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The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Nuclear protein‐1 (NUPR1) (also known as p8) is one of the genes associated with transcription factors that participate in various aspects of cancer initiation and development. However, the molecular mechanisms of NUPR1 in bladder cancer (BLCA) remain unclear. We conducted an analysis of the correlation between NUPR1 expression and related genes using the Gene Expression Omnibus (GEO) online database. We employed lentivirus‐mediated small interfering RNA (siRNA) to knockdown the expression of NUPR1 in two human BLCA cell lines. In vitro experiments were conducted to validate the impact of NUPR1 interference on BLCA and the influence of NUPR1 on the transcription of chemokine receptor‐2 (CCR2). Furthermore, transcription factors for CCR2 were predicted using the PROMO database. Co‐immunoprecipitation (Co‐IP) and immunofluorescence double staining were used to detect the binding between NUPR1 and CCAAT/enhancer binding protein γ (CEBPG). In vivo and in vitro experiments were conducted to validate that NUPR1 regulates CCR2 transcription through CEBPG. In vitro experiments indicate that the suppression of NUPR1 inhibited BLCA growth. Analysis of the GEO database revealed a positive correlation between the expression of NUPR1 and CCR2. Luciferase experiments confirmed that NUPR1 influences the transcription of CCR2. Online data indicates that CEBPG is a transcription factor for CCR2. Co‐IP and immunofluorescence double staining confirmed binding between NUPR1 and CEBPG. Luciferase assays and chromatin immunoprecipitation (ChIP) demonstrate that CEBPG regulates the transcription of CCR2. Additionally, rescue experiments at the cellular level and animal experiments validated the aforementioned mechanism. NUPR1 promotes a promotional role in BLCA, and interference with NUPR1 can inhibit the proliferation and invasive abilities of BLCA. There was a correlation between the expressions of NUPR1 and CCR2, and NUPR1 binds with CEBPG in the cell nucleus. Transcriptional regulation of CCR2 by NUPR1 may be achieved through the involvement of CEBPG. [ABSTRACT FROM AUTHOR]

Simple Summary: Our study investigated the molecular mechanism of co-cultivated Citrus sinensis in diminishing the resistance of Poncirus trifoliata to the Asian Citrus Psyllid (ACP). The presence of C. sinensis was found to weaken the resistance of P. trifoliata to ACP, shown by a significant increase in ACP survival after 14 days of incubation. RNA-seq analysis revealed differentially expressed genes (DEGs) between control samples and those under co-cultivation with C. sinensis, with significant changes in metabolic processes. Further analysis indicated that P. trifoliata co-cultivation with C. sinensis significantly impacted the expression of many genes, including those involved in polysaccharide metabolic processes, metabolic pathways, biosynthesis of secondary metabolites, and phenylpropanoid biosynthesis. The study also identified a hub gene, Ptrif_0004s_1029, a NAC gene from transcription factor genes, appearing crucial in transcriptional regulation related to potential ACP resistance. Additionally, DEGs related to ACP feeding were also identified. Our research has elucidated the molecular mechanisms that underlie the resistance of P. trifoliata to the ACP and identified potential genes associated with this resistance. Citrus huanglongbing (HLB) is a devastating disease for citrus production, largely caused by the Asian citrus psyllid (ACP). Poncirus trifoliata exhibits high resistance to ACP; however, this resistance is weakened when C. sinensis is co-cultivated. This study aimed to identify the differentially expressed genes (DEGs) during ACP feeding and to uncover potential ACP resistance genes in P. trifoliata. In comparison to independent cultivation, 1247 and 205 DEGs were identified in P. trifoliata when co-cultivated with C. sinensis after 7 and 14 days, respectively. Analysis of enriched Gene Ontology categories revealed that DEGs were significantly associated with the cell wall, glucometabolic activities, and secondary metabolites. Additionally, these genes were found to be involved in phytohormone signaling, cell wall metabolism, redox state homeostasis, and secondary metabolites, as well as a number of transcription factor genes (TFs). Furthermore, we examined the impact of the ACP feeding factor on the gene expression patterns in P. trifoliata. Results showed an increase in the JA signaling pathway and various TFs. The RNA-seq results were verified using reverse transcription quantitative PCR. Our findings shed light on the molecular basis of ACP resistance in P. trifoliata and identified potential genes associated with this resistance. [ABSTRACT FROM AUTHOR]

The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and regulatory elements, which are either not transcribed or transcribed at a very low level. To solve above problems, the Functional Annotation of the Animal Genomes (FAANG) project proposed a systemic approach to tissue collection, phenotyping, and data generation, adopting the blueprint laid out by the Encyclopedia of DNA Elements (ENCODE) project. Here we detail the first comprehensive overview of gene expression and regulation in the horse, presenting 39,625 novel transcripts, 84,613 candidate cis-regulatory elements (CRE) and their target genes, 332,115 open chromatin regions genome wide across a diverse set of tissues. We showed substantial concordance between chromatin accessibility, chromatin states in different genic features and gene expression. This comprehensive and expanded set of genomics resources will provide the equine research community ample opportunities for studies of complex traits in the horse.

