RESUMEN
RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
Asunto(s)
Factor de Unión a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas de Homeodominio/metabolismo , Recombinación V(D)J , Animales , Línea Celular , ADN Intergénico/genética , ADN Intergénico/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutagénesis , Señales de Clasificación de Proteína , ARN Guía de Kinetoplastida/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.
Asunto(s)
Caenorhabditis elegans/metabolismo , ADN Intergénico/metabolismo , Silenciador del Gen , Animales , Composición de Base , Caenorhabditis elegans/genética , Cromatina , Elementos Transponibles de ADN , ADN Viral/genética , Células Germinativas/metabolismo , Intrones , Regiones Promotoras Genéticas , ARN sin Sentido/metabolismo , ARN Mensajero/metabolismo , TransgenesRESUMEN
CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains â¼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA â¼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.
Asunto(s)
Proteína 9 Asociada a CRISPR/química , Sistemas CRISPR-Cas , ADN Intergénico/química , ADN/química , Streptococcus thermophilus/genética , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Clonación Molecular , Microscopía por Crioelectrón , ADN/genética , ADN/metabolismo , ADN Intergénico/genética , ADN Intergénico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus thermophilus/metabolismo , Especificidad por SustratoRESUMEN
To achieve adaptive and heritable immunity against viruses and other mobile genetic elements, CRISPR-Cas systems must capture and store short DNA fragments (spacers) from these foreign elements into host genomic CRISPR arrays. This process is catalyzed by conserved Cas1/Cas2 integration complexes, but the specific roles of another highly conserved protein linked to spacer acquisition, the Cas4 nuclease, are just now emerging. Here, we show that two Cas4 nucleases (Cas4-1 and Cas4-2) play critical roles in CRISPR spacer acquisition in Pyrococcus furiosus. The nuclease activities of both Cas4 proteins are required to process protospacers to the correct size. Cas4-1 specifies the upstream PAM (protospacer adjacent motif), while Cas4-2 specifies the conserved downstream motif. Both Cas4 proteins ensure CRISPR spacer integration in a defined orientation leading to CRISPR immunity. Collectively, these findings provide in vivo evidence for critical roles of Cas4 nucleases in protospacer generation and functional spacer integration at CRISPR arrays.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Bacteriano/genética , ADN Intergénico/genética , Edición Génica , Motivos de Nucleótidos , Pyrococcus furiosus/genética , Inmunidad Adaptativa , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , ADN Bacteriano/química , ADN Bacteriano/inmunología , ADN Bacteriano/metabolismo , ADN Intergénico/química , ADN Intergénico/metabolismo , Regulación Bacteriana de la Expresión Génica , Conformación de Ácido Nucleico , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/inmunologíaRESUMEN
Immunoglobulin heavy-chain (IgH) genes are assembled by DNA rearrangements that juxtapose a variable (VH), a diversity (DH), and a joining (JH) gene segment. Here, we report that in the absence of intergenic control region 1 (IGCR1), the intronic enhancer (Eµ) associates with the next available CTCF binding site located close to VH81X via putative heterotypic interactions involving YY1 and CTCF. The alternate Eµ/VH81X loop leads to formation of a distorted recombination center and altered DH rearrangements and disrupts chromosome conformation that favors distal VH recombination. Cumulatively, these features drive highly skewed, Eµ-dependent recombination of VH81X. Sequential deletion of CTCF binding regions on IGCR1-deleted alleles suggests that they influence recombination of single proximal VH gene segments. Our observations demonstrate that Eµ interacts differently with IGCR1- or VH-associated CTCF binding sites and thereby identify distinct roles for insulator-like elements in directing enhancer activity.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN Intergénico/genética , Elementos de Facilitación Genéticos , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Sitios Genéticos , Región Variable de Inmunoglobulina/genética , Células Precursoras de Linfocitos B/metabolismo , Recombinación Genética , Animales , Sitios de Unión , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Línea Celular , ADN Intergénico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Ratones de la Cepa 129 , Ratones Noqueados , Conformación de Ácido Nucleico , Células Precursoras de Linfocitos B/inmunología , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismoRESUMEN
Evolutionary analyses have estimated that â¼60% of nucleotides in intergenic regions of the Drosophila melanogaster genome are functionally relevant, suggesting that regulatory information may be encoded more densely in intergenic regions than has been revealed by most functional dissections of regulatory DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the â¼90â kb of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory information that drives svb expression in the abdominal pupal epidermis is organized in a different way than the elements that drive svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact enhancers separated by large inactive DNA regions, svb expression in the pupal epidermis is driven by regulatory information distributed over broader regions of svb cis-regulatory DNA. In the same vein, we observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved noncoding DNA of the Drosophila genome is contained within regions of open chromatin. These results suggest that part of the evolutionary constraint on noncoding DNA of Drosophila is explained by the density of regulatory information, which may be greater than previously appreciated.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Factores de Transcripción/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , ADN , ADN Intergénico/genética , ADN Intergénico/metabolismo , Elementos de Facilitación GenéticosRESUMEN
Initiation of translation on many viral mRNAs occurs by noncanonical mechanisms that involve 5' end-independent binding of ribosomes to an internal ribosome entry site (IRES). The â¼190-nt-long intergenic region (IGR) IRES of dicistroviruses such as cricket paralysis virus (CrPV) initiates translation without Met-tRNAi Met or initiation factors. Advances in metagenomics have revealed numerous dicistrovirus-like genomes with shorter, structurally distinct IGRs, such as nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV1). Like canonical IGR IRESs, the â¼165-nt-long NediV-like IGRs comprise three domains, but they lack key canonical motifs, including L1.1a/L1.1b loops (which bind to the L1 stalk of the ribosomal 60S subunit) and the apex of stem-loop V (SLV) (which binds to the head of the 40S subunit). Domain 2 consists of a compact, highly conserved pseudoknot (PKIII) that contains a UACUA loop motif and a protruding CrPV-like stem--loop SLIV. In vitro reconstitution experiments showed that NediV-like IRESs initiate translation from a non-AUG codon and form elongation-competent 80S ribosomal complexes in the absence of initiation factors and Met-tRNAi Met Unlike canonical IGR IRESs, NediV-like IRESs bind directly to the peptidyl (P) site of ribosomes leaving the aminoacyl (A) site accessible for decoding. The related structures of NediV-like IRESs and their common mechanism of action indicate that they exemplify a distinct class of IGR IRES.
Asunto(s)
Sitios Internos de Entrada al Ribosoma , Ribosomas , Sitios Internos de Entrada al Ribosoma/genética , ADN Intergénico/genética , ADN Intergénico/metabolismo , Ribosomas/metabolismo , Factores de Iniciación de Péptidos , ARN de Transferencia/química , ARN Viral/genética , ARN Viral/química , Biosíntesis de ProteínasRESUMEN
Enzymes that catalyse CpG methylation in DNA, including the DNA methyltransferases 1 (DNMT1), 3A (DNMT3A) and 3B (DNMT3B), are indispensable for mammalian tissue development and homeostasis1-4. They are also implicated in human developmental disorders and cancers5-8, supporting the critical role of DNA methylation in the specification and maintenance of cell fate. Previous studies have suggested that post-translational modifications of histones are involved in specifying patterns of DNA methyltransferase localization and DNA methylation at promoters and actively transcribed gene bodies9-11. However, the mechanisms that control the establishment and maintenance of intergenic DNA methylation remain poorly understood. Tatton-Brown-Rahman syndrome (TBRS) is a childhood overgrowth disorder that is defined by germline mutations in DNMT3A. TBRS shares clinical features with Sotos syndrome (which is caused by haploinsufficiency of NSD1, a histone methyltransferase that catalyses the dimethylation of histone H3 at K36 (H3K36me2)8,12,13), which suggests that there is a mechanistic link between these two diseases. Here we report that NSD1-mediated H3K36me2 is required for the recruitment of DNMT3A and maintenance of DNA methylation at intergenic regions. Genome-wide analysis shows that the binding and activity of DNMT3A colocalize with H3K36me2 at non-coding regions of euchromatin. Genetic ablation of Nsd1 and its paralogue Nsd2 in mouse cells results in a redistribution of DNMT3A to H3K36me3-modified gene bodies and a reduction in the methylation of intergenic DNA. Blood samples from patients with Sotos syndrome and NSD1-mutant tumours also exhibit hypomethylation of intergenic DNA. The PWWP domain of DNMT3A shows dual recognition of H3K36me2 and H3K36me3 in vitro, with a higher binding affinity towards H3K36me2 that is abrogated by TBRS-derived missense mutations. Together, our study reveals a trans-chromatin regulatory pathway that connects aberrant intergenic CpG methylation to human neoplastic and developmental overgrowth.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Intergénico/metabolismo , Histonas/metabolismo , Animales , Línea Celular , ADN Metiltransferasa 3A , Estudio de Asociación del Genoma Completo , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/fisiopatología , Humanos , Ratones , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Síndrome de Sotos/genética , Síndrome de Sotos/fisiopatologíaRESUMEN
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Intergénico/genética , Endonucleasas/genética , Edición Génica/métodos , Genoma , Animales , ADN Intergénico/metabolismo , Endonucleasas/metabolismo , Células Eucariotas/citología , Células Eucariotas/metabolismo , Ingeniería Genética , Biblioteca Genómica , Ensayos Analíticos de Alto Rendimiento , Humanos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application.
