Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms

Adipose tissue is a key metabolic organ for carbohydrate and lipid metabolism. Multiple nutrient sensing pathways and metabolic enzymes operate in adipocytes to control energy production and lipid mobilization in response to physiological conditions. Important regulators of glycolytic and lipid metabolism in many species are members of the estrogen-related receptor (ERR) family of nuclear receptors. We previously showed that ERR is required for transcriptional regulation of glycolytic and pentose phosphate pathway enzymes in adult Drosophila females, consistent with what is observed in ERR mutant males and larvae. However, the cell-type specific targets of ERR in adipose and other tissues have not been fully elucidated. Here, using a modified targeted DamID approach (NanoDam), we identified ERR occupancy specifically in adult adipocytes at the loci that encode genes involved in glycolysis, the pentose phosphate pathway, and fatty acid metabolism. Overall, our results predict that ERR centrally functions in adipose tissue to regulate multiple metabolic pathways.

Chromosomes in Drosophila melanogaster are organized into distinct topologically associated domains delimited by boundaries bound by insulator proteins. The insulator protein BEAF (Boundary Element-Associated Factor of 32kDa) plays roles in both chromatin organization and transcriptional regulation, yet its precise molecular mechanisms remain elusive. To examine the role of BEAF in insulator function we used yeast 2-hybrid assays, pull-down assays with bacterially expressed proteins, and bimolecular fluorescence complementation assays in S2 cells to characterize interactions between BEAF and three co-insulator proteins: CP190, Pzg, and Chro. Our studies pinpoint minimal regions of CP190, Pzg, and Chro that directly interact with BEAF, as well as parts of BEAF that are crucial for its interactions with these co-insulator proteins. Functional analyses in transfected S2 cells revealed distinct regulatory roles for BEAF in association with CP190, Pzg, and Chro. CP190 showed a weak interaction with BEAF, and CP190 bound 2.3 kb upstream of promoter-proximal BEAF could not loop out the intervening DNA to effectively communicate with BEAF for luciferase reporter gene activation. In contrast, more robust interactions were observed between BEAF and both Pzg and Chro. Both could also effectively interact with BEAF from a distance to activate the reporter gene. It is likely that the role of CP190 in long-range insulator interactions is mediated by insulator proteins other than BEAF, although BEAF could help stabilize interactions. On the other hand, we propose that BEAF can directly collaborate with Pzg and Chro to mediate long-range chromatin interactions.

Limited oxygen supply or hypoxia can impair fetal development and lead to developmental disorders, but the molecular mechanism underlying this phenomenon remains poorly understood. It is also well known that hypoxia results in transcriptomic alterations and epigenetic reprogramming. Drosophila melanogaster (fruit fly) has been used for decades as a powerful model to dissect the molecular mechanisms regulating development. To better understand the role of early hypoxic stress on development, we performed single-cell joint analysis of chromatin accessibility and transcriptome to characterize the influence of hypoxia on Drosophila embryonic development. We identified hypoxia-induced alterations in both gene expression and chromatin accessibility across 22 cell groups, especially in the genes regulating organogenesis and development of neuronal, tracheal, and muscular systems, including a reduction of germ cells, suggesting a long-lasting influence of hypoxic stress at an early embryonic stage on development and reproduction. In summary, this study demonstrates that early embryonic hypoxia induces cell type- and dose-dependent changes in chromatin accessibility and gene expression, leading to distinct developmental phenotypic responses, such as reduced number of germ cells under both 3% and 5% O2. We further conclude that the tramtrack (ttk) gene is critical in germ cell development and reproduction in Drosophila melanogaster.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Protein homeostasis - i.e., proteostasis - is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In this work, we show that the Rqc1p protein is necessary for the thermoadaptation of S. cerevisiae to heat shock, as RQC1-deficient yeast is sensitive to elevated temperatures. In silico approaches - such as multiple sequence alignment, structural analysis, and molecular dynamics simulations - confirmed earlier predictions that Rqc1p shares characteristics with the bHLH family of proteins. We also verified, through computational prediction of sub-cellular localization, that S. cerevisiae Rqc1p contains nuclear localization signals, suggesting that this protein can potentially be translocated toward the nucleus, thereby broadening its current range of recognized biological functions in this organism. Also, analysis of yeast transcriptomes subjected to heat shock showed that Rqc1p mRNA levels do not fluctuate in response to heat shock, suggesting that cellular concentrations of Rqc1p are already at optimal levels to elicit a rapid and effective response during thermal stress in S. cerevisiae.