Enhancing root development is pivotal for boosting crop yield and augmenting stress resilience. In this study, we explored the regulatory effects of xylooligosaccharides (XOSs) on lettuce root growth, comparing their impact with that of indole-3-butyric acid potassium salt (IBAP). Treatment with XOS led to a substantial increase in root dry weight (30.77%), total root length (29.40%), volume (21.58%), and surface area (25.44%) compared to the water-treated control. These enhancements were on par with those induced by IBAP. Comprehensive phytohormone profiling disclosed marked increases in indole-3-acetic acid (IAA), zeatin riboside (ZR), methyl jasmonate (JA-ME), and brassinosteroids (BRs) following XOS application. Through RNA sequencing, we identified 3807 differentially expressed genes (DEGs) in the roots of XOS-treated plants, which were significantly enriched in pathways associated with manganese ion homeostasis, microtubule motor activity, and carbohydrate metabolism. Intriguingly, approximately 62.7% of the DEGs responsive to XOS also responded to IBAP, underscoring common regulatory mechanisms. However, XOS uniquely influenced genes related to cutin, suberine, and wax biosynthesis, as well as plant hormone signal transduction, hinting at novel mechanisms of stress tolerance. Prominent up-regulation of genes encoding beta-glucosidase and beta-fructofuranosidase highlights enhanced carbohydrate metabolism as a key driver of XOS-induced root enhancement. Collectively, these results position XOS as a promising, sustainable option for agricultural biostimulation. [ABSTRACT FROM AUTHOR]

Rhizobium etli CFN42 proteome–transcriptome mixed data of exponential growth and nitrogen-fixing bacteroids, as well as Sinorhizobium meliloti 1021 transcriptome data of growth and nitrogen-fixing bacteroids, were integrated into transcriptional regulatory networks (TRNs). The one-step construction network consisted of a matrix-clustering analysis of matrices of the gene profile and all matrices of the transcription factors (TFs) of their genome. The networks were constructed with the prediction of regulatory network application of the RhizoBindingSites database (http://rhizobindingsites.ccg.unam.mx/). The deduced free-living Rhizobium etli network contained 1,146 genes, including 380 TFs and 12 sigma factors. In addition, the bacteroid R. etli CFN42 network contained 884 genes, where 364 were TFs, and 12 were sigma factors, whereas the deduced free-living Sinorhizobium meliloti 1021 network contained 643 genes, where 259 were TFs and seven were sigma factors, and the bacteroid Sinorhizobium meliloti 1021 network contained 357 genes, where 210 were TFs and six were sigma factors. The similarity of these deduced condition-dependent networks and the biological E. coli and B. subtilis independent condition networks segregates from the random Erdös–Rényi networks. Deduced networks showed a low average clustering coefficient. They were not scale-free, showing a gradually diminishing hierarchy of TFs in contrast to the hierarchy role of the sigma factor rpoD in the E. coli K12 network. For rhizobia networks, partitioning the genome in the chromosome, chromids, and plasmids, where essential genes are distributed, and the symbiotic ability that is mostly coded in plasmids, may alter the structure of these deduced condition-dependent networks. It provides potential TF gen–target relationship data for constructing regulons, which are the basic units of a TRN.

Exploration of gene expression variations is a potential source to unravel biological pathways involved in pathological changes in body and understand the mechanism underneath. Vitiligo patients were explored for gene expression changes transcriptionally at perilesional site in comparison to normal site of same patients for melanogenesis pathway (TYR, DCT & TYRP1) cell adhesion (MMPs & TIMP1), cell survival (BCL2 & BAX1) as well as proliferation, migration & development (SOX9, SOX10 & MITF) regulatory system, using skin biopsy samples. Results were also compared with changes in gene expression for melanocytes under stress after hydrogen peroxide treatment in-vitro. Gene amplification was carried out via real time PCR. We found increased expression of proliferation, migration & development regulatory genes as well as melanogenesis pathway genes at perilesional site of patients. In-vitro study also supports induced MITF expression and disturbed melanogenesis in melanocytes under stress. Expression level ratio of cell survival regulatory genes’ (BCL2/BAX1) as well as cell adhesion regulatory genes (MMPs/TIMP1) was observed upregulated at patient’s perilesional site however downregulated in hydrogen peroxide treated melanocytes in-vitro. Observed upregulated gene expression at perilesional site of patients may be via positive feedback loop in response to stress to increase cell tolerance power to survive against adverse conditions. Gene expression analysis suggests better cell survival and proliferation potential at perilesional site in vitiligo patients. It seems in-vivo conditions/growth factors supports cells to fight for survival to accommodate stressed conditions. [ABSTRACT FROM AUTHOR]

Introduction: Prevention or treatment for acoustic injury has been met with many translational challenges, resulting in the absence of FDA-approved interventions. Localized hypothermia following noise exposure mitigates acute cochlear injury and may serve as a potential avenue for therapeutic approaches. However, the mechanisms by which hypothermia results in therapeutic improvements are poorly understood. Methods: This study performs the transcriptomic analysis of cochleae from juvenile rats that experienced noise-induced hearing loss (NIHL) followed by hypothermia or control normothermia treatment. Results: Differential gene expression results from RNA sequencing at 24 h postexposure to noise suggest that NIHL alone results in increased inflammatory and immune defense responses, involving complement activation and cytokinemediated signaling. Hypothermia treatment post-noise, in turn, may mitigate the acute inflammatory response. Discussion: This study provides a framework for future research to optimize hypothermic intervention for ameliorating hearing loss and suggests additional pathways that could be targeted for NIHL therapeutic intervention. [ABSTRACT FROM AUTHOR]

