RESUMO
R-loops are three-stranded nucleotide structures consisting of a DNA:RNA hybrid and a displaced ssDNA non-template strand. Previous work suggests that R-loop formation is primarily determined by the thermodynamics of DNA:RNA binding, which are governed by base composition (e.g., GC skew) and transcription-induced DNA superhelicity. However, R-loops have been described at genomic locations that lack these properties, suggesting that they may serve other context-specific roles. To better understand the genetic determinants of R-loop formation, we have characterized the Drosophila melanogaster R-loop landscape across strains and between sexes using DNA:RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq). We find that R-loops are associated with sequence motifs that are G-rich or exhibit G/C skew, as well as highly expressed genes, tRNAs, and small nuclear RNAs, consistent with a role for DNA sequence and torsion in R-loop specification. However, we also find motifs associated with R-loops that are A/T-rich and lack G/C skew as well as a subset of R-loops that are enriched in polycomb-repressed chromatin. Differential enrichment analysis reveals a small number of sex-biased R-loops: while non-differentially enriched and male-enriched R-loops form at similar genetic features and chromatin states and contain similar sequence motifs, female-enriched R-loops form at unique genetic features, chromatin states, and sequence motifs and are associated with genes that show ovary-biased expression. Male-enriched R-loops are most abundant on the dosage-compensated X chromosome, where R-loops appear stronger compared to autosomal R-loops. R-loop-containing genes on the X chromosome are dosage-compensated yet show lower MOF binding and reduced H4K16ac compared to R-loop-absent genes, suggesting that H4K16ac or MOF may attenuate R-loop formation. Collectively, these results suggest that R-loop formation in vivo is not fully explained by DNA sequence and topology and raise the possibility that a distinct subset of these hybrid structures plays an important role in the establishment and maintenance of epigenetic differences between sexes.
Assuntos
Drosophila melanogaster , Estruturas R-Loop , Animais , Cromatina/genética , DNA/genética , Drosophila melanogaster/genética , Feminino , Masculino , RNA/genéticaRESUMO
Topologically associating domains (TADs) are thought to play an important role in preventing gene misexpression by spatially constraining enhancer-promoter contacts. The deleterious nature of gene misexpression implies that TADs should, therefore, be conserved among related species. Several early studies comparing chromosome conformation between species reported high levels of TAD conservation; however, more recent studies have questioned these results. Furthermore, recent work suggests that TAD reorganization is not associated with extensive changes in gene expression. Here, we investigate the evolutionary conservation of TADs among 11 species of Drosophila. We use Hi-C data to identify TADs in each species and employ a comparative phylogenetic approach to derive empirical estimates of the rate of TAD evolution. Surprisingly, we find that TADs evolve rapidly. However, we also find that the rate of evolution depends on the chromatin state of the TAD, with TADs enriched for developmentally regulated chromatin evolving significantly slower than TADs enriched for broadly expressed, active chromatin. We also find that, after controlling for differences in chromatin state, highly conserved TADs do not exhibit higher levels of gene expression constraint. These results suggest that, in general, most TADs evolve rapidly and their divergence is not associated with widespread changes in gene expression. However, higher levels of evolutionary conservation and gene expression constraints in TADs enriched for developmentally regulated chromatin suggest that these TAD subtypes may be more important for regulating gene expression, likely due to the larger number of long-distance enhancer-promoter contacts associated with developmental genes.