Asunto(s)
Inmunidad Adaptativa , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/inmunología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/inmunología , ADN Intergénico/genética , ADN Viral/genética , Endonucleasas/genética , Mutación , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Proteínas Asociadas a CRISPR/inmunología , Proteínas Asociadas a CRISPR/metabolismo , ADN Intergénico/inmunología , ADN Intergénico/metabolismo , ADN Viral/inmunología , ADN Viral/metabolismo , Endonucleasas/inmunología , Endonucleasas/metabolismo , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno , Fenotipo , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Staphylococcus aureus/virología , Especificidad por Sustrato , Factores de TiempoRESUMEN
Internal ribosomal entry sites (IRESs) engage with the eukaryotic translation apparatus to promote end-independent initiation. We identified a conserved class of â¼150 nt long intergenic region (IGR) IRESs in dicistrovirus genomes derived from members of the phyla Arthropoda, Bryozoa, Cnidaria, Echinodermata, Entoprocta, Mollusca and Porifera. These IRESs, exemplified by Wenling picorna-like virus 2, resemble the canonical cricket paralysis virus (CrPV) IGR IRES in comprising two nested pseudoknots (PKII/PKIII) and a 3'-terminal pseudoknot (PKI) that mimics a tRNA anticodon stem-loop base-paired to mRNA. However, they are â¼50 nt shorter than CrPV-like IRESs, and PKIII is an H-type pseudoknot that lacks the SLIV and SLV stem-loops that are primarily responsible for the affinity of CrPV-like IRESs for the 40S ribosomal subunit and that restrict initial binding of PKI to its aminoacyl (A) site. Wenling-class IRESs bound strongly to 80S ribosomes but only weakly to 40S subunits. Whereas CrPV-like IRESs must be translocated from the A site to the peptidyl (P) site by elongation factor 2 for elongation to commence, Wenling-class IRESs bound directly to the P site of 80S ribosomes, and decoding begins without a prior translocation step. A chimeric CrPV clone containing a Wenling-class IRES was infectious, confirming that the IRES functioned in cells.
Asunto(s)
Sitios Internos de Entrada al Ribosoma , Virus ARN , Secuencia de Bases , ADN Intergénico/genética , ADN Intergénico/metabolismo , Ribosomas/metabolismo , Virus ARN/genética , ARN Viral/metabolismo , Biosíntesis de ProteínasRESUMEN
SNPs associated with human stroke risk have been identified in the intergenic region between Forkhead family transcription factors FOXF2 and FOXQ1, but we lack a mechanism for the association. FoxF2 is expressed in vascular mural pericytes and is important for maintaining pericyte number and stabilizing small vessels in zebrafish. The stroke-associated SNPs are located in a previously unknown transcriptional enhancer for FOXF2, functional in human cells and zebrafish. We identify critical enhancer regions for FOXF2 gene expression, including binding sites occupied by transcription factors ETS1, RBPJ, and CTCF. rs74564934, a stroke-associated SNP adjacent to the ETS1 binding site, decreases enhancer function, as does mutation of RPBJ sites. rs74564934 is significantly associated with the increased risk of any stroke, ischemic stroke, small vessel stroke, and elevated white matter hyperintensity burden in humans. Foxf2 has a conserved function cross-species and is expressed in vascular mural pericytes of the vessel wall. Thus, stroke-associated SNPs modulate enhancer activity and expression of a regulator of vascular stabilization, FOXF2, thereby modulating stroke risk.