Citrus huanglongbing (HLB) is a devastating disease for citrus production, largely caused by the Asian citrus psyllid (ACP). Poncirus trifoliata exhibits high resistance to ACP; however, this resistance is weakened when C. sinensis is co-cultivated. This study aimed to identify the differentially expressed genes (DEGs) during ACP feeding and to uncover potential ACP resistance genes in P. trifoliata. In comparison to independent cultivation, 1247 and 205 DEGs were identified in P. trifoliata when co-cultivated with C. sinensis after 7 and 14 days, respectively. Analysis of enriched Gene Ontology categories revealed that DEGs were significantly associated with the cell wall, glucometabolic activities, and secondary metabolites. Additionally, these genes were found to be involved in phytohormone signaling, cell wall metabolism, redox state homeostasis, and secondary metabolites, as well as a number of transcription factor genes (TFs). Furthermore, we examined the impact of the ACP feeding factor on the gene expression patterns in P. trifoliata. Results showed an increase in the JA signaling pathway and various TFs. The RNA-seq results were verified using reverse transcription quantitative PCR. Our findings shed light on the molecular basis of ACP resistance in P. trifoliata and identified potential genes associated with this resistance.

We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP6) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.

Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of "kinetic proofreading". Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

IntroductionPrevention or treatment for acoustic injury has been met with many translational challenges, resulting in the absence of FDA-approved interventions. Localized hypothermia following noise exposure mitigates acute cochlear injury and may serve as a potential avenue for therapeutic approaches. However, the mechanisms by which hypothermia results in therapeutic improvements are poorly understood.MethodsThis study performs the transcriptomic analysis of cochleae from juvenile rats that experienced noise-induced hearing loss (NIHL) followed by hypothermia or control normothermia treatment.ResultsDifferential gene expression results from RNA sequencing at 24 h post-exposure to noise suggest that NIHL alone results in increased inflammatory and immune defense responses, involving complement activation and cytokine-mediated signaling. Hypothermia treatment post-noise, in turn, may mitigate the acute inflammatory response.DiscussionThis study provides a framework for future research to optimize hypothermic intervention for ameliorating hearing loss and suggests additional pathways that could be targeted for NIHL therapeutic intervention.

Climate change is expected to pose a global threat to forest health by intensifying extreme events like drought and insect attacks. Carbon allocation is a fundamental process that determines the adaptive responses of long-lived late-maturing organisms like trees to such stresses. However, our mechanistic understanding of how trees coordinate and set allocation priorities among different sinks (e.g., growth and storage) under severe source limitation remains limited. Using flux measurements, isotopic tracing, targeted metabolomics, and transcriptomics, we investigated how limitation of source supply influences sink activity, particularly growth and carbon storage, and their relative regulation in Norway spruce (Picea abies) clones. During photosynthetic deprivation, absolute rates of respiration, growth, and allocation to storage all decline. When trees approach neutral carbon balance, i.e., daytime net carbon gain equals nighttime carbon loss, genes encoding major enzymes of metabolic pathways remain relatively unaffected. However, under negative carbon balance, photosynthesis and growth are down-regulated while sucrose and starch biosynthesis pathways are up-regulated, indicating that trees prioritize carbon allocation to storage over growth. Moreover, trees under negative carbon balance actively increase the turnover rate of starch, lipids, and amino acids, most likely to support respiration and mitigate stress. Our study provides molecular evidence that trees faced with severe photosynthetic limitation strategically regulate storage allocation and consumption at the expense of growth. Understanding such allocation strategies is crucial for predicting how trees may respond to extreme events involving steep declines in photosynthesis, like severe drought, or defoliation by heat waves, late frost, or insect attack.

Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.

Enhancing root development is pivotal for boosting crop yield and augmenting stress resilience. In this study, we explored the regulatory effects of xylooligosaccharides (XOSs) on lettuce root growth, comparing their impact with that of indole-3-butyric acid potassium salt (IBAP). Treatment with XOS led to a substantial increase in root dry weight (30.77%), total root length (29.40%), volume (21.58%), and surface area (25.44%) compared to the water-treated control. These enhancements were on par with those induced by IBAP. Comprehensive phytohormone profiling disclosed marked increases in indole-3-acetic acid (IAA), zeatin riboside (ZR), methyl jasmonate (JA-ME), and brassinosteroids (BRs) following XOS application. Through RNA sequencing, we identified 3807 differentially expressed genes (DEGs) in the roots of XOS-treated plants, which were significantly enriched in pathways associated with manganese ion homeostasis, microtubule motor activity, and carbohydrate metabolism. Intriguingly, approximately 62.7% of the DEGs responsive to XOS also responded to IBAP, underscoring common regulatory mechanisms. However, XOS uniquely influenced genes related to cutin, suberine, and wax biosynthesis, as well as plant hormone signal transduction, hinting at novel mechanisms of stress tolerance. Prominent up-regulation of genes encoding beta-glucosidase and beta-fructofuranosidase highlights enhanced carbohydrate metabolism as a key driver of XOS-induced root enhancement. Collectively, these results position XOS as a promising, sustainable option for agricultural biostimulation.

Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone1,2. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.

Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how the development, physiology, and functions of CINs influence cortical circuit activity and behavior in model organisms such as mice and primates. While transgenic driver lines are powerful tools for studying CINs in mice, this technology is limited in other species. An alternative approach is to use viral vectors such as AAV, which can be used in multiple species including primates and also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell types. The present study describes the systematic genome-wide identification of putative REs (pREs) that are preferentially active in immature CINs by histone modification chromatin immunoprecipitation and sequencing (ChIP-seq). We evaluated two novel pREs in AAV vectors, alongside the well-established Dlx I12b enhancer, and found that they drove CIN-specific reporter expression in adult mice. We also showed that the identified Arl4d pRE could drive sufficient expression of channelrhodopsin for optogenetic rescue of behavioral deficits in the Dlx5/6+/- mouse model of fast-spiking CIN dysfunction.

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.

The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.

Agricultural biotechnology strategies often require the precise regulation of multiple genes to effectively modify complex plant traits. However, most efforts are hindered by a lack of characterized tools that allow for reliable and targeted expression of transgenes. We have successfully engineered a library of synthetic transcriptional regulators that modulate expression strength in planta. By leveraging orthogonal regulatory systems from Saccharomyces spp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their use in Nicotiana benthamiana and Arabidopsis thaliana. This characterization of contributing genetic elements that dictate gene expression represents a foundation for the rational design of refined synthetic regulators. Our findings demonstrate that these tools provide variation in transcriptional output while enabling the concerted expression of multiple genes in a tissue-specific and environmentally responsive manner, providing a basis for generating complex genetic circuits that process endogenous and environmental stimuli.

One of the primary aims of the Functional Annotation of ANimal Genomes (FAANG) initiative is to characterize tissue-specific regulation within animal genomes. To this end, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight prioritized tissues collected as part of the FAANG equine biobank from two thoroughbred mares. Data were generated according to optimized experimental parameters developed during quality control testing. To ensure that we obtained sufficient ChIP and successful peak-calling, data and peak-calls were assessed using six quality metrics, replicate comparisons, and site-specific evaluations. Tissue specificity was explored by identifying binding motifs within unique active regions, and motifs were further characterized by gene ontology (GO) and protein-protein interaction analyses. The histone marks identified in this study represent some of the first resources for tissue-specific regulation within the equine genome. As such, these publicly available annotation data can be used to advance equine studies investigating health, performance, reproduction, and other traits of economic interest in the horse.

The generation of protein coding mRNAs from pre-mRNA is a fundamental biological process that is required for gene expression. Alternative pre-mRNA splicing is responsible for much of the transcriptomic and proteomic diversity observed in higher order eukaryotes. Aberrations that disrupt regular alternative splicing patterns are known to cause human diseases, including various cancers. Alternative splicing is a combinatorial process, meaning many factors affect which two splice sites are ligated together. The features that dictate exon inclusion are comprised of splice site strength, intron-exon architecture, RNA secondary structure, splicing regulatory elements, promoter use and transcription speed by RNA polymerase and the presence of post-transcriptional nucleotide modifications. A comprehensive view of all of the factors that influence alternative splicing decisions is necessary to predict splicing outcomes and to understand the molecular basis of disease. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell-derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes.

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

The majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we perform saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitutions and deletions. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and comprise a rich dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.

Modern genetic studies indicate that human brain evolution is driven primarily by changes in gene regulation, which requires understanding the biological function of largely non-coding gene regulatory elements, many of which act in tissue specific manner. We leverage chromatin interaction profiles in human fetal and adult cortex to assign three classes of human-evolved elements to putative target genes. We find that human-evolved elements involving DNA sequence changes and those involving epigenetic changes are associated with human-specific gene regulation via effects on different classes of genes representing distinct biological pathways. However, both types of human-evolved elements converge on specific cell types and laminae involved in cerebral cortical expansion. Moreover, human evolved elements interact with neurodevelopmental disease risk genes, and genes with a high level of evolutionary constraint, highlighting a relationship between brain evolution and vulnerability to disorders affecting cognition and behavior. These results provide novel insights into gene regulatory mechanisms driving the evolution of human cognition and mechanisms of vulnerability to neuropsychiatric conditions.