Assuntos
Drosophila , Genoma , Animais , Drosophila/genética , Filogenia , Cromatina/genética , Evolução MolecularRESUMO
Coevolution between transposable elements (TEs) and their hosts can be antagonistic, where TEs evolve to avoid silencing and the host responds by reestablishing TE suppression, or mutualistic, where TEs are co-opted to benefit their host. The TART-A TE functions as an important component of Drosophila telomeres but has also reportedly inserted into the Drosophila melanogaster nuclear export factor gene nxf2. We find that, rather than inserting into nxf2, TART-A has actually captured a portion of nxf2 sequence. We show that TART-A produces abundant Piwi-interacting small RNAs (piRNAs), some of which are antisense to the nxf2 transcript, and that the TART-like region of nxf2 is evolving rapidly. Furthermore, in D. melanogaster, TART-A is present at higher copy numbers, and nxf2 shows reduced expression, compared to the closely related species Drosophila simulans. We propose that capturing nxf2 sequence allowed TART-A to target the nxf2 gene for piRNA-mediated repression and that these 2 elements are engaged in antagonistic coevolution despite the fact that TART-A is serving a critical role for its host genome.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , RNA Interferente Pequeno/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Evolução Molecular , Elementos Nucleotídeos Longos e Dispersos , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Topologically associating domains, or TADs, are functional units that organize chromosomes into 3D structures of interacting chromatin. TADs play an important role in regulating gene expression by constraining enhancer-promoter contacts and there is evidence that deletion of TAD boundaries leads to aberrant expression of neighboring genes. While the mechanisms of TAD formation have been well-studied, current knowledge on the patterns of TAD evolution across species is limited. Due to the integral role TADs play in gene regulation, their structure and organization is expected to be conserved during evolution. However, more recent research suggests that TAD structures diverge relatively rapidly. We use Hi-C chromosome conformation capture to measure evolutionary conservation of whole TADs and TAD boundary elements between D. melanogaster and D. triauraria, two early-branching species from the melanogaster species group which diverged â¼15 million years ago. We find that the majority of TADs have been reorganized since the common ancestor of D. melanogaster and D. triauraria, via a combination of chromosomal rearrangements and gain/loss of TAD boundaries. TAD reorganization between these two species is associated with a localized effect on gene expression, near the site of disruption. By separating TADs into subtypes based on their chromatin state, we find that different subtypes are evolving under different evolutionary forces. TADs enriched for broadly expressed, transcriptionally active genes are evolving rapidly, potentially due to positive selection, whereas TADs enriched for developmentally-regulated genes remain conserved, presumably due to their importance in restricting gene-regulatory element interactions. These results provide novel insight into the evolutionary dynamics of TADs and help to reconcile contradictory reports related to the evolutionary conservation of TADs and whether changes in TAD structure affect gene expression.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromossomos de Insetos/genética , Evolução Molecular , Genoma de Inseto , Animais , Sequência Conservada , Drosophila melanogaster , Rearranjo Gênico , Transcrição GênicaRESUMO
Illumina sequencing has allowed for population-level surveys of transposable element (TE) polymorphism via split alignment approaches, which has provided important insight into the population dynamics of TEs. However, such approaches are not able to identify insertions of uncharacterized TEs, nor can they assemble the full sequence of inserted elements. Here, we use nanopore sequencing and Hi-C scaffolding to produce de novo genome assemblies for two wild strains of Drosophila melanogaster from the Drosophila Genetic Reference Panel (DGRP). Ovarian piRNA populations and Illumina split-read TE insertion profiles have been previously produced for both strains. We find that nanopore sequencing with Hi-C scaffolding produces highly contiguous, chromosome-length scaffolds, and we identify hundreds of TE insertions that were missed by Illumina-based methods, including a novel micropia-like element that has recently invaded the DGRP population. We also find hundreds of piRNA-producing loci that are specific to each strain. Some of these loci are created by strain-specific TE insertions, while others appear to be epigenetically controlled. Our results suggest that Illumina approaches reveal only a portion of the repetitive sequence landscape of eukaryotic genomes and that population-level resequencing using long reads is likely to provide novel insight into the evolutionary dynamics of repetitive elements.
Assuntos
Elementos de DNA Transponíveis , DNA/genética , Drosophila melanogaster/genética , Genoma de Inseto , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Evolução Biológica , DNA/química , DNA/metabolismo , Drosophila melanogaster/metabolismo , Epigênese Genética , Feminino , Loci Gênicos , Heterocromatina/química , Heterocromatina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Mutagênese Insercional , Sequenciamento por Nanoporos/métodos , Ovário/metabolismo , Polimorfismo Genético , RNA Interferente Pequeno/metabolismoRESUMO
Dosage compensation has arisen in response to the evolution of distinct male (XY) and female (XX) karyotypes. In Drosophila melanogaster, the MSL complex increases male X transcription approximately twofold. X-specific targeting is thought to occur through sequence-dependent binding to chromatin entry sites (CESs), followed by spreading in cis to active genes. We tested this model by asking how newly evolving sex chromosome arms in Drosophila miranda acquired dosage compensation. We found evidence for the creation of new CESs, with the analogous sequence and spacing as in D. melanogaster, providing strong support for the spreading model in the establishment of dosage compensation.
