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1.
Mol Cell ; 84(11): 2017-2035.e6, 2024 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-38795706

RESUMEN

Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wild-type tissues is the segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a⋅H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, the self-association of pericentromeric regions is largely preserved despite the loss of H3K9 methylation and HP1a occupancy. Thus, the HP1a⋅H3K9 interaction contributes to but does not solely drive the segregation of euchromatin and heterochromatin inside the nucleus.


Asunto(s)
Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona , Proteínas de Drosophila , Drosophila melanogaster , Heterocromatina , Histonas , Heterocromatina/metabolismo , Heterocromatina/genética , Animales , Histonas/metabolismo , Histonas/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metilación , Eucromatina/metabolismo , Eucromatina/genética , Centrómero/metabolismo , Centrómero/genética , Unión Proteica , Genoma de los Insectos , Segregación Cromosómica , Procesamiento Proteico-Postraduccional
2.
Cell ; 159(6): 1249-51, 2014 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-25480289

RESUMEN

A high-resolution look at where histones touch DNA reveals a surprisingly intricate, dynamic, and modular nucleosome. Three advances in the study by Rhee et al. include unexpected interactions between the H3 tail and linker DNA, new evidence for existence of subnucleosomal particles, and asymmetric patterns of histone modification within a single nucleosome that correspond to the direction of transcription.


Asunto(s)
Nucleosomas/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética
3.
Proc Natl Acad Sci U S A ; 119(40): e2208935119, 2022 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-36161884

RESUMEN

Steroid hormones perform diverse biological functions in developing and adult animals. However, the mechanistic basis for their tissue specificity remains unclear. In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its nuclear receptor, a heterodimer of the EcR and ultraspiracle proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates morphogenesis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Finally, despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.


Asunto(s)
Cromatina , Proteínas de Drosophila , Drosophila melanogaster , Ecdisona , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Receptores de Esteroides , Animales , Cromatina/metabolismo , ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Ligandos , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo
4.
Genes Dev ; 31(9): 862-875, 2017 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-28536147

RESUMEN

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility.


Asunto(s)
Cromatina/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Ecdisona/metabolismo , Regulación del Desarrollo de la Expresión Génica , Alas de Animales/metabolismo , Animales , Cromatina/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos de Facilitación Genéticos/genética , Femenino , Pupa/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Alas de Animales/crecimiento & desarrollo
5.
Genes Dev ; 30(16): 1866-80, 2016 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-27566777

RESUMEN

A defining feature of heterochromatin is methylation of Lys9 of histone H3 (H3K9me), a binding site for heterochromatin protein 1 (HP1). Although H3K9 methyltransferases and HP1 are necessary for proper heterochromatin structure, the specific contribution of H3K9 to heterochromatin function and animal development is unknown. Using our recently developed platform to engineer histone genes in Drosophila, we generated H3K9R mutant flies, separating the functions of H3K9 and nonhistone substrates of H3K9 methyltransferases. Nucleosome occupancy and HP1a binding at pericentromeric heterochromatin are markedly decreased in H3K9R mutants. Despite these changes in chromosome architecture, a small percentage of H3K9R mutants complete development. Consistent with this result, expression of most protein-coding genes, including those within heterochromatin, is similar between H3K9R and controls. In contrast, H3K9R mutants exhibit increased open chromatin and transcription from piRNA clusters and transposons, resulting in transposon mobilization. Hence, transposon silencing is a major developmental function of H3K9.


Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Animales , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/química , Cromosomas/genética , Elementos Transponibles de ADN/genética , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Mutación , Nucleosomas/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética
6.
Development ; 147(6)2020 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-32094114

RESUMEN

How temporal cues combine with spatial inputs to control gene expression during development is poorly understood. Here, we test the hypothesis that the Drosophila transcription factor E93 controls temporal gene expression by regulating chromatin accessibility. Precocious expression of E93 early in wing development reveals that it can simultaneously activate and deactivate different target enhancers. Notably, the precocious patterns of enhancer activity resemble the wild-type patterns that occur later in development, suggesting that expression of E93 alters the competence of enhancers to respond to spatial cues. Genomic profiling reveals that precocious E93 expression is sufficient to regulate chromatin accessibility at a subset of its targets. These accessibility changes mimic those that normally occur later in development, indicating that precocious E93 accelerates the wild-type developmental program. Further, we find that target enhancers that do not respond to precocious E93 in early wings become responsive after a developmental transition, suggesting that parallel temporal pathways work alongside E93. These findings support a model wherein E93 expression functions as an instructive cue that defines a broad window of developmental time through control of chromatin accessibility.


Asunto(s)
Cromatina/metabolismo , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Elementos de Facilitación Genéticos/fisiología , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Cromatina/química , Ensamble y Desensamble de Cromatina/fisiología , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Metamorfosis Biológica/genética , Unión Proteica , Factores de Transcripción/genética , Alas de Animales/embriología , Alas de Animales/metabolismo
7.
Proc Biol Sci ; 289(1985): 20221764, 2022 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-36285495

RESUMEN

Increasing evidence suggests that many novel traits might have originated via plasticity-led evolution (PLE). Yet, little is known of the developmental processes that underpin PLE, especially in its early stages. One such process is 'phenotypic accommodation', which occurs when, in response to a change in the environment, an organism experiences adjustments across variable parts of its phenotype that improve its fitness. Here, we asked if environmentally induced changes in gene expression are enhanced or reversed during phenotypic accommodation of a novel, complex phenotype in spadefoot toad tadpoles (Spea multiplicata). More genes than expected were affected by both the environment and phenotypic accommodation in the liver and brain. However, although phenotypic accommodation primarily reversed environmentally induced changes in gene expression in liver tissue, it enhanced these changes in brain tissue. Thus, depending on the tissue, phenotypic accommodation may either minimize functional disruption via reversal of gene expression patterns or promote novelty via enhancement of existing expression patterns. Our study thereby provides insights into the developmental origins of a novel phenotype and the incipient stages of PLE.


Asunto(s)
Anuros , Evolución Biológica , Animales , Fenotipo , Anuros/fisiología , Larva/genética , Expresión Génica , Adaptación Fisiológica/genética
8.
PLoS Biol ; 17(9): e3000378, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31479438

RESUMEN

During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here, we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle-independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross talk between cell cycle exit and terminal differentiation during metamorphosis.


Asunto(s)
Ciclo Celular , Diferenciación Celular , Cromatina/metabolismo , Metamorfosis Biológica , Alas de Animales/crecimiento & desarrollo , Animales , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Elementos Reguladores de la Transcripción , Alas de Animales/ultraestructura
9.
PLoS Comput Biol ; 17(9): e1008991, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34570758

RESUMEN

Identification of biopolymer motifs represents a key step in the analysis of biological sequences. The MEME Suite is a widely used toolkit for comprehensive analysis of biopolymer motifs; however, these tools are poorly integrated within popular analysis frameworks like the R/Bioconductor project, creating barriers to their use. Here we present memes, an R package that provides a seamless R interface to a selection of popular MEME Suite tools. memes provides a novel "data aware" interface to these tools, enabling rapid and complex discriminative motif analysis workflows. In addition to interfacing with popular MEME Suite tools, memes leverages existing R/Bioconductor data structures to store the multidimensional data returned by MEME Suite tools for rapid data access and manipulation. Finally, memes provides data visualization capabilities to facilitate communication of results. memes is available as a Bioconductor package at https://bioconductor.org/packages/memes, and the source code can be found at github.com/snystrom/memes.


