Regulatory Sequences Research Articles

ABI3 regulates ABI1 function to control cell length in primary root elongation zone.

Post-embryonic primary root growth is effectively an interplay of several hormone signalling pathways. Here, we show that the ABA-responsive transcription factor ABI3 controls primary root growth through the regulation of JA signalling molecule JAZ1 along with ABA-responsive factor ABI1. In the absence of ABI3, the primary root elongation zone is shortened with significantly reduced cell length. Expression analyses and ChIP-based assays indicate that ABI3 negatively regulates JAZ1 expression by occupying its upstream regulatory sequence and enriching repressive histone modification mark H3K27 trimethylation, thereby occluding RNAPII occupancy. Previous studies have shown that JAZ1 interacts with ABI1, the protein phosphatase 2C, that works during ABA signalling. Our results indicate that in the absence of ABI3, when JAZ1 expression levels are high, the ABI1 protein shows increased stability, compared to when JAZ1 is absent, or ABI3 is overexpressed. Consequently, in the abi3-6 mutant, due to the higher stability of ABI1, reduced phosphorylation of plasma membrane H+-ATPase (AHA2) occurs. HPTS staining further indicated that abi3-6 root cell apoplasts show reduced protonation, compared to wild-type and ABI3 overexpressing seedlings. Such impeded proton extrusion negatively affects cell length in the primary root elongation zone. ABI3 therefore controls cell elongation in the primary root by affecting the ABI1-dependent protonation of root cell apoplasts. In summary, ABI3 controls the expression of JAZ1 and in turn modulates the function of ABI1 to regulate cell length in the elongation zone during primary root growth.

Intronic regulatory sequences regulate epicardial gene expression and EMT during heart development

Abstract Background During organogenesis, cells from the outer layer of the heart, the epicardium, undergo epithelial-to-mesenchymal transition (EMT), contributing essential paracrine signals and different cell types to the growing heart. Epicardial cells are integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties during heart development. Purpose Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for Wt1 in heart development is widely accepted, the upstream regulatory mechanisms underpinning its activation remain elusive. Our previous research has led to the identification of two intronic evolutionary conserved regions (ECRs) shared between mouse and human, located within intron 1 of the Wt1 locus, i.e. +4.0kbECR and +5.8kbECR. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development. Methods & Results Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR (Wt1Δ+4.0kbECR and Wt1Δ+5.8kbECR mice) or a deletion comprising both ECRs (Wt1Δ+4-5.8kbECR mouse). Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation in these models. Mendelian ratios indicated an overall underrepresentation of Wt1ΔECR/ΔECR mutants recovered in the offspring arising from heterozygous inter-crosses. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular or membranous ventricular septum defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal formation/patterning of coronary artery stems in ΔECR/ΔECR hearts. Expression of Wt1 was markedly reduced in ΔECR/ΔECR hearts, but not in the kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue-section immunostaining combined with confocal microscopy revealed abnormal coronary vessel and innervation extension and patterning along the subepicardial space, as well as reduced epicardial EMT and myocardial non-compaction/hyper-trabeculation in Wt1ΔECR/ΔECR hearts. Conclusions Together, we demonstrated a requirement for novel intronic enhancers regulating Wt1 locus activity and impacting on essential epicardial EMT and associated biological processes during normal heart development. Importantly, observation of septum and aortic valve defects in ΔECR mutants suggests an hitherto unrecognised role for WT1/epicardial EMT and opens new avenues of research to improve our understanding of congenital heart disease that affects at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.

Deletion upstream of MAB21L2 highlights the importance of evolutionarily conserved non-coding sequences for eye development