Many strains of the spirochete Leptospira interrogans serovar Pomona express the osmotically inducible sphingomyelinase gene sph2 at much higher levels than strains from other serovars. We developed a new green fluorescent protein (GFP) reporter plasmid to examine sph2 gene expression determinants. The vector enables the fusion of the test promoter to the ribosome-binding site and coding region of gfp We fused the sph2 promoters from the L. interrogans serovar Lai strain 56601 and from the L. interrogans serovar Pomona strain LC82-25 to gfp to examine the molecular determinants of differential sph2 expression between the two strains. Similar to what was observed with the native sph2 genes, the introduction of the plasmids into the Lai 56601 strain resulted in near background levels of gfp expression from the Lai sph2 promoter, while the expression from the Pomona sph2 promoter was high. The expression of both fusions increased at physiologic levels of osmolarity achieved by adding sodium chloride to the culture medium. We examined the role of a 17-bp upstream element found in all L. interrogans strains expressing low basal levels of sph2 and missing from Pomona strains that express sph2 at high levels. When the 17-bp sequence present upstream of the Lai sph2 promoter was deleted or scrambled, the fusion expression increased substantially. Conversely, the insertion of the 17-bp sequence upstream of the Pomona sph2 promoter diminished fusion expression. In contrast, the removal of an insertion sequence-like element that is found only in the Pomona sph2 upstream sequence had no effect on the expression from the Pomona sph2 fusion in the Lai strain. These findings demonstrate the utility of the gfp reporter plasmid in analyzing gene expression in L. interrogansIMPORTANCE Genetic tools are needed to examine gene expression in the pathogen Leptospira interrogans We developed a reporter plasmid that replicates in L. interrogans with green fluorescent protein (GFP) as the readout of promoter activity. We demonstrated an application of the new reporter plasmid by identifying an upstream element responsible for the poor basal expression of the sph2 sphingomyelinase gene in an L. interrogans serovar Lai strain. This new tool is useful for the discovery of the molecular determinants of L. interrogans gene expression.

BackgroundHomeodomain (HD) transcription factor (TF) NKX2-1 critical for the regional specification of the medial ganglionic eminence (MGE) as well as promoting the GABAergic and cholinergic neuron fates via the induction of TFs such as LHX6 and LHX8. NKX2-1 defines MGE regional identity in large part through transcriptional repression, while specification and maturation of GABAergic and cholinergic fates is mediated in part by transcriptional activation via TFs such as LHX6 and LHX8. Here we analyze the signaling and TF pathways, downstream of NKX2-1, required for GABAergic and cholinergic neuron fate maturation.MethodsDifferential ChIP-seq analysis was used to identify regulatory elements (REs) where chromatin state was sensitive to change in the Nkx2-1cKO MGE at embryonic day (E) 13.5. TF motifs in the REs were identified using RSAT. CRISPR-mediated genome editing was used to generate enhancer knockouts. Differential gene expression in these knockouts was analyzed through RT-qPCR and in situ hybridization. Functional analysis of motifs within hs623 was analyzed via site directed mutagenesis and reporter assays in primary MGE cultures.ResultsWe identified 4782 activating REs (aREs) and 6391 repressing REs (rREs) in the Nkx2-1 conditional knockout (Nkx2-1cKO) MGE. aREs are associated with basic-Helix-Loop-Helix (bHLH) TFs. Deletion of hs623, an intragenic Tcf12 aRE, caused a reduction of Tcf12 expression in the sub-ventricular zone (SVZ) and mantle zone (MZ) of the MGE. Mutation of LHX, SOX and octamers, within hs623, caused a reduction of hs623 activity in MGE primary cultures.ConclusionsTcf12 expression in the SVZ of the MGE is mediated through aRE hs623. The activity of hs623 is dependent on LHX6, SOX and octamers. Thus, maintaining the expression of Tcf12 in the SVZ involves on TF pathways parallel and genetically downstream of NKX2-1.

Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this 'PAC-K' silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.

Cell-specific patterns of gene expression are determined by combinatorial actions of sequence-specific transcription factors at cis-regulatory elements. Studies indicate that relatively simple combinations of lineage-determining transcription factors (LDTFs) play dominant roles in the selection of enhancers that establish cell identities and functions. LDTFs require collaborative interactions with additional transcription factors to mediate enhancer function, but the identities of these factors are often unknown. We have shown that natural genetic variation between individuals has great utility for discovering collaborative transcription factors. Here, we introduce MMARGE (Motif Mutation Analysis of Regulatory Genomic Elements), the first publicly available suite of software tools that integrates genome-wide genetic variation with epigenetic data to identify collaborative transcription factor pairs. MMARGE is optimized to work with chromatin accessibility assays (such as ATAC-seq or DNase I hypersensitivity), as well as transcription factor binding data collected by ChIP-seq. Herein, we provide investigators with rationale for each step in the MMARGE pipeline and key differences for analysis of datasets with different experimental designs. We demonstrate the utility of MMARGE using mouse peritoneal macrophages, liver cells, and human lymphoblastoid cells. MMARGE provides a powerful tool to identify combinations of cell type-specific transcription factors while simultaneously interpreting functional effects of non-coding genetic variation.

Developmental patterning involves the progressive subdivision of tissue into different cell types by invoking different genetic programs. In particular, cell-cell signaling is a universally deployed means of specifying distinct cell fates in adjacent cells. For this mechanism to be effective, it is essential that an asymmetry be established in the signaling and responding capacities of the participating cells. Here we focus on the regulatory mechanisms underlying the role of the neuralized gene and its protein product in establishing and maintaining asymmetry of signaling through the Notch pathway. The context is the classical process of "lateral inhibition" within Drosophila proneural clusters, which is responsible for distinguishing the sensory organ precursor (SOP) and non-SOP fates among adjacent cells. We find that neur is directly regulated in proneural clusters by both proneural transcriptional activators and Enhancer of split basic helix-loop-helix repressors (bHLH-Rs), via two separate cis-regulatory modules within the neur locus. We show that this bHLH-R regulation is required to prevent the early, pre-SOP expression of neur from being maintained in a subset of non-SOPs following SOP specification. Lastly, we demonstrate that Neur activity in the SOP is required to inhibit, in a cell non-autonomous manner, both neur expression and Neur function in non-SOPs, thus helping to secure the robust establishment of distinct cell identities within the developing proneural cluster.