Asunto(s)
Secuencias de Aminoácidos , Biología Computacional/métodos , Motivos de Nucleótidos , Programas Informáticos , Animales , Secuenciación de Inmunoprecipitación de Cromatina/estadística & datos numéricos , Biología Computacional/estadística & datos numéricos , Interpretación Estadística de Datos , Humanos
10.
Optom Vis Sci ; 99(9): 725-729, 2022 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-35678637

RESUMEN

SIGNIFICANCE: These cases highlight the importance of monitoring choroidal nevi with benign imaging characteristics and the potential to quantify horizontal growth using optical coherence tomography (OCT), in the absence of color fundus photography. PURPOSE: This study aimed to present reports of two patients with pigmented choroidal tumors with low malignant potential based on their multimodal imaging features at the time of referral, but access to prior OCT imaging confirmed horizontal growth consistent with melanoma. CASE REPORTS: Two patients with pigmented, dome-shaped, subfoveal tumors were referred. Both tumors had basal diameters greater than 5 mm but no other risk factor for growth at the time of referral. Screening OCT scans had been taken of each patient's macula more than 5 years before referral, but color fundus photography was not available for either. Repeat OCT scanning at the time of referral showed horizontal growth of the tumors consistent with melanoma. As per the "To Find Small Ocular Melanoma-Do Imaging" risk factor assessment, the 5-year risk of growth of both tumors would be estimated at 11% at the time of referral, and in the absence of the documented horizontal growth on OCT scanning, the patients would have been monitored for growth. After discussion of the risks and benefits, both patients elected for their tumors to be managed as choroidal melanomas and underwent ruthenium plaque brachytherapy. CONCLUSIONS: Horizontal growth of choroidal tumors can be established using sequential OCT scans in the absence of color fundus photography. Access to prior imaging can expedite the diagnosis of choroidal melanoma, potentially allowing patients to be treated earlier.


Asunto(s)
Neoplasias de la Coroides , Melanoma , Nevo Pigmentado , Neoplasias Cutáneas , Neoplasias de la Coroides/diagnóstico , Angiografía con Fluoresceína/métodos , Humanos , Melanoma/diagnóstico , Nevo Pigmentado/diagnóstico por imagen , Nevo Pigmentado/patología , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodos , Neoplasias de la Úvea
11.
Proc Natl Acad Sci U S A ; 116(20): 9893-9902, 2019 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-31019084

RESUMEN

The ecdysone pathway was among the first experimental systems employed to study the impact of steroid hormones on the genome. In Drosophila and other insects, ecdysone coordinates developmental transitions, including wholesale transformation of the larva into the adult during metamorphosis. Like other hormones, ecdysone controls gene expression through a nuclear receptor, which functions as a ligand-dependent transcription factor. Although it is clear that ecdysone elicits distinct transcriptional responses within its different target tissues, the role of its receptor, EcR, in regulating target gene expression is incompletely understood. In particular, EcR initiates a cascade of transcription factor expression in response to ecdysone, making it unclear which ecdysone-responsive genes are direct EcR targets. Here, we use the larval-to-prepupal transition of developing wings to examine the role of EcR in gene regulation. Genome-wide DNA binding profiles reveal that EcR exhibits widespread binding across the genome, including at many canonical ecdysone response genes. However, the majority of its binding sites reside at genes with wing-specific functions. We also find that EcR binding is temporally dynamic, with thousands of binding sites changing over time. RNA-seq reveals that EcR acts as both a temporal gate to block precocious entry to the next developmental stage as well as a temporal trigger to promote the subsequent program. Finally, transgenic reporter analysis indicates that EcR regulates not only temporal changes in target enhancer activity but also spatial patterns. Together, these studies define EcR as a multipurpose, direct regulator of gene expression, greatly expanding its role in coordinating developmental transitions.