Anophthalmia, microphthalmia and coloboma (AMC) comprise a spectrum of developmental eye disorders, accounting for approximately 20% of childhood visual impairment. While non-coding regulatory sequences are increasingly recognised as contributing to disease burden, characterising their impact on gene function and phenotype remains challenging. Furthermore, little is known of the nature and extent of their contribution to AMC phenotypes. We report two families with variants in or near MAB21L2, a gene where genetic variants are known to cause AMC in humans and animal models. The first proband, presenting with microphthalmia and coloboma, has a likely pathogenic missense variant (c.338 G > C; p.[Trp113Ser]), segregating within the family. The second individual, presenting with microphthalmia, carries an ~ 113.5 kb homozygous deletion 19.38 kb upstream of MAB21L2. Modelling of the deletion results in transient small lens and coloboma as well as midbrain anomalies in zebrafish, and microphthalmia and coloboma in Xenopus tropicalis. Using conservation analysis, we identify 15 non-coding conserved elements (CEs) within the deleted region, while ChIP-seq data from mouse embryonic stem cells demonstrates that two of these (CE13 and 14) bind Otx2, a protein with an established role in eye development. Targeted disruption of CE14 in Xenopus tropicalis recapitulates an ocular coloboma phenotype, supporting its role in eye development. Together, our data provides insights into regulatory mechanisms underlying eye development and highlights the importance of non-coding sequences as a source of genetic diagnoses in AMC.

C/EBPα-mediated ACSL4-dependent ferroptosis exacerbates tubular injury in diabetic kidney disease

Diabetic kidney disease (DKD) is a prevalent and debilitating complication of diabetes characterized by progressive renal function decline and a lack of effective treatment options. Here, we investigated the role of the transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) in DKD pathogenesis. Analysis of renal biopsy samples revealed increased C/EBPα expression in patients with DKD. Using RNA sequencing and proteomics, we explored the mechanisms through which the C/EBPα contributes to DKD. Our findings demonstrated that C/EBPα exacerbated tubular injury by promoting acyl-CoA synthetase long-chain family member 4 (ACSL4)-dependent ferroptosis. We identified that C/EBPα upregulated ACSL4 expression by binding to its transcription regulatory sequence (TRS), leading to elevated lipid peroxidation and ferroptosis. Furthermore, inhibition or genetic ablation of C/EBPα attenuated ferroptosis and mitigated tubular injury in DKD. These results highlighted the C/EBPα-ACSL4-ferroptosis pathway as a promising therapeutic target for DKD treatment.

Differential expression of the yfj operon in a Bacillus subtilis biofilm.

Type VII protein secretion systems play an important role in the survival and virulence of pathogens and in the competition among some microbes. Potential polymorphic toxin substrates of the type VII secretion system (T7SS) in Bacillus subtilis are important for competition in the context of biofilm communities. Within a biofilm, there is significant physiological heterogeneity as cells within the population take on differential cell fates. Which cells express and deploy the various T7SS substrates is still unknown. To identify which cells express at least one of the T7SS substrates, we investigated the yfj operon. The yfjABCDEF operon encodes at least one predicted T7SS substrate. Starting with an in silico analysis of the yfj operon promoter region, we identified potential regulatory sequences. Using a yfj promoter-reporter fusion, we then identified several regulators that impact expression of the operon, including a regulator of biofilm formation, DegU. In a degU deletion mutant, yfj expression is completely abolished. Mutation of predicted DegU binding sites also results in a significant reduction in yfj reporter levels. Further analysis of yfj regulation reveals that deletion of spo0A has the opposite effect of the degU deletion. Following the yfj reporter by microscopy of cells harvested from biofilms, we find that the yfj operon is expressed specifically in the subset of cells undergoing sporulation. Together, our results define cells entering sporulation as the subpopulation most likely to express products of the yfj operon in B. subtilis.IMPORTANCEDifferential expression of genes in a bacterial community allows for the division of labor among cells in the community. The toxin substrates of the type VII secretions system (T7SS) are known to be active in Bacillus subtilis biofilm communities. This work describes the expression of one of the T7SS-associated operons, the yfj operon, which encodes the YFJ toxin, in the sporulating subpopulation within a biofilm. The evidence that the YFJ toxin may be deployed specifically in cells at the early stages of sporulation provides a potential role for deployment of T7SS in community-associated activities, such as cannibalism.

AncestryGrapher toolkit: Python command-line pipelines to visualize global- and local- ancestry inferences from the RFMIX version 2 software.

