RESUMO
Recent evidence suggests that human gene promoters display gene expression regulatory mechanisms beyond the typical single gene local transcription modulation. In mammalian genomes, genes with an associated bidirectional promoter are abundant; bidirectional promoter architecture serves as a regulatory hub for a gene pair expression. However, it has been suggested that its contribution to transcriptional regulation might exceed local transcription initiation modulation. Despite their abundance, the functional consequences of bidirectional promoter architecture remain largely unexplored. This work studies the long-range gene expression regulatory role of a long non-coding RNA gene promoter using chromosome conformation capture methods. We found that this particular bidirectional promoter contributes to distal gene expression regulation in a target-specific manner by establishing promoter-promoter interactions. In particular, we validated that the promoter-promoter interactions of this regulatory element with the promoter of distal gene BBX contribute to modulating the transcription rate of this gene; removing the bidirectional promoter from its genomic context leads to a rearrangement of BBX promoter-enhancer interactions and to increased gene expression. Moreover, long-range regulatory functionality is not directly dependent on its associated non-coding gene pair expression levels.
Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Regulação da Expressão Gênica/genética , Transcrição Gênica , Elementos Facilitadores GenéticosRESUMO
Background: Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Methods: Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells. Results: Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Conclusion: Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.
RESUMO
Alu elements are primate-specific repeats and represent the most abundant type of transposable elements (TE) in the human genome. Genome-wide analysis of the enrichment of histone post-translational modifications suggests that human Alu sequences could function as transcriptional enhancers; however, no functional experiments have evaluated the role of Alu sequences in the control of transcription in situ. The present study analyses the regulatory activity of a human Alu sequence from the AluSx family located in the second intron of the long intergenic non-coding RNA Linc00441, found in divergent orientation to the RB1 gene. We observed that the Alu sequence acts as an enhancer element based on reporter gene assays while CRISPR-Cas9 deletions of the Alu sequence in K562 cells resulted in a marked transcriptional upregulation of Linc00441 and a decrease in proliferation. Our results suggest that an intragenic Alu sequence with enhancer activity can act as a transcriptional attenuator of its host lincRNA.
RESUMO
Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.
Assuntos
Fator de Ligação a CCCTC/genética , Desenvolvimento Embrionário/genética , Animais , Apoptose/genética , Sistemas CRISPR-Cas/genética , Sobrevivência Celular/genética , Cromatina/genética , Técnicas de Inativação de Genes/métodos , Humanos , RNA Mensageiro/genética , Peixe-ZebraRESUMO
As a part of an ongoing project to inventory the helminth parasites of rodents in Mexico, 85 specimens of 2 families of rodents were collected from the Mexican Plateau: Cricetidae ( Neotoma sp., Neotoma leucodon , Onychomys arenicola , Peromyscus sp., Peromyscus eremicus , and Reithrodontomys sp.) and Heteromyidae ( Chaetodipus sp., Chaetodipus eremicus , Chaetodipus hispidus , Dipodomys merriami , Dipodomys ordii , Dipodomys ornatus, Dipodomys spectabilis , Liomys irroratus , Perognathus sp., and Perognathus flavus ). A total of 13 taxa of helminths were found: Heteromyoxyuris longejector, Heteromyoxyuris otomii, Heteromyoxyuris sp., Onchocercidae gen. sp. 1 and sp. 2, Physalopteridae gen. sp., Protospirura dipodomis, Pterygodermatites dipodomis, Subulura sp., Syphacia sp., Trichuris dipodomis, Vexillata liomyos, and Vexillata armande. The highest species richness was recorded in D. merriami (7 taxa). This study is the first report of nematodes from O. arenicola (Physalopteridae gen. sp.) and C. eremicus (H. longejector) and for V. liomyos from D. merriami . All reports of these species of nematodes represent new collection localities in Mexico.
