Análisis transcripcional de genes de respuesta a Interferon de tipo 1 en pacientes con Aritritis Reumatoide temprana y familiares consanguíneos

"La artritis reumatoide (AR) es una enfermedad autoinmune de etiología desconocida caracterizada por la presencia de autoanticuerpos de tipo IgM e IgG. Se ha observado que en sujetos sanos los autoanticuerpos, en particular contra péptidos citrulinados (ACCP), aumentan el riesgo a desarrollar AR. El riesgo es aún mayor en familiares de primer grado de pacientes con AR. Por otra parte, los genes inducidos por Interferon de tipo I predicen el éxito o fracaso de la terapia biológica, pero no se sabe si puedan predecir el desarrollo de la AR o si se relacionan con la seropositividad a ACCP. Algunos autores sugieren que la sobreexpresión de 7 genes (MXA, MXB, LY6E, ISG15, EPSTI1, RSAD2 y HERC5) se asocia con la progresión a la fase clínica temprana de AR. En este estudio evaluamos la expresión relativa de estos 7 genes en sangre periférica de sujetos en diferente estado de evolución de esta enfermedad mediante qPCR. En particular comparamos sujetos en estado preclínico y aquellos en la etapa temprana del desarrollo de AR. Se tomó sangre a 16 pacientes con AR, a 20 familiares de primer grado de pacientes con AR y a 10 sujetos sanos. Se separó el suero para medir la presencia de anticuerpos ACCP y se aisló RNA de sangre para producir cDNA. Con el cDNA de cada sujeto se cuantificaron los niveles de expresión relativa de los genes MXA, MXB, LY6E, ISG15, EPSTI1, RSAD2 y HERC5 por el método de ΔΔCt. La expresión de LY6E, ISG15 y RSAD2 estan elevados únicamente en los familiares de pacientes con AR con respecto a los sanos (p<0.05); la expresión de HERC5 se encuentra aumentada en pacientes con AR temprana con respecto a los sanos y respecto a los pacientes con AR crónica (p<0.05) y MXB solo se eleva en pacientes con ARt y familiares ACCP+ en comparación a los familiares ACCP- y sujetos sanos, respectivamente (p<0.05). Los niveles de expresión de MXB y HERC5 se relacionan con la seropositividad a ACCP y pueden predecir el desarrollo de las diferentes etapas de evolución de la AR. Por su parte LY6E, ISG15 y RSAD2 pueden predecir la evolución a una fase pre-clínica en sujetos ACCP+, pero no a la fase de enfermedad de AR temprana.""Rheumatoid Arthritis (RA) is an autoimmune disease of unknow etiology, distinguished bye the presence of IgM and IgG autoantibodies in serum. It has been reported that the presence of autoantibodies, specially against citrullinated peptides (ACCP), in healthy subjects increase the risk to developing RA. This risk is even greater in first-degree relatives of patients with RA. The Interferon type I response genes, have been related to predict the therapeutic outcome in RA with biologic treatment, however, is not clear if can predict the development of RA or their association with in ACCP seropositivity. We sugest that overexpression of seven IFN type I response genes (MXA, MXB, LY6E, ISG15, EPSTI1, RSAD2 y HERC5) are involved in the early clinic phase progression. In this study, we evaluated the relative expression of those seven genes in periferal blood of subjects of different disease stage. In particular, compared between the preclinic stage and the early stage of RA. Whole blood samples were collected and total RNA extracted from 16 RA patients, 20 first degree relatives and 10 healthy subjects. We determined the serum levels of ACCP antibodies. The relative expression of MXA, MXB, LY6E, ISG15, EPSTI1, RSAD 2 and HERC5 was calculated by the ΔΔCt method. LY6E, ISG15 and RSAD2 is up-regulated only in the first-degree relatives of RA patients compared to healthy subjects (p<0.05); HERC5 is up-regulated in early RA compared to healthy subjects and patients with chronic RA(p<0.05); and MXB is up-regulated in patients with early RA and ACCP+ relatives compared to ACCP- relatives and healthy subjects. The expression of MXB and HERC5 seems to be related with the seropositivity to ACCP presence and might predict the development in different stages of RA. On the other hand, LY6E, ISG15 and RSAD2 are useful to predict the transition to a pre-clinical stage with ACCP+, but not to the early RA stage.

Dissection de la réponse coordonnée de l'interféron par des éléments promoteurs avec une activité enhancer