Assuntos
Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Cromossomos Sexuais/genética , Animais , Evolução Molecular , Feminino , Cariótipo , Masculino , Dados de Sequência Molecular , Cromossomos Sexuais/metabolismoRESUMO
Research over the past two decades shows that both recombination and clonality are likely to contribute to the reproduction of all fungi. This view of fungi is different from the historical and still commonly held view that a large fraction of fungi are exclusively clonal and that some fungi have been exclusively clonal for hundreds of millions of years. Here, we first will consider how these two historical views have changed. Then we will examine the impact on fungal research of the concept of restrained recombination [Tibayrenc M, Ayala FJ (2012) Proc Natl Acad Sci USA 109 (48):E3305-E3313]. Using animal and human pathogenic fungi, we examine extrinsic restraints on recombination associated with bottlenecks in genetic variation caused by geographic dispersal and extrinsic restraints caused by shifts in reproductive mode associated with either disease transmission or hybridization. Using species of the model yeast Saccharomyces and the model filamentous fungus Neurospora, we examine intrinsic restraints on recombination associated with mating systems that range from strictly clonal at one extreme to fully outbreeding at the other and those that lie between, including selfing and inbreeding. We also consider the effect of nomenclature on perception of reproductive mode and a means of comparing the relative impact of clonality and recombination on fungal populations. Last, we consider a recent hypothesis suggesting that fungi thought to have the most severe intrinsic constraints on recombination actually may have the fewest.
Assuntos
Fungos/fisiologia , Animais , Células Clonais , Fungos/genética , Genética Populacional , Genótipo , Humanos , Micorrizas/fisiologia , Recombinação Genética/genética , ReproduçãoRESUMO
Recent advancements in sequencing technology allowed researchers to better address the patterns and mechanisms involved in microbial environmental adaptation at large spatial scales. Here we investigated the genomic basis of adaptation to climate at the continental scale in Suillus brevipes, an ectomycorrhizal fungus symbiotically associated with the roots of pine trees. We used genomic data from 55 individuals in seven locations across North America to perform genome scans to detect signatures of positive selection and assess whether temperature and precipitation were associated with genetic differentiation. We found that S. brevipes exhibited overall strong population differentiation, with potential admixture in Canadian populations. This species also displayed genomic signatures of positive selection as well as genomic sites significantly associated with distinct climatic regimes and abiotic environmental parameters. These genomic regions included genes involved in transmembrane transport of substances and helicase activity potentially involved in cold stress response. Our study sheds light on large-scale environmental adaptation in fungi by identifying putative adaptive genes and providing a framework to further investigate the genetic basis of fungal adaptation.
Assuntos
Adaptação Fisiológica/genética , Agaricales/genética , Genética Populacional , Seleção Genética , Basidiomycota/genética , Canadá , Clima , Resposta ao Choque Frio/genética , DNA Fúngico/genética , Genoma Fúngico , Genótipo , Desequilíbrio de Ligação , Micorrizas/genética , América do Norte , Pinus/microbiologia , Chuva , Neve , TemperaturaRESUMO
Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.
Assuntos
Mecanismo Genético de Compensação de Dose , Drosophila/genética , Heterocromatina/genética , Cromossomos Sexuais/genética , Animais , Sequência de Bases , Sítios de Ligação , Evolução Molecular , Feminino , Expressão Gênica , Masculino , Anotação de Sequência Molecular , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Fungi are an omnipresent and highly diverse group of organisms, making up a significant part of eukaryotic diversity. Little is currently known about the drivers of fungal population differentiation and subsequent divergence of species, particularly in symbiotic, mycorrhizal fungi. Here, we investigate the population structure and environmental adaptation in Suillus brevipes (Peck) Kuntze, a wind-dispersed soil fungus that is symbiotic with pine trees. We assembled and annotated the reference genome for Su. brevipes and resequenced the whole genomes of 28 individuals from coastal and montane sites in California. We detected two clearly delineated coast and mountain populations with very low divergence. Genomic divergence was restricted to few regions, including a region of extreme divergence containing a gene encoding for a membrane Na(+) /H(+) exchanger known for enhancing salt tolerance in plants and yeast. Our results are consistent with a very recent split between the montane and coastal Su. brevipes populations, with few small genomic regions under positive selection and a pattern of dispersal and/or establishment limitation. Furthermore, we identify a putatively adaptive gene that motivates further functional analyses to link genotypes and phenotypes and shed light on the genetic basis of adaptive traits.