Admixture is a fundamental process that has shaped levels and patterns of genetic variation in human populations. RFMIX version 2 (RFMIX2) utilizes a robust modeling approach to identify the genetic ancestries in admixed populations. However, this software does not have a built-in method to visually summarize the results of analyses. Here, we introduce the AncestryGrapher toolkit, which converts the numerical output of RFMIX2 into graphical representations of global and local ancestry (i.e., the per-individual ancestry components and the genetic ancestry along chromosomes, respectively). To demonstrate the utility of our methods, we applied the AncestryGrapher toolkit to visualize the global and local ancestry of individuals in the North African Mozabite Berber population from the Human Genome Diversity Panel (HGDP). Our results showed that the Mozabite Berbers derived their ancestry from the Middle East, Europe, and sub-Saharan Africa (global ancestry). We also found that the population origin of ancestry varied considerably along chromosomes (local ancestry). For example, we observed variance in local ancestry in the genomic region on chromosome 2 containing the regulatory sequence in the MCM6 gene associated with lactase persistence (LP), a human adaptive trait tied to the cultural development of adult milk consumption. Overall, the AncestryGrapher toolkit facilitates the exploration, interpretation, and reporting of ancestry patterns in human populations. The AncestryGrapher toolkit is free and open source on https://github.com/alisi1989/RFmix2-Pipeline-to-plot. Supplementary data are available at Bioinformatics online.

The Induction of Drug Uptake Transporter Organic Anion Transporting Polypeptide 1A2 by Radiation Is Mediated by the Nonreceptor Tyrosine Kinase v-YES-1 Yamaguchi Sarcoma Viral Oncogene Homolog 1.

Organic anion transporting polypeptides (OATP, gene symbol SLCO) are well-recognized key determinants for the absorption, distribution, and excretion of a wide spectrum of endogenous and exogenous compounds including many antineoplastic agents. It was therefore proposed as a potential drug target for cancer therapy. In our previous study, it was found that low-dose X-ray and carbon ion irradiation both upregulated the expression of OATP family member OATP1A2 and in turn, led to a more dramatic killing effect when cancer cells were cotreated with antitumor drugs such as methotrexate. In the present study, the underlying mechanism of the phenomenon was explored in breast cancer cell line MCF-7. It was found that the nonreceptor tyrosine kinase v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES-1) was temporally coordinated with the change of OATP1A2 after irradiation. The overexpression of YES-1 significantly increased OATP1A2 both at the mRNA and protein level. The signal transducer and activator of transcription 3 (STAT3) pathway is likely the downstream target of YES-1 because phosphorylation and nuclear accumulation of STAT3 were both enhanced after overexpressing YES-1 in MCF-7 cells. Further investigation revealed that there are two possible binding sites of STAT3 localized at the upstream sequence of SLCO1A2, the encoding gene of OATP1A2. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis suggested that these two sites bound to STAT3 specifically and the overexpression of YES-1 significantly increased the association of the transcription factor with the putative binding sites. Finally, inhibition or knockdown of YES-1 attenuated the induction effect of radiation on the expression of OATP1A2. SIGNIFICANCE STATEMENT: The present study found that the effect of X-rays on v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES-1) and organic anion transporting polypeptides (OATP)1A2 was temporally coordinated. YES-1 phosphorylates and increases the nuclear accumulation of signal transducer and activator of transcription 3, which in turn binds to the upstream regulatory sequences of SLCO1A2, the coding gene for OATP1A2. Hence, inhibitors of YES-1 may suppress the radiation induction effect on OATP1A2.

Circadian plasticity evolves through regulatory changes in a neuropeptide gene.

Many organisms, including cosmopolitan drosophilids, show circadian plasticity, varying their activity with changing dawn-dusk intervals1. How this behaviour evolves is unclear. Here we compare Drosophila melanogaster with Drosophila sechellia, an equatorial, ecological specialist that experiences minimal photoperiod variation, to investigate the mechanistic basis of circadian plasticity evolution2. D. sechellia has lost the ability to delay its evening activity peak time under long photoperiods. Screening of circadian mutants in D. melanogaster/D. sechellia hybrids identifies a contribution of the neuropeptide pigment-dispersing factor (Pdf) to this loss. Pdf exhibits species-specific temporal expression, due in part to cis-regulatory divergence. RNA interference and rescue experiments in D. melanogaster using species-specific Pdf regulatory sequences demonstrate that modulation of this neuropeptide's expression affects the degree of behavioural plasticity. The Pdf regulatory region exhibits signals of selection in D. sechellia and across populations of D. melanogaster from different latitudes. We provide evidence that plasticity confers a selective advantage for D. melanogaster at elevated latitude, whereas D. sechellia probably suffers fitness costs through reduced copulation success outside its range. Our findings highlight this neuropeptide gene as a hotspot locus for circadian plasticity evolution that might have contributed to both D. melanogaster's global distribution and D. sechellia's specialization.

Regulatory genome annotation of 33 insect species

Annotation of newly sequenced genomes frequently includes genes, but rarely covers important non-coding genomic features such as the cis-regulatory modules—e.g., enhancers and silencers—that regulate gene expression. Here, we begin to remedy this situation by developing a workflow for rapid initial annotation of insect regulatory sequences, and provide a searchable database resource with enhancer predictions for 33 genomes. Using our previously developed SCRMshaw computational enhancer prediction method, we predict over 2.8 million regulatory sequences along with the tissues where they are expected to be active, in a set of insect species ranging over 360 million years of evolution. Extensive analysis and validation of the data provides several lines of evidence suggesting that we achieve a high true-positive rate for enhancer prediction. One, we show that our predictions target specific loci, rather than random genomic locations. Two, we predict enhancers in orthologous loci across a diverged set of species to a significantly higher degree than random expectation would allow. Three, we demonstrate that our predictions are highly enriched for regions of accessible chromatin. Four, we achieve a validation rate in excess of 70% using in vivo reporter gene assays. As we continue to annotate both new tissues and new species, our regulatory annotation resource will provide a rich source of data for the research community and will have utility for both small-scale (single gene, single species) and large-scale (many genes, many species) studies of gene regulation. In particular, the ability to search for functionally related regulatory elements in orthologous loci should greatly facilitate studies of enhancer evolution even among distantly related species.

Regulatory genome annotation of 33 insect species.

Annotation of newly sequenced genomes frequently includes genes, but rarely covers important non-coding genomic features such as the cis-regulatory modules-e.g., enhancers and silencers-that regulate gene expression. Here, we begin to remedy this situation by developing a workflow for rapid initial annotation of insect regulatory sequences, and provide a searchable database resource with enhancer predictions for 33 genomes. Using our previously developed SCRMshaw computational enhancer prediction method, we predict over 2.8 million regulatory sequences along with the tissues where they are expected to be active, in a set of insect species ranging over 360 million years of evolution. Extensive analysis and validation of the data provides several lines of evidence suggesting that we achieve a high true-positive rate for enhancer prediction. One, we show that our predictions target specific loci, rather than random genomic locations. Two, we predict enhancers in orthologous loci across a diverged set of species to a significantly higher degree than random expectation would allow. Three, we demonstrate that our predictions are highly enriched for regions of accessible chromatin. Four, we achieve a validation rate in excess of 70% using in vivo reporter gene assays. As we continue to annotate both new tissues and new species, our regulatory annotation resource will provide a rich source of data for the research community and will have utility for both small-scale (single gene, single species) and large-scale (many genes, many species) studies of gene regulation. In particular, the ability to search for functionally related regulatory elements in orthologous loci should greatly facilitate studies of enhancer evolution even among distantly related species.

Evolutionary Modes of wtf Meiotic Driver Genes in Schizosaccharomyces pombe.

Killer meiotic drivers are a class of selfish genetic elements that bias inheritance in their favor by destroying meiotic progeny that do not carry them. How killer meiotic drivers evolve is not well understood. In the fission yeast, Schizosaccharomyces pombe, the largest gene family, known as the wtf genes, is a killer meiotic driver family that causes intraspecific hybrid sterility. Here, we investigate how wtf genes evolve using long-read-based genome assemblies of 31 distinct S. pombe natural isolates, which encompass the known genetic diversity of S. pombe. Our analysis, involving nearly 1,000 wtf genes in these isolates, yields a comprehensive portrayal of the intraspecific diversity of wtf genes. Leveraging single-nucleotide polymorphisms in adjacent unique sequences, we pinpoint wtf gene-containing loci that have recently undergone gene conversion events and infer their ancestral state. These events include the revival of wtf pseudogenes, lending support to the notion that gene conversion plays a role in preserving this gene family from extinction. Moreover, our investigation reveals that solo long terminal repeats of retrotransposons, frequently found near wtf genes, can act as recombination arms, influencing the upstream regulatory sequences of wtf genes. Additionally, our exploration of the outer boundaries of wtf genes uncovers a previously unrecognized type of directly oriented repeats flanking wtf genes. These repeats may have facilitated the early expansion of the wtf gene family in S. pombe. Our findings enhance the understanding of the mechanisms influencing the evolution of this killer meiotic driver gene family.

Semi-supervised learning with pseudo-labeling compares favorably with large language models for regulatory sequence prediction.

Predicting molecular processes using deep learning is a promising approach to provide biological insights for non-coding single nucleotide polymorphisms identified in genome-wide association studies. However, most deep learning methods rely on supervised learning, which requires DNA sequences associated with functional data, and whose amount is severely limited by the finite size of the human genome. Conversely, the amount of mammalian DNA sequences is growing exponentially due to ongoing large-scale sequencing projects, but in most cases without functional data. To alleviate the limitations of supervised learning, we propose a novel semi-supervised learning (SSL) based on pseudo-labeling, which allows to exploit unlabeled DNA sequences from numerous genomes during model pre-training. We further improved it incorporating principles from the Noisy Student algorithm to predict the confidence in pseudo-labeled data used for pre-training, which showed improvements for transcription factor with very few binding (very small training data). The approach is very flexible and can be used to train any neural architecture including state-of-the-art models, and shows in most cases strong predictive performance improvements compared to standard supervised learning. Moreover, small models trained by SSL showed similar or better performance than large language model DNABERT2.

A Synthetic Biology Approach to Transgene Expression in Insects.

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most nonmodel and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3' untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and 5' regulatory sequences, representing a significant advancement in mosquito synthetic biology gene expression tools.

Transdifferentiation occurs without resetting development-specific DNA methylation, a key determinant of full-function cell identity

A number of studies have demonstrated that it is possible to directly convert one cell type to another by factor-mediated transdifferentiation, but in the vast majority of cases, the resulting reprogrammed cells are unable to maintain their new cell identity for prolonged culture times and have a phenotype only partially similar to their endogenous counterparts. To better understand this phenomenon, we developed an analytical approach for better characterizing trans-differentiation-associated changes in DNA methylation, a major determinant of long-term cell identity. By examining various models of transdifferentiation both in vitro and in vivo, our studies indicate that despite convincing expression changes, transdifferentiated cells seem unable to alter their original developmentally mandated methylation patterns. We propose that this blockage is due to basic developmental limitations built into the regulatory sequences that govern epigenetic programming of cell identity. These results shed light on the molecular rules necessary to achieve complete somatic cell reprogramming.

Widespread position-dependent transcriptional regulatory sequences in plants

Much of what we know about eukaryotic transcription stems from animals and yeast; however, plants evolved separately for over a billion years, leaving ample time for divergence in transcriptional regulation. Here we set out to elucidate fundamental properties of cis-regulatory sequences in plants. Using massively parallel reporter assays across four plant species, we demonstrate the central role of sequences downstream of the transcription start site (TSS) in transcriptional regulation. Unlike animal enhancers that are position independent, plant regulatory elements depend on their position, as altering their location relative to the TSS significantly affects transcription. We highlight the importance of the region downstream of the TSS in regulating transcription by identifying a DNA motif that is conserved across vascular plants and is sufficient to enhance gene expression in a dose-dependent manner. The identification of a large number of position-dependent enhancers points to fundamental differences in gene regulation between plants and animals.

Adjusting methylation levels with nucleus proportions highlights functional significance of differentially methylated cytosines associated with pre-eclampsia.

Studies on DNA methylation alterations associated with pre-eclampsia (PE) have improved our understanding of the mechanisms underlying this disorder. However, differentially methylated cytosines (DMCs) have not been adjusted for cell-type heterogeneity, hampering the identification of alterations that drive disease risk. Using a reference-based, cell-type deconvolution approach, we estimated the nuclear proportions of 335 placental samples based on DNA methylation data. We found that the nuclei of total trophoblast lineages accounted for more than 80% of the placental samples, with a significant increase in PE placentas. The nuclear proportions of stromal and Hofbauer cells decreased in PE placentas. Our nuclear proportion estimation reflected previous histological knowledge on the changes in cell type proportions in PE placentas. We corrected 2125 DMCs associated with early-onset PE for cell-type heterogeneity by adjusting for the nuclear proportions and observed a notable reduction in the association signals, with 145 probes not reaching epigenome-wide significance. After correction, the top 200 significant DMCs were strongly enriched in active enhancers in trophoblast lineages, whereas 145 non-significant probes were enriched in regions with a quiescent state of chromatin. Our results suggest that future epigenetic studies of PE should focus on functional regulatory sequences.

CRISPR-CLEAR: Nucleotide-Resolution Mapping of Regulatory Elements via Allelic Readout of Tiled Base Editing.

CRISPR tiling screens have advanced the identification and characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing. This study introduces CRISPR-CLEAR , an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. This approach enables the dissection of regulatory elements at nucleotide resolution, facilitating a direct assessment of genotype-phenotype effects.

Identification and functional analysis of recent IS transposition events in rhizobia

Rhizobia are alpha- and beta- Proteobacteria that, through the establishment of symbiotic interactions with leguminous plants, are able to fix atmospheric nitrogen as ammonium. The successful establishment of a symbiotic interaction is highly dependent on the availability of nitrogen sources in the soil, and on the specific rhizobia strain. Insertion sequences (ISs) are simple transposable genetic elements that can move to different locations within the host genome and are known to play an important evolutionary role, contributing to genome plasticity by acting as recombination hot-spots, and disrupting coding and regulatory sequences. Disruption of coding sequences may have occurred either in a common ancestor of the species or more recently. By means of ISComapare, we identified Differentially Located ISs (DLISs) in nearly related rhizobial strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium. Our results revealed that recent IS transposition could have a role in adaptation by enabling the activation and inactivation of genes that could dynamically affect the competition and survival of rhizobia in the rhizosphere.Graphical

Mechanisms of transcriptional regulation in Anopheles gambiae revealed by allele-specific expression.

Malaria control relies on insecticides targeting the mosquito vector, but this is increasingly compromised by insecticide resistance, which can be achieved by elevated expression of detoxifying enzymes that metabolize the insecticide. In diploid organisms, gene expression is regulated both in cis, by regulatory sequences on the same chromosome, and by trans acting factors, affecting both alleles equally. Differing levels of transcription can be caused by mutations in cis-regulatory modules (CRM), but few of these have been identified in mosquitoes. We crossed bendiocarb-resistant and susceptible Anopheles gambiae strains to identify cis-regulated genes that might be responsible for the resistant phenotype using RNAseq, and CRM sequences controlling gene expression in insecticide resistance relevant tissues were predicted using machine learning. We found 115 genes showing allele-specific expression (ASE) in hybrids of insecticide susceptible and resistant strains, suggesting cis-regulation is an important mechanism of gene expression regulation in A. gambiae. The genes showing ASE included a higher proportion of Anopheles-specific genes on average younger than genes with balanced allelic expression.

A STAR for the ages: a 30-year historical perspective of the role of transcription factors in the regulation of steroidogenic acute regulatory gene expression.

The steroidogenic acute regulatory (STAR) protein is an essential cholesterol transporter that shuttles cholesterol from the outer to the inner mitochondrial membrane in the major steroidogenic endocrine organs. It is a key player in the acute regulation of steroid hormone biosynthesis in response to tropic hormone stimulation. Its discovery 30 years ago sparked immediate interest in understanding how STAR action is controlled. Since increased STAR gene expression is a classic feature of the acute regulation of steroidogenesis, a special emphasis was placed on defining the transcriptional regulatory mechanisms that underlie its rapid induction in response to tropic hormone stimulation. These mechanisms include the effects of enhancers, the regulation of chromatin accessibility, the impact of epigenetic factors, and the role of transcription factors. Over the past three decades, understanding the transcription factors that regulate STAR gene expression has been the subject of more than 170 independent scientific publications, making it one of, and if not the best, studied genes in the steroidogenic pathway. This intense research effort has led to the identification of dozens of transcription factors and their related binding sites in STAR 5' flanking (promoter) sequences across multiple species. STAR gene transcription appears to be complex in that a large number of transcription factors have been proposed to interact with either isolated or overlapping regulatory sequences that are tightly clustered over a relatively short promoter region upstream of the STAR transcription start site. Many of these transcription factors appear to work in unique combinatorial codes and are impacted by diverse hormonal and intracellular signaling pathways. This review provides a retrospective overview of the transcription factors proposed to regulate both basal and acute (hormonal) STAR gene expression, and how insights in this area have evolved over the past 30 years.