BackgroundHeterochromatic long arm of mouse Y chromosome harbors the multicopy species-specific sequences Ssty, Sly, Asty and Orly that are transcribed in testis and have known functions in male fertility. Of these Ssty and Sly encode proteins - yet all the transcripts are not translated. To investigate the roles of these Y-heterochromatic transcripts further, we analyzed them. MethodsMice with 2/3rd deletion of the Y-chromosome (XYRIIIqdel) and its wild type (XYRIII) were used in this study. Bioinformatic approaches, small RNA northern blots, Electrophoretic Mobility Shift Assays, Luciferase reporter assays, dPCR analysis, RT-qPCR assays and western blotting techniques were used to identify piRNAs that regulate autosomal genes. ResultsWe demonstrate that the multicopy gene families from mouse Y-long arm generate piRNAs predominantly in testis. We observed sequences homologous to these piRNAs in the UTRs of a few autosomal genes, which are differentially expressed in the sperms of XYRIIIqdel mice. Furthermore, the Endogenous Retrovirus Element (ERV) LTR, found in the Orly1 transcript identified piRNAs in the database, showed homology to UTRs and associated genomic regions of a few autosomal genes. Orly1 showed a reduction in genomic copy number by digital PCR in XYRIIIqdel mice. One of the four autosomal genes containing the ERV segment in their UTRs, showed a differential testicular protein expression in the mutant mice. ConclusionsThus, we further elucidate that different classes of repeats from Y-chromosome regulate autosomal gene expression via piRNAs. Besides, this study also identified novel roles for a Y-derived ERV in autosomal gene regulation in testis.

The Tbx1 gene is haploinsufficient in mice and in humans, where it causes a DiGeorge syndrome phenotype characterized by developmental deficits of the pharyngeal apparatus. TBX1 plays a critical role in the differentiation and regionalization of the cardiopharyngeal mesoderm lineage and its derivatives. Nevertheless, its regulation is incompletely understood. Here we used a combination of computational and wet-lab approaches to identify regulatory sequences of the Tbx1 gene, and we use single-cell molecular analysis as a read-out and to establish the consequences of their deletion. Results revealed a cluster of regulatory sequences with at least three distinct elements. Elimination of the entire cluster caused a near shut down of the gene, while individual deletions had milder, quantitative effects. Transcriptomic analyses of the deletion mutants revealed the down regulation of genes related to cardiopharyngeal lineage specification and, more surprisingly, up regulation and anteriorization of genes related to embryonic patterning, thereby providing a rationale for the severe dysmorphogenesis of the posterior pharyngeal apparatus observed in Tbx1 mutant mice.

Trehalose is a primary circulating sugar in insects and essential for energy homeostasis, yet its non-coding RNA-based regulatory circuitry remains enigmatic. Here, we characterize a conserved long non-coding RNA, lncRNA1, as a post-transcriptional regulator of trehalose-glucose homeostasis in Lepidoptera. lncRNA1 encodes a structurally stable, pseudoknot-containing transcript that is strongly induced upon trehalose pathway perturbation and exhibits a reciprocal developmental expression pattern relative to the trehalose metabolism enzymes. RNAi-mediated silencing of lncRNA1 in Helicoverpa armigera elevates TPS/TPP and Treh transcript abundance, increases enzyme activities, reduces haemolymph trehalose, raises glucose. This drives broad transcriptomic and metabolomic reprogramming of carbohydrate, lipid, and growth-signalling pathways, resulting in accelerated larval growth. Overexpression of lncRNA1 reverses these phenotypes. Mechanistically, lncRNA1 physically associates with TPS/TPP and Treh mRNAs through evolutionarily conserved sequence motifs, modulating their post-transcriptional dynamics. Targeted deletion of these motifs abolishes regulatory activity and disrupts metabolic homeostasis. This regulatory axis is functionally conserved in Spodoptera frugiperda, validated across loss-of-function, gain-of-function, and cell-based systems. Our findings reveal a conserved lncRNA-based layer of post-transcriptional control over insect energy metabolism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/738560v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@f1c170org.highwire.dtl.DTLVardef@1d2a9c7org.highwire.dtl.DTLVardef@1886786org.highwire.dtl.DTLVardef@44b83b_HPS_FORMAT_FIGEXP M_FIG C_FIG