Assuntos
Basidiomycota/genética , Especiação Genética , Genética Populacional , Isolamento Reprodutivo , California , DNA Fúngico/genética , Ecossistema , Genoma Fúngico , Funções Verossimilhança , Micorrizas/genética , Pinus/microbiologia , Seleção Genética , Análise de Sequência de DNA , Microbiologia do Solo , SimbioseRESUMO
Elucidating the connection between genotype, phenotype, and adaptation in wild populations is fundamental to the study of evolutionary biology, yet it remains an elusive goal, particularly for microscopic taxa, which comprise the majority of life. Even for microbes that can be reliably found in the wild, defining the boundaries of their populations and discovering ecologically relevant phenotypes has proved extremely difficult. Here, we have circumvented these issues in the microbial eukaryote Neurospora crassa by using a "reverse-ecology" population genomic approach that is free of a priori assumptions about candidate adaptive alleles. We performed Illumina whole-transcriptome sequencing of 48 individuals to identify single nucleotide polymorphisms. From these data, we discovered two cryptic and recently diverged populations, one in the tropical Caribbean basin and the other endemic to subtropical Louisiana. We conducted high-resolution scans for chromosomal regions of extreme divergence between these populations and found two such genomic "islands." Through growth-rate assays, we found that the subtropical Louisiana population has a higher fitness at low temperature (10 °C) and that several of the genes within these distinct regions have functions related to the response to cold temperature. These results suggest the divergence islands may be the result of local adaptation to the 9 °C difference in average yearly minimum temperature between these two populations. Remarkably, another of the genes identified using this unbiased, whole-genome approach is the well-known circadian oscillator frequency, suggesting that the 2.4°-10.6° difference in latitude between the populations may be another important environmental parameter.
Assuntos
Adaptação Biológica/fisiologia , Evolução Biológica , Variação Genética , Genética Populacional , Neurospora crassa/genética , Temperatura , Adaptação Biológica/genética , Sequência de Bases , Teorema de Bayes , Demografia , Aptidão Genética/genética , Genômica/métodos , Geografia , Louisiana , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Especificidade da Espécie , Índias OcidentaisRESUMO
Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and sequencing of immunoprecipitated double-stranded RNA were used to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions and maintaining intestinal health.
Assuntos
Histona Acetiltransferases , Interferons , Camundongos Knockout , RNA de Cadeia Dupla , Transdução de Sinais , Células-Tronco , Animais , RNA de Cadeia Dupla/metabolismo , Camundongos , Células-Tronco/metabolismo , Células-Tronco/citologia , Interferons/metabolismo , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citologia , Mitocôndrias/metabolismo , Autorrenovação Celular/genética , Intestinos/citologiaRESUMO
Transposable elements (TEs) are ubiquitous among eukaryotic species. Their evolutionary persistence is likely due to a combination of tolerogenic, evasive/antagonistic, and cooperative interactions with their host genomes. Here, we focus on metazoan species and review recent advances related to the harmful effects of TE insertions, including how epigenetic effects and TE-derived RNAs can damage host cells. We discuss new findings related to host pathways that silence TEs, such as the piRNA pathway and the APOBEC3 and Kruppel-associated box zinc finger gene families. Finally, we summarize novel strategies used by TEs to evade host silencing, including the Y chromosome as a permissive niche for TE mobilization and TE counterdefense strategies to block host silencing factors.
Assuntos
Elementos de DNA Transponíveis , Inativação Gênica , Animais , Elementos de DNA Transponíveis/genética , RNA Interferente Pequeno/genética , Evolução Molecular , Evolução BiológicaRESUMO
Flow cytometry estimates of genome sizes among species of Drosophila show a 3-fold variation, ranging from â¼127 Mb in Drosophila mercatorum to â¼400 Mb in Drosophila cyrtoloma. However, the assembled portion of the Muller F element (orthologous to the fourth chromosome in Drosophila melanogaster) shows a nearly 14-fold variation in size, ranging from â¼1.3 Mb to >18 Mb. Here, we present chromosome-level long-read genome assemblies for 4 Drosophila species with expanded F elements ranging in size from 2.3 to 20.5 Mb. Each Muller element is present as a single scaffold in each assembly. These assemblies will enable new insights into the evolutionary causes and consequences of chromosome size expansion.