Asunto(s)
Drosophila/fisiología , Ecdisona/fisiología , Metamorfosis Biológica , Receptores de Esteroides/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genoma de los Insectos , Factores de Transcripción/metabolismo , Alas de Animales/metabolismo
12.
PLoS Genet ; 15(1): e1007932, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30699116

RESUMEN

Proper determination of cell fates depends on epigenetic information that is used to preserve memory of decisions made earlier in development. Post-translational modification of histone residues is thought to be a central means by which epigenetic information is propagated. In particular, modifications of histone H3 lysine 27 (H3K27) are strongly correlated with both gene activation and gene repression. H3K27 acetylation is found at sites of active transcription, whereas H3K27 methylation is found at loci silenced by Polycomb group proteins. The histones bearing these modifications are encoded by the replication-dependent H3 genes as well as the replication-independent H3.3 genes. Owing to differential rates of nucleosome turnover, H3K27 acetylation is enriched on replication-independent H3.3 histones at active gene loci, and H3K27 methylation is enriched on replication-dependent H3 histones across silenced gene loci. Previously, we found that modification of replication-dependent H3K27 is required for Polycomb target gene silencing, but it is not required for gene activation. However, the contribution of replication-independent H3.3K27 to these functions is unknown. Here, we used CRISPR/Cas9 to mutate the endogenous replication-independent H3.3K27 to a non-modifiable residue. Surprisingly, we find that H3.3K27 is also required for Polycomb target gene silencing despite the association of H3.3 with active transcription. However, the requirement for H3.3K27 comes at a later stage of development than that found for replication-dependent H3K27, suggesting a greater reliance on replication-independent H3.3K27 in post-mitotic cells. Notably, we find no evidence of global transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription.


Asunto(s)
Epigénesis Genética/genética , Histonas/genética , Lisina/genética , Proteínas del Grupo Polycomb/genética , Alelos , Animales , Sistemas CRISPR-Cas/genética , Cromatina/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Silenciador del Gen , Código de Histonas/genética , Humanos , Metilación , Activación Transcripcional/genética
13.
Clin Exp Ophthalmol ; 50(1): 50-61, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34714583

RESUMEN

BACKGROUND: Prognostic cytological and molecular features of uveal melanoma have been well researched and are essential in management. Samples can be obtained in vivo through fine needle aspirate biopsy, vitrector cutter, forceps or post-enucleation for off-site testing. This study aims to examine cytological and chromosome microarray yields of these samples. METHODS: A retrospective cohort analysis of 119 uveal melanoma biopsies submitted to our laboratory. Samples included those taken in vivo (n = 57) and post-enucleation (n = 62). Patient and tumour features were collected including age, sex, primary tumour location, basal diameter and tumour height. Prognostic outcomes measured include cell morphology, chromosomal status and immunohistochemistry. RESULTS: Post-enucleation biopsies accounted for just over half of our samples (52%). Post-enucleation samples had a more successful genetic yield than in vivo biopsies (77% vs. 50%, p = 0.04) though there was no difference for cytological yields. There was no difference in cytological or microarray yields between instruments. The vitrector biopsy group had the smallest tumour thickness (5 mm vs. 10 mm [fine-needle aspirate biopsy], p = 0.003). There was a strong correlation between monosomy 3, BAP1 aberrancy and epithelioid cell type in post-enucleation samples (Tb  = 0.742, p = 0.005). However, epithelioid morphology was not associated with either monosomy 3 (p = 0.07) or BAP1 aberrancy (p = 0.24) for in vivo biopsies. CONCLUSIONS: All three biopsy instruments provide similar cytological yields as post-enucleation sampling, although post-enucleation samples had a more successful chromosome microarray yield. Epithelioid cytomorphology alone is insufficient for prognostication in in vivo biopsies, immunohistochemistry would be a useful surrogate test.


