and mediates both NTD-LBD interaction and selective co-activator engagements to drive NTD-mediated transcriptional activity. O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/724725v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@5a37aorg.highwire.dtl.DTLVardef@2fa9e1org.highwire.dtl.DTLVardef@13a19daorg.highwire.dtl.DTLVardef@1775ed2_HPS_FORMAT_FIGEXP M_FIG C_FIG