In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and -independent pathways.

Robust and predictably performing synthetic circuits rely on the use of well-characterized regulatory parts across different genetic backgrounds and environmental contexts. Here we report the large-scale metagenomic mining of thousands of natural 5' regulatory sequences from diverse bacteria, and their multiplexed gene expression characterization in industrially relevant microbes. We identified sequences with broad and host-specific expression properties that are robust in various growth conditions. We also observed substantial differences between species in terms of their capacity to utilize exogenous regulatory sequences. Finally, we demonstrate programmable species-selective gene expression that produces distinct and diverse output patterns in different microbes. Together, these findings provide a rich resource of characterized natural regulatory sequences and a framework that can be used to engineer synthetic gene circuits with unique and tunable cross-species functionality and properties, and also suggest the prospect of ultimately engineering complex behaviors at the community level.

The assembly of sequence-specific enhancer-binding transcription factors (TFs) at cis-regulatory elements in the genome has long been regarded as the fundamental mechanism driving cell type-specific gene expression. However, despite extensive biochemical, genetic, and genomic studies in the past three decades, our understanding of molecular mechanisms underlying enhancer-mediated gene regulation remains incomplete. Recent advances in imaging technologies now enable direct visualization of TF-driven regulatory events and transcriptional activities at the single-cell, single-molecule level. The ability to observe the remarkably dynamic behavior of individual TFs in live cells at high spatiotemporal resolution has begun to provide novel mechanistic insights and promises new advances in deciphering causal-functional relationships of TF targeting, genome organization, and gene activation. In this review, we review current transcription imaging techniques and summarize converging results from various lines of research that may instigate a revision of models to describe key features of eukaryotic gene regulation.

CRISPR-Cas-based transcriptional activation (CRISPRa) and interference (CRISPRi) enable transient programmable gene regulation by recruitment or fusion of transcriptional regulators to nuclease-deficient Cas (dCas). Here, we expand on the emerging area of transcriptional engineering and RNA delivery by benchmarking combinations of RNA-delivered dCas and transcriptional modulators. We utilize dCas9 from Staphylococcus aureus and Streptococcus pyogenes for orthogonal transcriptional modulation to upregulate one set of genes while downregulating another. We also establish trimodal genetic engineering by combining orthogonal transcriptional regulation with gene knockout by Cas12a (Acidaminococcus; AsCas12a) ribonucleoprotein delivery. To simplify trimodal engineering, we explore optimal parameters for implementing truncated single guide RNAs (sgRNAs) to make use of SpCas9 for knockout and CRISPRa. We find the Cas9 protein/sgRNA ratio to be crucial for avoiding sgRNA cross-complexation and for balancing knockout and activation efficiencies. We demonstrate high efficiencies of trimodal genetic engineering in primary human T cells while preserving basic T cell health and functionality. This study highlights the versatility and potential of complex genetic engineering using multiple CRISPR-Cas systems in a simple one-step process yielding transient transcriptome modulation and permanent DNA changes. We believe such elaborate engineering can be implemented in regenerative medicine and therapies to facilitate more sophisticated treatments., Competing Interests: Declaration of interests R.O.B. holds equity in UNIKUM Tx and is a consultant to UNIKUM Tx. T.I.J. is an employee of and holds equity in UNIKUM Tx. R.O.B. is inventor on patents or patent applications related to CRISPR-Cas and engineering of cellular products. R.O.B. reports research funding from Novo Nordisk., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Temperature-dependent sex determination (TSD) decides the sex fate of an individual based on incubation temperature. However, other environmental factors, such as pollutants, could derail TSD sexual development. Cadmium is one such contaminant of soils and water bodies known to affect DNA methylation, an epigenetic DNA modification with a key role in sexual development of TSD vertebrate embryos. Yet, whether cadmium alters DNA methylation of genes underlying gonadal formation in turtles remains unknown. Here, we investigated the effects of cadmium on the expression of two gene regulators of TSD in the painted turtle, Chrysemys picta, incubated at male-producing and female-producing temperatures using qPCR. Results revealed that cadmium alters transcription of Dmrt1 and aromatase, overriding the normal thermal effects during embryogenesis, which could potentially disrupt the sexual development of TSD turtles. Results from a preliminary DNA methylation-sensitive PCR assay implicate changes in DNA methylation of Dmrt1 as a potential cause that requires further testing (aromatase methylation assays were precluded). [ABSTRACT FROM AUTHOR]

Panax notoginseng (Burk) F.H. Chen is a rare and valuable Chinese herb, but root rot mainly caused by Fusarium solani severely affects the yield and quality of P. notoginseng herbal materials. In this study, we isolated 30 P. notoginseng WRKY transcription factors (TFs), which were divided into three groups (I, II, and III) on the basis of a phylogenetic analysis. The expression levels of 10 WRKY genes, including PnWRKY9, in P. notoginseng roots increased in response to a methyl jasmonate (MeJA) treatment and the following F. solani infection. Additionally, PnWRKY9 was functionally characterized. The PnWRKY9 protein was localized to the nucleus. The overexpression of PnWRKY9 in tobacco (Nicotiana tabacum) considerably increased the resistance to F. solani, whereas an RNAi-mediated decrease in the PnWRKY9 expression level in P. notoginseng leaves increased the susceptibility to F. solani. The RNA sequencing and hormone content analyses of PnWRKY9-overexpression tobacco revealed that PnWRKY9 and the jasmonic acid (JA) signaling pathway synergistically enhance disease resistance. The PnWRKY9 recombinant protein was observed to bind specifically to the W-box sequence in the promoter of a JA-responsive and F. solani resistance-related defensin gene (PnDEFL1). A yeast one-hybrid assay indicated that PnWRKY9 can activate the transcription of PnDEFL1. Furthermore, a co-expression assay in tobacco using b-glucuronidase (GUS) as a reporter further verified that PnWRKY9 positively regulates PnDEFL1 expression. Overall, in this study, we identified P. notoginseng WRKY TFs and demonstrated that PnWRKY9 positively affects plant defenses against the root rot pathogen. The data presented herein provide researchers with fundamental information regarding the regulatory mechanism mediating the coordinated activities of WRKY TFs and the JA signaling pathway in P. notoginseng responses to the root rot pathogen. [ABSTRACT FROM AUTHOR]

Lean body mass, consisting mostly of skeletal muscle, is important for healthy aging. We performed a genome-wide association study for whole body (20 cohorts of European ancestry with n = 38,292) and appendicular (arms and legs) lean body mass (n = 28,330) measured using dual energy X-ray absorptiometry or bioelectrical impedance analysis, adjusted for sex, age, height, and fat mass. Twenty-one single-nucleotide polymorphisms were significantly associated with lean body mass either genome wide (p

Endothelial cells (ECs) are critical determinants of vascular homeostasis and inflammation, but transcriptional mechanisms specifying their identities and functional states remain poorly understood. Here, we report a genome-wide assessment of regulatory landscapes of primary human aortic endothelial cells (HAECs) under basal and activated conditions, enabling inference of transcription factor networks that direct homeostatic and pro-inflammatory programs. We demonstrate that 43% of detected enhancers are EC-specific and contain SNPs associated to cardiovascular disease and hypertension. We provide evidence that AP1, ETS, and GATA transcription factors play key roles in HAEC transcription by co-binding enhancers associated with EC-specific genes. We further demonstrate that exposure of HAECs to oxidized phospholipids or pro-inflammatory cytokines results in signal-specific alterations in enhancer landscapes and associate with coordinated binding of CEBPD, IRF1, and NFκB. Collectively, these findings identify cis-regulatory elements and corresponding trans-acting factors that contribute to EC identity and their specific responses to pro-inflammatory stimuli.

Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.

Chromatin structure plays a pivotal role in facilitating proper control of gene expression. Transcription factor (TF) binding of cis-elements is often associated with accessible chromatin regions. Therefore, the ability to identify these accessible regions throughout plant genomes will advance understanding of the relationship between TF binding, chromatin status and the regulation of gene expression. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is a recently developed technique used to map open chromatin zones in animal genomes. However, in plants, the existence of cell walls, subcellular organelles and the lack of stable cell lines have prevented routine application of this technique. Here, we describe an assay combining ATAC-seq with fluorescence-activated nuclei sorting (FANS) to identify and map open chromatin and TF-binding sites in plant genomes. FANS-ATAC-seq compares favorably with published DNaseI sequencing (DNase-seq) results and it requires less than 50 000 nuclei for accurate identification of accessible genomic regions.SummaryApplication of ATAC-seq to sorted nuclei identifies accessible regions genome-wide.

Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic-enhancer inactivation, and pluripotency enhancer selection. Together, our data indicate that collaborative interactions among OSK and with stage-specific TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.

• Temperature variations significantly influence the growth and metabolite production of C. militaris. • Responsive mechanisms in acclimatization to temperature conditions identified in C. militaris. • Low temperature induced transcriptional upregulation of lipid biosynthetic pathways indicating an adaptive metabolic response of C. militaris. Cordyceps militaris is a medicinal entomopathogenic fungus containing valuable biometabolites for pharmaceutical applications. Its genetic inheritance and environmental factors play a crucial role in the production of biomass enriched with cordycepin. While temperature is a crucial controlled parameter for fungal cultivation, its impacts on growth and metabolite biosynthesis remains poorly characterized. This study aimed to investigate the metabolic responses and cordycepin production of C. militaris strain TBRC6039 under various temperature conditions through transcriptome analysis. Among 9599 expressed genes , 576 genes were significantly differentially expressed at culture temperatures of 15 and 25 °C. The changes in the transcriptional responses induced by these temperatures were found in several metabolisms involved in nutrient assimilation and energy source, including amino acids metabolism (e.g., glycine, serine and threonine metabolism) and lipid metabolism (e.g., biosynthesis of unsaturated fatty acids and steroid biosynthesis). At the lower temperature (15 °C), the biosynthetic pathways of lipids, specifically ergosterol and squalene, were the target for maintaining membrane function by transcriptional upregulation. Our study revealed the responsive mechanisms of C. militaris in acclimatization to temperature conditions that provide an insight on physiological manipulation for the production of metabolites by C. militaris. [ABSTRACT FROM AUTHOR]