Asunto(s)
Neoplasias de la Úvea , Biopsia con Aguja Fina , Humanos , Melanoma , Monosomía , Pronóstico , Estudios Retrospectivos , Medición de Riesgo , Neoplasias de la Úvea/diagnóstico , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo
14.
Genome Res ; 28(11): 1688-1700, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30279224

RESUMEN

Chromatin structure has emerged as a key contributor to spatial and temporal control over the initiation of DNA replication. However, despite genome-wide correlations between early replication of gene-rich, accessible euchromatin and late replication of gene-poor, inaccessible heterochromatin, a causal relationship between chromatin structure and replication initiation remains elusive. Here, we combined histone gene engineering and whole-genome sequencing in Drosophila to determine how perturbing chromatin structure affects replication initiation. We found that most pericentric heterochromatin remains late replicating in H3K9R mutants, even though H3K9R pericentric heterochromatin is depleted of HP1a, more accessible, and transcriptionally active. These data indicate that HP1a loss, increased chromatin accessibility, and elevated transcription do not result in early replication of heterochromatin. Nevertheless, a small amount of pericentric heterochromatin with increased accessibility replicates earlier in H3K9R mutants. Transcription is de-repressed in these regions of advanced replication but not in those regions of the H3K9R mutant genome that replicate later, suggesting that transcriptional repression may contribute to late replication. We also explored relationships among chromatin, transcription, and replication in euchromatin by analyzing H4K16R mutants. In Drosophila, the X Chromosome gene expression is up-regulated twofold and replicates earlier in XY males than it does in XX females. We found that H4K16R mutation prevents normal male development and abrogates hyperexpression and earlier replication of the male X, consistent with previously established genome-wide correlations between transcription and early replication. In contrast, H4K16R females are viable and fertile, indicating that H4K16 modification is dispensable for genome replication and gene expression.


Asunto(s)
Ensamble y Desensamble de Cromatina , Momento de Replicación del ADN , Animales , Cromosomas de Insectos/genética , Drosophila , Femenino , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Masculino , Mutación , Activación Transcripcional , Cromosoma X/genética
15.
Development ; 145(7)2018 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-29540499

RESUMEN

Evolution of cis-regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised cis-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using nubbin in the wing and hunchback in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in Tribolium, and in a cross-species context. Together, these advances will facilitate investigation of cis-evolution and morphological diversity in Tribolium and other insects.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Genes Reporteros/genética , Tribolium/genética , Animales , Clonación de Organismos , Drosophila/genética , Técnicas de Transferencia de Gen , Inmunohistoquímica , Hibridación in Situ
16.
J Exp Zool B Mol Dev Evol ; 336(6): 482-495, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34142757

RESUMEN

Polyphenism-in which multiple distinct phenotypes are produced from a single genotype owing to differing environmental conditions-is commonplace, but its molecular bases are poorly understood. Here, we examine the transcriptomic bases of a polyphenism in Mexican spadefoot toads (Spea multiplicata). Depending on their environment, their tadpoles develop into either a default "omnivore" morph or a novel "carnivore" morph. We compared patterns of gene expression among sibships that exhibited high versus low production of carnivores when reared in conditions that induce the carnivore morph versus those that do not. We found that production of the novel carnivore morph actually involved changes in fewer genes than did the maintenance of the default omnivore morph in the inducing environment. However, only body samples showed this pattern; head samples showed the opposite pattern. We also found that changes to lipid metabolism (especially cholesterol biosynthesis) and peroxisome contents and function might be crucial for establishing and maintaining differences between the morphs. Thus, our findings suggest that carnivore phenotype might have originally evolved following the breakdown of robustness mechanisms that maintain the default omnivore phenotype, and that the carnivore morph is developmentally regulated by lipid metabolism and peroxisomal form, function, and/or signaling. This study also serves as a springboard for further exploration into the nature and causes of plasticity in an emerging model system.


Asunto(s)
Adaptación Fisiológica , Anuros/crecimiento & desarrollo , Anuros/genética , Genotipo , Animales , Anuros/metabolismo , Conducta Alimentaria , Larva/metabolismo , Metabolismo de los Lípidos , Peroxisomas , Transcriptoma
17.
PLoS Genet ; 14(4): e1007339, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29641560

RESUMEN

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.


Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Complejo de Señalización de la Axina/metabolismo , Proteínas de Drosophila/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , Animales , Animales Modificados Genéticamente , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/química , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/genética , Proteína Axina/química , Proteína Axina/genética , Proteína Axina/metabolismo , Complejo de Señalización de la Axina/química , Complejo de Señalización de la Axina/genética , Línea Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
18.
J Comput Aided Mol Des ; 34(12): 1219-1228, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32918236

RESUMEN

SARS-CoV-2 recently jumped species and rapidly spread via human-to-human transmission to cause a global outbreak of COVID-19. The lack of effective vaccine combined with the severity of the disease necessitates attempts to develop small molecule drugs to combat the virus. COVID19_GIST_HSA is a freely available online repository to provide solvation thermodynamic maps of COVID-19-related protein small molecule drug targets. Grid inhomogeneous solvation theory maps were generated using AmberTools cpptraj-GIST, 3D reference interaction site model maps were created with AmberTools rism3d.snglpnt and hydration site analysis maps were created using SSTMap code. The resultant data can be applied to drug design efforts: scoring solvent displacement for docking, rational lead modification, prioritization of ligand- and protein- based pharmacophore elements, and creation of water-based pharmacophores. Herein, we demonstrate the use of the solvation thermodynamic mapping data. It is hoped that this freely provided data will aid in small molecule drug discovery efforts to defeat SARS-CoV-2.


Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Modelos Químicos , Simulación de Dinámica Molecular , Terapia Molecular Dirigida , Pandemias , Neumonía Viral/tratamiento farmacológico , Termodinámica , Proteínas no Estructurales Virales/efectos de los fármacos , Antivirales/química , Betacoronavirus/química , Sitios de Unión , COVID-19 , Dominio Catalítico , Humanos , Ligandos , Modelos Moleculares , Conformación Proteica , SARS-CoV-2 , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/química , Agua , Tratamiento Farmacológico de COVID-19
19.
Development ; 143(20): 3723-3732, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27802137

RESUMEN

One of the key issues in studying transcriptional regulation during development is how to employ genome-wide assays that reveals sites of open chromatin and transcription factor binding to efficiently identify biologically relevant genes and enhancers. Analysis of Drosophila CNS midline cell development provides a useful system for studying transcriptional regulation at the genomic level due to a large, well-characterized set of midline-expressed genes and in vivo validated enhancers. In this study, FAIRE-seq on FACS-purified midline cells was performed and the midline FAIRE data were compared with whole-embryo FAIRE data. We find that regions of the genome with a strong midline FAIRE peak and weak whole-embryo FAIRE peak overlap with known midline enhancers and provide a useful predictive tool for enhancer identification. In a complementary analysis, we compared a large dataset of fragments that drive midline expression in vivo with the FAIRE data. Midline enhancer fragments with a midline FAIRE peak tend to be near midline-expressed genes, whereas midline enhancers without a midline FAIRE peak were often distant from midline-expressed genes and unlikely to drive midline transcription in vivo.


Asunto(s)
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Animales , Animales Modificados Genéticamente , Cromatina/genética , Inmunoprecipitación de Cromatina , Drosophila , Proteínas de Drosophila/genética , Elementos de Facilitación Genéticos/genética , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica/genética
20.
BMC Genomics ; 19(1): 157, 2018 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-29466941

RESUMEN

BACKGROUND: High-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis- and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. Here, we present a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis. RESULTS: The data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. We confirm the paucity of divergent transcription units in D. melanogaster, but also identify notable exceptions. Furthermore, we identify thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for "enhancer RNAs" (eRNAs) in defining developmental transcription programs. CONCLUSIONS: High-depth TSS mapping is a powerful strategy for identifying and characterizing low-abundance and/or low-stability RNAs. Global analysis of transcription initiation patterns in a developing organism reveals a vast number of novel initiation events that identify potential eRNAs as well as other non-coding transcripts critical for animal development.


Asunto(s)
Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , ARN/genética , Sitio de Iniciación de la Transcripción , Animales , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Nucleosomas , Regiones Promotoras Genéticas , Transcripción Genética
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