In TMPRSS2 fusion-positive prostate cancer, ERR is involved in regulation of ERG and promotes the androgen receptor independent signaling in the cancer progression. The ERR binds to the ERREs (estrogen-related receptor response elements) present at -5042 bp of the TMPRSS2- promoter and enhances the ERG overexpression that causes prostate cancer progression. To dissect the structural basis of the ERR recognition to the TMPRSS2 promoter DNA, we have purified the full-length ERR (ERRFL), NTD deleted construct (ERR{Delta}NTD), and the DNA-binding domain (ERRDBD) proteins and performed the binding analysis with 30 bp TMPRSS2-promoter DNA (5' -AGTCCAAGGTCGGTGGATC ACAAGGTCAGG-3'). Circular dichroism analysis showed that all three ERR proteins adopt native secondary structures. DNA binding induced subtle changes in the secondary structures, while enhancing the thermal stability (Tm) of all ERRa proteins. Binding analysis showed that ERRDBD bound weakly to the DNA, whereas ERRFL and ERR{Delta}NTD exhibited substantially higher affinities ~120-fold and ~131-fold than ERRaDBD, respectively. Small-angle X-ray scattering (SAXS) analyses revealed a dimeric ERRFL structure and an ERRFL-DNA complex (2:1) structure in solution and fitted well with Alpha Fold model of apo and DNA bound complex of ERRFL. Furthermore, 100 ns dynamics simulations on apo and DNA-bound ERRa proteins showed that all proteins remained structurally stable, with flexibility largely confined to loop regions of ERRa proteins. Our biophysical, DNA binding and structural analyses have revealed the mechanism involved in ERR recognition of the TMPRSS2- promoter DNA, which provides insight into ERR-mediated transcriptional regulation and development of anticancer drugs against ERR-driven prostate cancer.

Epigenetic regulation of chromatin dynamics via histone acetylation is one of several mechanisms by which eukaryotes regulate gene expression, DNA replication and repair, and maintain genome stability. This function is performed by histone acetyltransferases (HATs). Rtt109 is one such cytoplasmically localized HAT required for H3K56 acetylation found exclusively in fungi. Using recombinantly expressed Candida albicans Rtt109 and its chaperones, Vps75 and Asf1, we show that it can acetylate a 20-residue N-terminal H3 peptide in a coupled HAT assay only in the presence of Vps75, but not in the presence of Asf1 in vitro. This appears to be due to the fact that Rtt109-Vps75 is a high affinity stable complex, as estimated by biolayer interferometry (BLI) and gel filtration studies. The HAT activity of the Rtt109-Vps75 complex necessarily requires a flexible 118-160 residue loop of Rtt109 but not the C-terminal domain of Vps75. These results are comparable with what has been observed for the Saccharomyces cerevisiae Rtt109 homolog. In silico screening of 1,350,000 molecules from Life Chemicals Databases identified some likely inhibitors of C. albicans Rtt109 and six of them tested for binding to Rtt109 using BLI. The best ligand, F2368-0266, was used to study its effect on steady state enzyme kinetics, and found to be a competitive inhibitor of the peptide substrate but not of acetyl-CoA. Given the importance of Rtt109 in regulating virulence attributes such as hyphal morphogenesis and GPI biosynthesis in Candida albicans, and its effect on fungal pathogenesis, these results have significant clinical implications.