L'expression génique est contrôlée par l'implication des éléments régulateurs du gène proximal (promoteurs) et distal (enhancer). Des résultats antérieurs ont démontré qu'un sous-ensemble de promoteurs de gènes, également appelés Epromoteurs, fonctionne comme des enhancers de bona fide et régule l'expression distale des gènes. Nous avons émis l'hypothèse que les Epromoteurs pourraient jouer un rôle clé dans la coordination de l'induction rapide des gènes dans la réponse au stress, en particulier pendant l'inflammation. En utilisant un test de reporter à haut débit, nous avons exploré la fonction d'Epromoter en réponse à l'interféron de type I. Nous avons constaté que les facteurs de transcription STAT1/STAT2 et IRF se lient préférentiellement aux Epromoteurs induits par l'IFNa et régulent de manière distale l'activation des gènes de réponse à l'interféron. Des observations similaires ont été faites dans d'autres types de réponse inflammatoire. Nos résultats suggèrent que les Epromoteurs pourraient fonctionner comme une plaque tournante pour recruter les TF clés nécessaires à la régulation coordonnée des grappes de gènes pendant la réponse inflammatoire, et plus généralement sur la réponse cellulaire aux signaux intra et extracellulaires.Gene expression is controlled by the involvement of gene-proximal (promoters) and distal (enhancers) regulatory elements. Previous results have demonstrated that a subset of gene promoters also termed Epromoters, works as bona fide enhancers and regulate distal gene expression. We hypothesised that Epromoters might play a key role in the coordination of rapid gene induction in the stress response, in particular during inflammation. Using a high-throughput reporter assay we explored the function of Epromoter in response to type I interferon. We found that STAT1/2 and IRF transcription factors preferentially bind to IFNa-induced Epromoters and distally regulate the activation of interferon-response genes. Similar observations were made in other types of inflammatory response. Our findings suggest that Epromoters might function as a hub to recruit key TFs required for coordinated regulation of gene clusters during the inflammatory response, and more generally upon cellular response to intra- and extra-cellular signals

Approches haut débit pour l’étude des séquences

La régulation de la transcription des gènes chez les eucaryotes supérieurs implique l’action d’éléments régulateurs proximaux (promoteurs) ou distaux (amplificateurs ou enhancers) du site d’initiation de la transcription (Transcription Start Site, TSS). Il est aujourd’hui bien connu que les éléments enhancers jouent un rôle essentiel dans le développement et la différenciation cellulaire. Les altérations génétiques de ces éléments sont une cause majeure de pathologies humaines. De nombreuses stratégies ont été développées pour identifier et caractériser les enhancers. Ici, nous discutons des progrès récents pour évaluer de façon systématique l’activité des enhancers allant des approches haut débit de type « gène rapporteur » aux récentes technologies basées sur le système CRISPR/Cas9. Nous soulignons comment ces approches contribuent à une meilleure compréhension de la fonction des enhancers en conduisant à la découverte de nouveaux types de séquences de régulation et comment l’altération des enhancers peut affecter la régulation transcriptionnelle

Widespread Enhancer Activity from Core Promoters

International audienceGene expression in higher eukaryotes is precisely regulated in time and space through the interplay between promoters and gene-distal regulatory regions, known as enhancers. The original definition of enhancers implies the ability to activate gene expression remotely, while promoters entail the capability to locally induce gene expression. Despite the conventional distinction between them, promoters and enhancers share many genomic and epigenomic features. One intriguing finding in the gene regulation field comes from the observation that many core promoter regions display enhancer activity. Recent high-throughput reporter assays along with CRISPR/Cas9-related approaches have indicated that this phenomenon is relatively common and might have strong impact in our global understanding of genome organization and gene expression regulation.

Recent advances in high-throughput approaches to dissect enhancer function

International audienceno abstrac

Epromoters function as a hub to recruit key transcription factors required for the inflammatory response

International audienceGene expression is controlled by the involvement of gene-proximal (promoters) and distal (enhancers) regulatory elements. Our previous results demonstrated that a subset of gene promoters, termed Epromoters, work as bona fide enhancers and regulate distal gene expression. Here, we hypothesized that Epromoters play a key role in the coordination of rapid gene induction during the inflammatory response. Using a high-throughput reporter assay we explored the function of Epromoters in response to type I interferon. We find that clusters of IFNa-induced genes are frequently associated with Epromoters and that these regulatory elements preferentially recruit the STAT1/2 and IRF transcription factors and distally regulate the activation of interferon-response genes. Consistently, we identified and validated the involvement of Epromoter-containing clusters in the regulation of LPS-stimulated macrophages. Our findings suggest that Epromoters function as a local hub recruiting the key TFs required for coordinated regulation of gene clusters during the inflammatory response

Integration of high-throughput reporter assays identify a critical enhancer of the Ikzf1 gene

International audienceThe Ikzf1 locus encodes the lymphoid specific transcription factor Ikaros, which plays an essential role in both T and B cell differentiation, while deregulation or mutation of IKZF1/ Ikzf1 is involved in leukemia. Tissue-specific and cell identity genes are usually associated with clusters of enhancers, also called super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. Several potential regulatory regions have been identified in close proximity of Ikzf1, however, the full extent of the regulatory landscape of the Ikzf1 locus is not yet established. In this study, we combined epigenomics and transcription factor binding along with high-through-put enhancer assay and 4C-seq to prioritize an enhancer element located 120 kb upstream of the Ikzf1 gene. We found that deletion of the E120 enhancer resulted in a significant reduction of Ikzf1 mRNA. However, the epigenetic landscape and 3D topology of the locus were only slightly affected, highlighting the complexity of the regulatory landscape regulating the Ikzf1 locus

A critical regulator of Bcl2 revealed by systematic transcript discovery of lncRNAs associated with T-cell differentiation

Abstract Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8- double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion of Robnr, an inducible lncRNA located downstream of the anti-apoptotic gene Bcl2, demonstrated a critical role of the Robnr locus in the induction of Bcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation