RESUMEN
It is now established that Bcl11b specifies T cell fate. Here, we show that in developing T cells, the Bcl11b enhancer repositioned from the lamina to the nuclear interior. Our search for factors that relocalized the Bcl11b enhancer identified a non-coding RNA named ThymoD (thymocyte differentiation factor). ThymoD-deficient mice displayed a block at the onset of T cell development and developed lymphoid malignancies. We found that ThymoD transcription promoted demethylation at CTCF bound sites and activated cohesin-dependent looping to reposition the Bcl11b enhancer from the lamina to the nuclear interior and to juxtapose the Bcl11b enhancer and promoter into a single-loop domain. These large-scale changes in nuclear architecture were associated with the deposition of activating epigenetic marks across the loop domain, plausibly facilitating phase separation. These data indicate how, during developmental progression and tumor suppression, non-coding transcription orchestrates chromatin folding and compartmentalization to direct with high precision enhancer-promoter communication.
Asunto(s)
Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , ARN no Traducido/genética , Proteínas Represoras/genética , Linfocitos T/citología , Proteínas Supresoras de Tumor/genética , Animales , Factor de Unión a CCCTC , Cromatina/metabolismo , Leucemia/genética , Región de Control de Posición , Linfoma/genética , Ratones , Lámina Nuclear/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Timo/citología , Timo/metabolismo , Transcripción GenéticaRESUMEN
Somatic hypermutation (SHM) requires not only the expression of activation-induced cytidine deaminase, but also transcription in the target regions. However, how transcription guides activation-induced cytidine deaminase in targeting SHM to the Ig genes is not fully understood. Here, we found that the "facilitates chromatin transcription" (FACT) complex promotes SHM by RNAi screening of transcription elongation factors. Furthermore, FACT and histone H3.3, a hallmark of transcription-coupled histone turnover, are enriched at the V(D)J region, 5' flanking sequence of the Sµ switch region and the light chain Jκ 5 segment region in the Ig loci. The regions with the most abundant deposition of FACT and H3.3 were also the most efficient targets of SHM. These results demonstrate the importance of histone-exchanging dynamics at the chromatin of SHM targets, especially in Ig genes.
Asunto(s)
Cromatina/química , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/genética , Hipermutación Somática de Inmunoglobulina , Factores de Elongación Transcripcional/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Cinamatos/metabolismo , Citidina Desaminasa/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Genes Reporteros , Marcadores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Higromicina B/análogos & derivados , Higromicina B/metabolismo , Cambio de Clase de Inmunoglobulina , Inmunoglobulinas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Análisis de Secuencia de ADN , Transcripción Genética , Factores de Elongación Transcripcional/genéticaRESUMEN
An ortholog of activation-induced cytidine deaminase (AID) was, evolutionarily, the first enzyme to generate acquired immune diversity by catalyzing gene conversion and probably somatic hypermutation (SHM). AID began to mediate class switch recombination (CSR) only after the evolution of frogs. Recent studies revealed that the mechanisms for generating immune and genetic diversity share several critical features. Meiotic recombination, V(D)J recombination, CSR, and SHM all require H3K4 trimethyl histone modification to specify the target DNA. Genetic instability related to dinucleotide or triplet repeats depends on DNA cleavage by topoisomerase 1, which also initiates DNA cleavage in both SHM and CSR. These similarities suggest that AID hijacked the basic mechanism for genome instability when AID evolved in jawless fish. Thus, the risk of introducing genome instability into nonimmunoglobulin loci is unavoidable but tolerable compared with the advantage conferred on the host of being protected against pathogens by the enormous Ig diversification.
Asunto(s)
Inmunidad Adaptativa , Citidina Desaminasa , Variación Genética/inmunología , Genoma Humano/inmunología , Inmunoglobulinas/inmunología , Inmunidad Adaptativa/genética , Inmunidad Adaptativa/inmunología , Animales , Linfocitos B/inmunología , Evolución Biológica , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/inmunología , Conversión Génica , Inestabilidad Genómica/genética , Inestabilidad Genómica/inmunología , Histonas/genética , Histonas/inmunología , Humanos , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Inmunoglobulinas/genética , Hipermutación Somática de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/inmunología , Linfocitos T/inmunología , Recombinación V(D)J/genética , Recombinación V(D)J/inmunologíaRESUMEN
Ig class switch recombination (CSR) requires expression of activation-induced cytidine deaminase (AID) and transcription through target switch (S) regions. Here we show that knockdown of the histone chaperone facilitates chromatin transcription (FACT) completely inhibited S region cleavage and CSR in IgA-switch-inducible CH12F3-2A B cells. FACT knockdown did not reduce AID or S region transcripts but did decrease histone3 lysine4 trimethylation (H3K4me3) at both the Sµ and Sα regions. Because knockdown of FACT or H3K4 methyltransferase cofactors inhibited DNA cleavage in H3K4me3-depleted S regions, H3K4me3 may serve as a mark for recruiting CSR recombinase. These findings revealed an unexpected evolutionary conservation between CSR and meiotic recombination.
Asunto(s)
Linfocitos B/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Histonas/metabolismo , Cambio de Clase de Inmunoglobulina/fisiología , Transcripción Genética/fisiología , Animales , Línea Celular , Cromatina/genética , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/genética , Lisina/genética , Lisina/metabolismo , Meiosis/fisiología , Metilación , Ratones , VDJ Recombinasas/genética , VDJ Recombinasas/metabolismoRESUMEN
To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.
Asunto(s)
Citidina Desaminasa/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN/química , ADN/metabolismo , Cambio de Clase de Inmunoglobulina , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Camptotecina/farmacología , Línea Celular , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , ADN/genética , ADN-Topoisomerasas de Tipo I/genética , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Inhibidores de Topoisomerasa IRESUMEN
Recent estimates indicate that approximately 60% of human genes include alternative polyadenylation sites. Hence, control of alternative polyadenylation can have a great impact on gene expression and cellular function. Cleavage factor (CF) Im is a 3'-end processing factor that is essential for in vitro processing. CFIm purified from HeLa cells is associated with three polypeptides of 25, 59 and 68 kD, and it is generally thought to be a heterodimer composed of the 25-kD subunit and one of the larger subunits. Previously, we serendipitously discovered that knockdown of CFIm25 causes an upstream shift in the utilization of alternative polyadenylation sites. Here, we investigated whether this is because of an inherent property of the CFIm complex and, if so, what structural elements are important for its function. The major conclusions of this study are that (i) contrary to previous assumptions, CFIm forms stable heterotetramers through dimerization of CFIm25 and (ii) the CFIm complex per se is responsible for the control of alternative polyadenylation. (iii) However, the structurally related CFIm68 and CFIm59 are functionally redundant and (iv) CFIm68 appears to have a higher specific activity. Thus, this study establishes that CFIm not only plays a general role in 3'-end processing but also plays a regulatory role in poly(A) site selection.
Asunto(s)
Poli A/genética , Multimerización de Proteína , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Empalme Alternativo , Animales , Sitios de Unión/genética , Línea Celular , Expresión Génica , Células HeLa , Humanos , Immunoblotting , Peso Molecular , Poliadenilación , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Spodoptera , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Activation-induced cytidine deaminase (AID) is essential and sufficient to accomplish class-switch recombination and somatic hypermutation, which are two genetic events required for the generation of antibody-mediated memory responses. However, AID can also introduce genomic instability, giving rise to chromosomal translocation and/or mutations in proto-oncogenes. It is therefore important for cells to suppress AID expression unless B lymphocytes are stimulated by pathogens. The mechanisms for avoiding the accidental activation of AID and thereby avoiding genomic instability can be classified into three types: (i) transcriptional regulation, (ii) post-transcriptional regulation and (iii) target specificity. This review summarizes the recently elucidated comprehensive transcriptional regulation mechanisms of the AID gene and the post-transcriptional regulation that may be critical for preventing excess AID activity. Finally, we discuss why AID targets not only Igs but also other proto-oncogenes. AID targets many genes but it is not totally promiscuous and the criteria that specify its targets are unclear. A recent finding that a non-B DNA structure forms upon a decrease in topoisomerase 1 expression may explain this paradoxical target specificity determination. Evolution has chosen AID as a mutator of Ig genes because of its efficient DNA cleavage activity, even though its presence increases the risk of genomic instability. This is probably because immediate protection against pathogens is more critical for species survival than complete protection from the slower acting consequences of genomic instability, such as tumor formation.
Asunto(s)
Diversidad de Anticuerpos/genética , Linfocitos B/inmunología , Citidina Desaminasa/metabolismo , Inestabilidad Genómica , Inmunoglobulina G/inmunología , Animales , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , ADN-Topoisomerasas de Tipo I/metabolismo , Elementos de Facilitación Genéticos , Silenciador del Gen , Humanos , Inmunoglobulina G/genética , Japón , Proto-Oncogenes/genética , Transcripción GenéticaRESUMEN
Human 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) negatively regulate transcription elongation by RNA polymerase II (RNAPII) in vitro. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF- and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position +50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF- and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.
Asunto(s)
Regulación de la Expresión Génica , Genes Inmediatos-Precoces/genética , Genes jun/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Células Cultivadas , Humanos , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Elongación TranscripcionalRESUMEN
Intestinal epithelial cell-derived interleukin (IL)-7 functions as a pleiotropic and nonredundant cytokine in the human intestinal mucosa; however, the molecular basis of its production has remained totally unknown. We here showed that human intestinal epithelial cells both constitutively and when induced by gamma interferon (IFN-gamma) produced IL-7, while several other factors we tested had no effect. Transcriptional regulation via an IFN regulatory factor element (IRF-E) on the 5' flanking region, which lacks canonical core promoter sequences, was pivotal for both modes of IL-7 expression. IRF-1 and IRF-2, the latter of which is generally known as a transcriptional repressor, were shown to interact with IRF-E and transactivate IL-7 gene expression in an IFN-gamma-inducible and constitutive manner, respectively. Indeed, tetracycline-inducible expression experiments revealed that both of these IRF proteins up-regulated IL-7 protein production, and their exclusive roles were further confirmed by small interfering RNA-mediated gene silencing systems. Moreover, these IRFs displayed distinct properties concerning the profile of IL-7 transcripts upon activation and expression patterns within human colonic epithelial tissues. These results suggest that the functional interplay between IRF-1 and IRF-2 serves as an elaborate and cooperative mechanism for timely as well as continuous regulation of IL-7 production that is essential for local immune regulation within human intestinal mucosa.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Epiteliales/fisiología , Regulación de la Expresión Génica , Interleucina-7/metabolismo , Mucosa Intestinal/citología , Fosfoproteínas/metabolismo , Regulación hacia Arriba , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Línea Celular Tumoral , Células Epiteliales/citología , Genes Reporteros , Humanos , Factor 1 Regulador del Interferón , Factor 2 Regulador del Interferón , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-7/genética , Mucosa Intestinal/metabolismo , Datos de Secuencia Molecular , Unión Proteica , ARN Interferente Pequeño/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.