BACH1 is a heme-regulated basic-leucine-zipper containing transcriptional repressor that binds its DNA recognition elements as a heterodimer with MAFK. Heme-binding is thought to be mediated by several Cys-Proline (CP) motifs and this results in dissociation of the heterodimer from DNA. The mechanism of heme-binding and heme-mediated DNA dissociation remains unresolved. We have used UV-visible spectroscopy, 2D-NMR and DNA-binding assays to explore both heme-binding and DNA dissociation of a minimal BACH1 construct containing 2 CP motifs (C492(CP5) and C646(CP6)) flanking the DNA-binding domain. We find that heme is able to bind to both CP motifs, but also to other non-CP cysteines and histidines in the construct. Using NMR spectroscopy, we identify a structured binding pocket in which heme interacts with both C646(CP6) and Cys621. However, DNA-binding assays show that C646(CP6) is not required for heme-mediated DNA dissociation of the BACH1:MAFK heterodimer. Using UV-visible spectroscopy we show that C492(CP5) also recruits heme with a second ligand, a conserved histidine, His559, in the BACH1 DNA-recognition helix. Mutation of C492(CP5) reduces but does not abolish heme-mediated dissociation from DNA. Our findings suggest a mechanism for heme-binding to BACH1 and heme-mediated dissociation from DNA.

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

HighlightsO_LIIdentified hundreds of potential chromatin-associated circRNAs in HEK293 cells, H9, and HeLa cells C_LIO_LIThe first report suggesting global interaction of circular RNAs with chromatin C_LIO_LIChromatin-associated circular RNAs interact with various RBPs involved in RNA splicing or processing C_LI Circular RNAs (circRNAs) have emerged as novel regulators of gene expression by interacting with various proteins and RNAs in a spatiotemporal manner. CircRNAs localized in the cytoplasm regulate mRNA translation or stability by binding to microRNAs and RNA-binding proteins (RBPs), while circRNAs in the nucleus regulate transcription and pre-mRNA splicing by associating with transcription factors and splicing factors. In this study, we sought to explore the interaction between circRNAs and chromatin. Analyzing published RNA-seq data from chromatin fractions identified hundreds of chromatin-associated circRNAs (cacRNAs) in various human cells. We validated the enrichment of a subset of circRNAs in the chromatin fraction and established the direct interaction of circDYNC1H1 and circKIF2C with chromatin in HEK293T cells. Furthermore, cacRNAs were found to interact with RBPs. Together, our research demonstrates the global association of hundreds of circRNAs with chromatin and expands our understanding of novel functional aspects of the circRNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/739476v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@1fc8831org.highwire.dtl.DTLVardef@516cdcorg.highwire.dtl.DTLVardef@1c20395org.highwire.dtl.DTLVardef@79313a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Thermostable DNA polymerases are indispensable tools in molecular biology, yet enzymes from the most extreme hyperthermophiles remain largely uncharacterized. Here, we report the biochemical and structural characterization of a family B DNA polymerase from Pyrolobus fumarii A1 (Pyrfu pol), one of the most thermoresistant archaea described to date. The enzyme was efficiently overproduced in E. coli Rosetta 2(DE3)[pLysS] and purified to homogeneity using a two-step protocol that combined heat treatment with immobilized metal affinity chromatography (IMAC). Bioinformatic analysis confirmed the canonical family B architecture, while AlphaFold-based structural modeling and comparative analysis with mesophilic RB69 DNA polymerase revealed a well-conserved structural core alongside thermoadaptive features. Radiolabel incorporation assays demonstrated enzymatic activity over a broad ionic strength range and an absolute requirement for Mg ions. PCR-based optimization confirmed these findings and revealed broad pH tolerance (6.5-11.0). Notably, Tris inhibited radiolabel-based assays (pH 7.0) yet proved essential for efficient PCR amplification (pH 8.5), suggesting a context-dependent role of buffer composition in polymerase activity. Processivity assays confirmed amplification of DNA fragments up to approximately 8,000 bp. Replication fidelity, assessed by the lacZ-based assay, showed a 2.9-fold improvement over Taq polymerase. Urea-nanoDSF yielded an exceptional melting temperature of 105.9 {+/-} 0.08 {degrees}C. Pyrfu pol also demonstrated tolerance to common PCR inhibitors, highlighting its potential utility in molecular biology applications.

RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remain unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes or transcriptomes of four snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus, and Lymnaea staginalis. Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all four species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient (SID) genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for dsRNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2, and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in trematode intermediate hosts as an experimental tool and potential control method.

Kissing complexes involve interactions between complementary loops of two RNA hairpins. These complexes are continously found in RNA structures and are essential for several biological processes, including the dimerization of retroviral RNA genomes, regulation of plasmid copy number, and small RNA-based control of mRNA translation. We previously identified novel RNA-RNA and RNA-DNA kissing complexes through the in vitro selection of oligonucleotides from synthetic libraries. In order to expand the use of kissing complexes to aptamers and DNA nanotechnology, we describe the selection and characterization of DNA-DNA kissing complexes and their application in the development of DNA adenosine aptasensors. Four DNA/DNA kissing complexes were identified from SELEX and characterized by EMSA and fluorescence anisotropy. The couple dk25/dk26 was further modified to generate an adenosine-triggered aptaswitch, where the addition of adenosine selectively induces the dissociation of loop-loop interactions. This paves the way for a new class of nucleic acid modules, which could be useful for the design of controlled nanostructure assembly and disassembly.

Precise regulation of Drosophila Myc is essential for growth and homeostasis, yet regulation of its transcriptional control remains incompletely understood. We investigated the Myc cis-regulatory landscape using in vivo reporter assays, EMSA, and LC-MS/MS-based identification of DNA-associated proteins. By truncating Myc cis-regulatory modules (CRMs), we delineated the activity of conserved non-coding elements across adult female tissues and larval stages. Specific DNA-protein interactions were confirmed by EMSA using nuclear embryonic extracts. We developed a Solid Surface Magnetic Enrichment protocol (SSMEP) to pull down DNA-protein complexes formed on Myc cis-elements. Affinity purification followed by LC-MS/MS enabled the identification of candidate transcriptional regulators associated with Myc-CRMs. This integrative approach provides new insights into promoter structure and trans-regulatory architecture of Myc and their roles in developmental gene expression programs. Our study identifies a distal enhancer required for larval and pupal patterning, with activation dependent on specific spacing relative to the TATA-box core promoter, a strong enhancer cluster within 5'-UTR cis-elements active in ovaries and embryos, and a DPE-core promoter requiring nearby enhancer action. The DPE-linked enhancer can function with both Inr-DPE and TATA-box promoters.The identified Myc-CRMs interact with conserved signaling pathways to tightly control Myc during development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/736081v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@fa98a9org.highwire.dtl.DTLVardef@3c82d3org.highwire.dtl.DTLVardef@b1025corg.highwire.dtl.DTLVardef@111e972_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMyc Oocyte Element: an eRNA-producing enhancer in ovaries and early embryos C_LIO_LIDPE promoter synergizes with nearby enhancer to drive Drosophila Myc transcription C_LIO_LILate enhancer licenses TATA promoter for larval tissue-specific Myc transcription C_LIO_LIPromoter-enhancer dynamics differentially drive Myc during development C_LIO_LISSMEP protocol helps purify and enrich low-abundance DNA-protein complexes C_LI