Assuntos
Drosophila melanogaster , Drosophila , Animais , Drosophila/genética , Drosophila melanogaster/genética , Cromossomos/genética , GenomaRESUMO
Flow cytometry estimates of genome sizes among species of Drosophila show a 3-fold variation, ranging from â¼127 Mb in Drosophila mercatorum to â¼400 Mb in Drosophila cyrtoloma . However, the assembled portion of the Muller F Element (orthologous to the fourth chromosome in Drosophila melanogaster ) shows a nearly 14-fold variation in size, ranging from â¼1.3 Mb to > 18 Mb. Here, we present chromosome-level long read genome assemblies for four Drosophila species with expanded F Elements ranging in size from 2.3 Mb to 20.5 Mb. Each Muller Element is present as a single scaffold in each assembly. These assemblies will enable new insights into the evolutionary causes and consequences of chromosome size expansion.
RESUMO
Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and dsRIP-seq were employed to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions as well as maintaining intestinal health.
RESUMO
Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of Drosophila testis are not well understood. Here, we reanalyze publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We develop a method for identification of TE and host gene expression modules and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are specifically enriched on the Y chromosome and depleted on the X chromosome, relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y chromosome-specific transcriptional programs.
Assuntos
Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Espermatogênese/genética , Cromossomo Y/genética , Animais , Drosophila melanogaster/citologia , Evolução Molecular , Expressão Gênica , Redes Reguladoras de Genes , Genes Ligados ao Cromossomo Y/genética , Masculino , Mutagênese Insercional , RNA Interferente Pequeno/genética , Espermatócitos/metabolismo , Testículo/citologia , Testículo/metabolismo , Cromossomo Y/metabolismoRESUMO
Cells in renewing tissues exhibit dramatic transcriptional changes as they differentiate. The contribution of chromatin looping to tissue renewal is incompletely understood. Enhancer-promoter interactions could be relatively stable as cells transition from progenitor to differentiated states; alternatively, chromatin looping could be as dynamic as the gene expression from their loci. The intestinal epithelium is the most rapidly renewing mammalian tissue. Proliferative cells in crypts of Lieberkühn sustain a stream of differentiated cells that are continually shed into the lumen. We apply chromosome conformation capture combined with chromatin immunoprecipitation (HiChIP) and sequencing to measure enhancer-promoter interactions in progenitor and differentiated cells of the intestinal epithelium. Despite dynamic gene regulation across the differentiation axis, we find that enhancer-promoter interactions are relatively stable. Functionally, we find HNF4 transcription factors are required for chromatin looping at target genes. Depletion of HNF4 disrupts local chromatin looping, histone modifications, and target gene expression. This study provides insights into transcriptional regulatory mechanisms governing homeostasis in renewing tissues.
Assuntos
Fator 4 Nuclear de Hepatócito/genética , Mucosa Intestinal/fisiologia , Regiões Promotoras Genéticas/genética , Diferenciação Celular/genética , Cromatina/genética , Elementos Facilitadores Genéticos , Humanos , Mucosa Intestinal/citologiaRESUMO
The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.
Assuntos
Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Mucosa Intestinal/metabolismo , Rim/metabolismo , Microvilosidades/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Saco Vitelino/metabolismo , Animais , Epitélio/metabolismo , Perfilação da Expressão Gênica/métodos , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Intestinos/ultraestrutura , Rim/ultraestrutura , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Receptores Citoplasmáticos e Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
N6-methyladenine (6mA or m6dA) is a DNA modification that has long been known to play an important role in a variety of biological functions in prokaryotes. This modification has only recently been described in eukaryotes, where it seems to have evolved species-specific functions ranging from nucleosome positioning to transposon repression. In Drosophila, 6mA has been shown to be important for enforcing the tissue specificity of neuronal genes in the brain and suppressing transposable element expression in the ovaries. In this study, we have analyzed the raw signal data from nanopore sequencing to identify 6mA positions in the D. melanogaster genome at single-base resolution. We find that this modification is enriched upstream from transcription start sites, within the introns and 3' UTRs of genes, as well as in simple repeats. These 6mA positions are enriched for sequence motifs that are recognized by known transcriptional activators involved in development, such as Bicoid and Caudal, and the genes that carry this modification are enriched for functions involved in development, regulation of transcription, and neuronal activity. These genes show high expression specificity in a variety of tissues besides the brain, suggesting that this modification may play a more general role in enforcing the specificity of gene expression across many tissues, throughout development, and between the sexes.