Fungal spores, germlings, and mycelia adhere to substrates, including host tissues. The adhesive forces depend on the substrate and on the adhesins, the fungal cell surface proteins. Attachment is often a prerequisite for the invasion of the host, hence its importance. Adhesion visibly precedes colonization of root surfaces and outer cortex layers, but little is known about the molecular details. We propose that by starting from what is already known from other fungi, including yeast and other filamentous pathogens and symbionts, the mechanism and function of Trichoderma adhesion will become accessible. There is a sequence, and perhaps functional, homology to other rhizosphere-competent Sordariomycetes. Specifically, Verticillium dahliae is a soil-borne pathogen that establishes itself in the xylem and causes destructive wilt disease. Metarhizium species are best-known as insect pathogens with biocontrol potential, but they also colonize roots. Verticillium orthologs of the yeast Flo8 transcription factor, Som1, and several other relevant genes are already under study for their roles in adhesion. Metarhizium encodes relevant adhesins. Trichoderma virens encodes homologs of Som1, as well as adhesin candidates. These genes should provide exciting leads toward the first step in the establishment of beneficial interactions with roots in the rhizosphere. [ABSTRACT FROM AUTHOR]

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology.

STUDY QUESTION: What are the functional characteristics and transcriptional regulators of human trophoblast progenitor cells (TBPCs)? SUMMARY ANSWER: TBPC lines established from the human smooth chorion by cell sorting for integrin α4 expressed markers of stemness and trophoblast (TB) stage-specific antigens, invaded Matrigel substrates and contributed to the cytotrophoblasts (CTBs) layer of smooth chorion explants with high-mobility group protein HMGI-C (HMGA2) and transcription factor GATA-4 (GATA4) controlling their progenitor state and TB identity. WHAT IS KNOWN ALREADY: Previously, we reported the derivation of TBPC lines by trypsinization of colonies that formed in cultures of chorionic mesenchyme cells that were treated with an activin nodal inhibitor. Microarray analyses showed that, among integrins, α4 was most highly expressed, and identified HMGA2 and GATA4 as potential transcriptional regulators. STUDY DESIGN, SIZE, DURATION: The aim of this study was to streamline TBPC derivation across gestation. High-cell surface expression of integrin α4 enabled the use of a fluorescence-activated cell sorter (FACS) approach for TBPC isolation from the human smooth chorion (n = 6 lines). To confirm their TBPC identity, we profiled their expression of stemness and TB markers, and growth factor receptors. At a functional level, we assayed their invasive capacity (n = 3) and tropism for the CTB layer of the smooth chorion (n = 3). At a molecular level, we studied the roles of HMGA2 and GATA4. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Cells were enzymatically disassociated from the human smooth chorion across gestation. FACS was used to isolate the integrin α4-positive population. In total, we established six TBPC lines, two per trimester. Their identity was determined by immunolocalization of a suite of antigens. Function was assessed via Matrigel invasion and co-culture with explants of the human smooth chorion. An siRNA approach was used to down-regulate HMGA2 and GATA4 expression and the results were confirmed by immunoblotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The endpoints analyzed included proliferation, as determined by 5-bromo-2-deoxyuridine (BrDU) incorporation, and the expression of stage-specific antigens and hormones, as determined by qRT-PCR and immunostaining approaches. MAIN RESULTS AND THE ROLE OF CHANCE: As with the original cell lines, the progenitors expressed a combination of human embryonic stem cell and TB markers. Upon differentiation, they primarily formed CTBs, which were capable of Matrigel invasion. Co-culture of the cells with smooth chorion explants enabled their migration through the mesenchyme after which they intercalated within the chorionic CTB layer. Down-regulation of HMGA2 showed that this DNA-binding protein governed their self-renewal. Both HMGA2 and GATA4 had pleitropic effects on the cells progenitor state and TB identity. LIMITATIONS, REASONS FOR CAUTION: This study supported our hypothesis that TBPCs from the chorionic mesenchyme can contribute to the subpopulation of CTBs that reside in the smooth chorion. In the absence of in vivo data, which is difficult to obtain in humans, the results have the limitations common to all in vitro studies. WIDER IMPLICATIONS OF THE FINDINGS: The accepted view is that progenitors reside among the villous CTB subpopulation. Here, we show that TBPCs also reside in the mesenchymal layer of the smooth chorion throughout gestation. We theorize that they can contribute to the CTB layer in this region. This phenomenon may be particularly important in pathological situations when CTBs of the smooth chorion might provide a functional reserve for CTBs of the placenta proper. STUDY FUNDING/COMPETING INTERESTS: Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award P50HD055764. O.G., N.L., K.O., A.P., T.G.-G., M.K., A.B., M.G. have nothing to disclose. S.J.F. received licensing fees and royalties from SeraCare Life Sciences for trisomic TBPC lines that were derived according to the methods described in this manuscript. TRIAL REGISTRATION NUMBER: N/A.