Assuntos
Arvicolinae/parasitologia , Nematoides/isolamento & purificação , Infecções por Nematoides/veterinária , Doenças dos Roedores/parasitologia , Animais , Dipodomys/parasitologia , México/epidemiologia , Nematoides/classificação , Nematoides/crescimento & desenvolvimento , Infecções por Nematoides/epidemiologia , Infecções por Nematoides/parasitologia , Prevalência , Doenças dos Roedores/epidemiologia , RoedoresRESUMO
The three-dimensional architecture of genomes provides new insights about genome organization and function, but many aspects remain unsolved at the local genomic scale. Here we investigate the regulation of two erythroid-specific loci, a folate receptor gene (FOLR1) and the ß-globin gene cluster, which are separated by 16kb of constitutive heterochromatin. We found that in early erythroid differentiation the FOLR1 gene presents a permissive chromatin configuration that allows its expression. Once the transition to the next differentiation state occurs, the heterochromatin spreads into the FOLR1 domain, concomitant with the dissociation of CTCF from a novel binding site, thereby resulting in irreversible silencing of the FOLR1 gene. We demonstrate that the sequences surrounding the CTCF-binding site possess classical insulator properties in vitro and in vivo. In contrast, the chicken cHS4 ß-globin insulator present on the other side of the heterochromatic segment is in a constitutive open chromatin configuration, with CTCF constantly bound from the early stages of erythroid differentiation. Therefore, this study demonstrates that the 16kb of constitutive heterochromatin contributes to silencing of the FOLR1 gene during erythroid differentiation.
Assuntos
Receptor 1 de Folato/genética , Loci Gênicos , Elementos Isolantes/fisiologia , Globinas beta/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Transformada , Embrião de Galinha , Galinhas , Cromatina/genética , Cromatina/metabolismo , Eritropoese/genética , Receptor 1 de Folato/metabolismo , Regulação da Expressão Gênica , Heterocromatina/genética , Heterocromatina/metabolismoRESUMO
Heteromyoxyuris otomii n. sp., which inhabits the intestinal caecum of Perognathus flavus (Heteromyidae), in Zaragoza, Hidalgo, Mexico, is described. This new species differs from the 2 other congeneric species in the morphology and length of lateral alae in males. Heteromyoxyuris deserti has simple lateral alae located at both sides of the body, whereas in the new species, these structures are double at both sides; in contrast, lateral alae of Heteromyoxyuris longejector begin at the posterior half of the body, whereas they arise in the first third in the new species. Heteromyoxyuris longejector was found in 2 new host species, i.e., Perognathus amplus and Chaetodipus hispidus. This record represents the first record for the species in Mexico, increasing its geographic distribution.
Assuntos
Oxiuríase/veterinária , Oxyuroidea/classificação , Doenças dos Roedores/parasitologia , Animais , Feminino , Masculino , México , Oxiuríase/parasitologia , Oxyuroidea/ultraestrutura , RoedoresRESUMO
At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.
Assuntos
Cromatina/química , Epigênese Genética , Globinas/genética , Neoplasias/genética , Transcrição Gênica , Animais , Globinas/química , Globinas/metabolismo , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
Con el objetivo de conocer la frecuecia de discrepancia entre los diagnósticos clínicos y los anatómicos en casos autopsiados y de conocer el grado de discrepancia de los mismos, se examinaron los protocolos de 1,000 autopsias consecutivas realizadas en el HGM. De cada caso se recabaron los siguientes datos: edad, sexo, servicio, días de estancia, análisis de laboratorio y gabinete, diagnósticos clínicos (DC) y diagnósticos anatómicos (DA). Se compararon los DA, las discrepancias entre ellos se clasificaron como leves (L) cuando la diferencia no influyó en la evolución del caso y como importantes (l) cuando ésta repercutió importantemente en la evolución del mismo. La frecuencia de discrepancia fue de 28.6 por ciento. De los casos discrepantes el 61.2 por ciento tuvieron l y el 38.8 por ciento fue L. La única variable que resultó estadísticamente significativa en la presencia o no de discrepancia fue la duración de la hospitalización. Los servicios en los que hubo mayor proporción de casos discrepantes fueron: Terapia Intensiva, Urgencias, Cirugía General, Neumología y Medicina Interna