Meiosis is a fundamental process in producing both sperm and eggs, yet recombination landscapes often exhibit sexual differences, known as heterochiasmy. Since meiotic proteins are generally expressed in both sexes, the molecular mechanism driving heterochiasmy remains elusive. The -kleisin subunit gene of meiotic cohesin, Rec8, is expressed bisexually in mammals, while its putative teleost ortholog, rec8a, is expressed in a female-biased manner, presumably due to the presence of its paralog originating from the teleost-specific whole-genome duplication (TGD). Here, we elucidated the evolutionary history and expression dynamics of -kleisin genes across teleost lineages. Through comprehensive phylogenetic and synteny analyses, we revealed that major teleost lineages retain two copies of rec8 and rad21, with rec8 loci experiencing drastic chromosomal rearrangements immediately after the TGD. Using in situ hybridization and single-cell transcriptome data in medaka and zebrafish, we demonstrated a conserved sexually biased expression pattern: rec8a is predominantly female-biased, whereas rec8b exhibits male-biased expression during gametogenesis. Furthermore, comparative epigenetic analyses revealed that the conserved sexually biased expression is driven by lineage-specific cis-regulatory elements, rather than conserved ones. Motif analyses imply that regulatory rewiring by transcription factors, including foxl2l in particular, might have played a crucial role in the establishment and maintenance of this paralog divergence. Our findings highlight how whole-genome duplication and subsequent genomic and epigenetic rewiring subdivided the bisexual function of rec8, offering insights into sexually distinct meiotic regulation. HighlightsO_LITeleosts possess a unique -kleisin repertoire originating from the TGD. C_LIO_LITeleost rec8 paralogs exhibit conserved sex-biased expression during meiosis. C_LIO_LIDrastic genomic rearrangements after the duplication rewired the teleost rec8 loci. C_LIO_LIThe conserved expression pattern is governed by lineage-specific CREs. C_LIO_LIThose CREs harbor similar types of TFBSs such as Fox-family TFs. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/731870v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@c8c84dorg.highwire.dtl.DTLVardef@1d65668org.highwire.dtl.DTLVardef@c2d732org.highwire.dtl.DTLVardef@1be54a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Protein-protein interactions (PPIs) play a crucial role in enabling proteins to carry out their functions within various biological processes (Hui et al., 2003). Since the introduction of the yeast two-hybrid (Y2H) method for PPI detection in 1989 (Fields and Song, 1989), the identification of PPIs has become a significant focus in modern biological research. PPI goes beyond examining individual proteins, allowing researchers to establish a comprehensive network that regulates biological processes. Rice, as a key model organism in plant biological studies, has been at the forefront of PPI research. In 2008, prominent rice scientists in China called for concerted efforts to define a comprehensive protein-protein interaction network experimentally, which aimed to facilitate the prediction of the functional mechanisms operating throughout a plants lifecycle (Zhang et al., 2008). With efforts for 2 decades, the experimentally identified rice PPIs have reached over ten thousand. Several public databases have been established to systematically collate and store PPIs, including STRING (Szklarczyk et al., 2019), BioGRID (Oughtred et al., 2020), IntAct (del Toro et al., 2022), PRIN (Gu et al., 2011), RicePPINet (Liu et al., 2017) and RiceNet v2 (Lee et al., 2015). However, most PPI datasets in rice stem from computational predictions, while experiment-based rice PPI datasets are fragmented due to the lack of systematic profiling at the rice PPIome level, which largely hinders information sharing in the rice research community. To bridge this gap, we constructed the Port of Protein-Protein Interactomes (POPPIN; https://riceome.hzau.edu.cn/poppin/), an integrated database dedicated to sharing experimentally verified PPIs and functional clues in rice. Empowered by high-throughput PPIome profiling technologies and text mining assisted by a large language model (Huang et al., 2025; Liu et al., 2025), POPPIN currently has deposited over 150,451 pieces of rice PPI-related information. Additionally, POPPIN provides detailed protein information, including GO annotations, subcellular localizations, domains, trait ontology (TO) information, and hyperlinks to external biological databases. Through offering a user-friendly web interface for search and dynamic network visualization, POPPIN serves as the first large-scale, experiment-based database for searchable PPIs in rice, and has the potential to be extended to other species under this structural framework.

The specificity protein 7 (SP7), also known as Osterix (OSX), is a zinc-finger transcription factor essential for osteoblast differentiation and skeletal development. Hereafter, the protein is referred to as OSX/SP7 throughout the manuscript. Although OSX/SP7 has been extensively studied in mammals and teleost fish, its developmental expression pattern in chicken (Gallus gallus) has not been described. Using an experimentally isolated chicken OSX/SP7 sequence, we examined OSX/SP7 mRNA expression during embryogenesis by in situ hybridization. As expected, OSX/SP7 expression was detected in developing skeletal elements undergoing ossification. Unexpectedly, transcripts were also observed in the neuroepithelium, retina, central nervous system, embryonic muscles and integument. Notably, OSX/SP7 expression was present in the neural tube from Hamburger and Hamilton stage 9 and persisted in the developing central nervous system until at least HH40, suggesting that OSX/SP7 functions during avian development may extend beyond osteogenesis. In addition, we reconstructed the chicken OSX/SP7 coding sequence and inferred its associated untranslated regions. The reconstructed ORF was independently supported by maternal and paternal haplotype-resolved chicken genome assemblies and retained the characteristic domain architecture of vertebrate OSX/SP7 proteins despite substantial divergence outside the DNA-binding domain. Taken together, these findings broaden the developmental landscape of OSX/SP7 expression in birds and provide new molecular resources for future studies of OSX/SP7 regulation and function during vertebrate development.