Asunto(s)
Factores de Transcripción Forkhead , Pericitos , Accidente Cerebrovascular , Animales , ADN Intergénico/genética , ADN Intergénico/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Variación Estructural del Genoma/genética , Humanos , Pericitos/metabolismo , Polimorfismo de Nucleótido Simple , Riesgo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Activación Transcripcional/genéticaRESUMEN
Enhancers activate gene transcription remotely, which requires tissue specific transcription factors binding to them. GATA1 and TAL1 are hematopoietic/erythroid-specific factors and often bind together to enhancers, activating target genes. Interestingly, we found that some hematopoietic/erythroid genes are transcribed in a GATA1-dependent but TAL1-independnet manner. They appear to have enhancers within a relatively short distance. In this study, we paired highly transcribed hematopoietic/erythroid genes with the nearest GATA1/TAL1-binding enhancers and analyzed these putative enhancer-gene pairs depending on distance between them. Enhancers located at various distances from genes in the pairs, which was not related to transcription level of the genes. However, genes with enhancers at short distances away tended to be transcriptionally unaffected by TAL1 depletion. Histone H3K27ac extended from the enhancers to target genes. The H3K27ac extension was maintained without TAL1, even though it disappeared owing to the loss of GATA1. Intergenic RNA was highly transcribed from the enhancers to nearby target genes, independent of TAL1. Taken together, TAL1-independent transcription of hematopoietic/erythroid genes appears to be promoted by enhancers present in a short distance. These enhancers are likely to activate nearby target genes by tracking the intervening regions.
Asunto(s)
ADN Intergénico , Elementos de Facilitación Genéticos , Hematopoyesis , Histonas , ADN Intergénico/genética , ADN Intergénico/metabolismo , Hematopoyesis/genética , Histonas/genética , Histonas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismoRESUMEN
Sense-antisense mRNA pairs generated by convergent transcription is a way of gene regulation. c-fms gene is closely juxtaposed to the HMGXB3 gene in the opposite orientation, in chromosome 5. The intergenic region (IR) between c-fms and HMGXB3 genes is 162 bp. We found that a small portion (â¼4.18%) of HMGXB3 mRNA is transcribed further downstream, including the end of the c-fms gene generating antisense mRNA against c-fms mRNA. Similarly, a small portion (â¼1.1%) of c-fms mRNA is transcribed further downstream, including the end of the HMGXB3 gene generating antisense mRNA against the HMGXB3 mRNA. Insertion of the strong poly(A) signal sequence in the IR results in decreased c-fms and HMGXB3 antisense mRNAs, resulting in up-regulation of both c-fms and HMGXB3 mRNA expression. miR-324-5p targets HMGXB3 mRNA 3' UTR, and as a result, regulates c-fms mRNA expression. HuR stabilizes c-fms mRNA, and as a result, down-regulates HMGXB3 mRNA expression. UALCAN analysis indicates that the expression pattern between c-fms and HMGXB3 proteins are opposite in vivo in breast cancer tissues. Together, our results indicate that the mRNA encoded by the HMGXB3 gene can influence the expression of adjacent c-fms mRNA, or vice versa.
Asunto(s)
Proteína 1 Similar a ELAV/genética , Regulación Neoplásica de la Expresión Génica , Genes fms , Proteínas del Grupo de Alta Movilidad/genética , MicroARNs/genética , Regiones no Traducidas 3' , Sistemas CRISPR-Cas , Línea Celular Tumoral , Cromosomas Humanos Par 5 , ADN Intergénico/genética , ADN Intergénico/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Femenino , Edición Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , MicroARNs/metabolismo , Polimorfismo Genético , Proto-Oncogenes Mas , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
FOXC1 encodes a forkhead-domain transcription factor associated with several ocular disorders. Correct FOXC1 dosage is critical to normal development, yet the mechanisms controlling its expression remain unknown. Together with FOXQ1 and FOXF2, FOXC1 is part of a cluster of FOX genes conserved in vertebrates. CRISPR-Cas9-mediated dissection of genomic sequences surrounding two zebrafish orthologs of FOXC1 was performed. This included five zebrafish-human conserved regions, three downstream of foxc1a and two remotely upstream of foxf2a/foxc1a or foxf2b/foxc1b clusters, as well as two intergenic regions between foxc1a/b and foxf2a/b lacking sequence conservation but positionally corresponding to the area encompassing a previously reported glaucoma-associated SNP in humans. Removal of downstream sequences altered foxc1a expression; moreover, zebrafish carrying deletions of two or three downstream elements demonstrated abnormal phenotypes including enlargement of the anterior chamber of the eye reminiscent of human congenital glaucoma. Deletions of distant upstream conserved elements influenced the expression of foxf2a/b or foxq1a/b but not foxc1a/b within each cluster. Removal of either intergenic sequence reduced foxc1a or foxc1b expression during late development, suggesting a role in transcriptional regulation despite the lack of conservation at the nucleotide level. Further studies of the identified regions in human patients may explain additional individuals with developmental ocular disorders.
Asunto(s)
Glaucoma , Pez Cebra , Animales , Humanos , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Sistemas CRISPR-Cas/genética , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Glaucoma/genética , Genómica , ADN Intergénico/genética , ADN Intergénico/metabolismo , Nucleótidos/metabolismoRESUMEN
The availability of genomes for many species has advanced our understanding of the non-protein-coding fraction of the genome. Comparative genomics has proven itself to be an invaluable approach for the systematic, genome-wide identification of conserved non-protein-coding elements (CNEs). However, for many non-mammalian model species, including chicken, our capability to interpret the functional importance of variants overlapping CNEs has been limited by current genomic annotations, which rely on a single information type (e.g. conservation). We here studied CNEs in chicken using a combination of population genomics and comparative genomics. To investigate the functional importance of variants found in CNEs we develop a ch(icken) Combined Annotation-Dependent Depletion (chCADD) model, a variant effect prediction tool first introduced for humans and later on for mouse and pig. We show that 73 Mb of the chicken genome has been conserved across more than 280 million years of vertebrate evolution. The vast majority of the conserved elements are in non-protein-coding regions, which display SNP densities and allele frequency distributions characteristic of genomic regions constrained by purifying selection. By annotating SNPs with the chCADD score we are able to pinpoint specific subregions of the CNEs to be of higher functional importance, as supported by SNPs found in these subregions are associated with known disease genes in humans, mice, and rats. Taken together, our findings indicate that CNEs harbor variants of functional significance that should be object of further investigation along with protein-coding mutations. We therefore anticipate chCADD to be of great use to the scientific community and breeding companies in future functional studies in chicken.
Asunto(s)
Pollos/genética , ADN Intergénico/genética , Genómica/métodos , Alelos , Animales , Secuencia Conservada/genética , ADN Intergénico/metabolismo , Evolución Molecular , Frecuencia de los Genes/genética , Variación Genética/genética , Genoma/genética , Intrones/genética , Metagenómica/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia/métodosRESUMEN
The Cre-loxP gene editing tool enables site-specific editing of DNA without leaving lesions that must be repaired by error-prone cellular processes. Cre recombines two 34-bp loxP DNA sites that feature a pair of palindromic recombinase-binding elements flanking an asymmetric 8-bp spacer region, via assembly of a tetrameric intasome complex and formation of a Holliday junction intermediate. Recombination proceeds by coordinated nucleophilic attack by pairs of catalytic tyrosine residues on specific phosphodiester bonds in the spacer regions of opposing strands. Despite not making base-specific contacts with the asymmetric spacer region of the DNA, Cre exhibits a preference for initial cleavage on one of the strands, suggesting that intrinsic properties of the uncontacted 8-bp spacer region give rise to this preference. Furthermore, little is known about the structural and dynamic features of the loxP spacer that make it a suitable target for Cre. To enable NMR spectroscopic studies of the spacer, we have aimed to identify a fragment of the 34-bp loxP site that retains the structural features of the spacer while minimizing the spectral crowding and line-broadening seen in longer oligonucleotides. Sequence-specific chemical shift differences between spacer oligos of different lengths, and of a mutant that inverts strand cleavage order, reveal how both nearest-neighbor and next-nearest-neighbor effects dominate the chemical environment experienced by the spacer. We have identified a 16-bp oligonucleotide that preserves the structural environment of the spacer, setting the stage for NMR-based structure determination and dynamics investigations.
Asunto(s)
Bacteriófago P1/química , ADN Intergénico/química , Oligonucleótidos/química , Bacteriófago P1/metabolismo , Secuencia de Bases , ADN Intergénico/metabolismo , Integrasas/química , Integrasas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Organisms use endogenous clocks to adapt to the rhythmicity of the environment and to synchronize social activities. Although the circadian cycle is implicated in aging, it is unknown whether natural variation in its function contributes to differences in lifespan between populations and whether the circadian clock of specific tissues is key for longevity. We have sequenced the genomes of Drosophila melanogaster strains with exceptional longevity that were obtained via multiple rounds of selection from a parental strain. Comparison of genomic, transcriptomic, and proteomic data revealed that changes in gene expression due to intergenic polymorphisms are associated with longevity and preservation of skeletal muscle function with aging in these strains. Analysis of transcription factors differentially modulated in long-lived versus parental strains indicates a possible role of circadian clock core components. Specifically, there is higher period and timeless and lower cycle expression in the muscle of strains with delayed aging compared to the parental strain. These changes in the levels of circadian clock transcription factors lead to changes in the muscle circadian transcriptome, which includes genes involved in metabolism, proteolysis, and xenobiotic detoxification. Moreover, a skeletal muscle-specific increase in timeless expression extends lifespan and recapitulates some of the transcriptional and circadian changes that differentiate the long-lived from the parental strains. Altogether, these findings indicate that the muscle circadian clock is important for longevity and that circadian gene variants contribute to the evolutionary divergence in longevity across populations.
Asunto(s)
Factores de Transcripción ARNTL/genética , Relojes Circadianos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genoma de los Insectos , Longevidad/genética , Músculo Esquelético/metabolismo , Proteínas Circadianas Period/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Evolución Biológica , Ritmo Circadiano/genética , ADN Intergénico/genética , ADN Intergénico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Genética de Población , Genómica , Músculo Esquelético/crecimiento & desarrollo , Proteínas Circadianas Period/metabolismo , Polimorfismo Genético , Transcriptoma , Secuenciación Completa del GenomaRESUMEN
Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-γ (IFN-γ)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.
Asunto(s)
Apoptosis/inmunología , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Interleucinas/inmunología , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Western Blotting , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , ADN Intergénico/genética , ADN Intergénico/inmunología , ADN Intergénico/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Citometría de Flujo , Galactosilceramidas/inmunología , Galactosilceramidas/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucinas/genética , Interleucinas/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Interleucina-21/deficiencia , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunologíaRESUMEN
The mechanism for how internal ribosome entry sites (IRESs) recruit ribosomes to initiate translation of an mRNA is not completely understood. We investigated how a 40S subunit was recruited by the cricket paralysis virus intergenic region (CrPV IGR) IRES to form a stable 40S-IRES complex. Kinetic binding studies revealed that formation of the complex between the CrPV IGR and the 40S subunit consisted of two-steps: an initial fast binding step of the IRES to the 40S ribosomal subunit, followed by a slow unimolecular reaction consistent with a conformational change that stabilized the complex. We further showed that the ribosomal protein S25 (eS25), which is required by functionally and structurally diverse IRESs, impacts both steps of the complex formation. Mutations in eS25 that reduced CrPV IGR IRES activity either decreased 40S-IRES complex formation, or increased the rate of the conformational change that was required to form a stable 40S-IRES complex. Our data are consistent with a model in which eS25 facilitates initial binding of the CrPV IGR IRES to the 40S while ensuring that the conformational change stabilizing the 40S-IRES complex does not occur prematurely.