Abstract
The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes.
Highlights
The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium
We applied the RNA-seq approach to systematically analyze the effect of etoposide, which blocks the religation activity of the topoisomerase 2 (TOP2) enzymes, in the doxycyclineing; IP, immunoprecipitation; FAIRE-seq, formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing; 3C, chromosome conformation capture; CEACAM, carcinoembryonic antigenlike cellular adhesion molecule; etoposide alone (Etop), etoposide; Dox, doxycycline; DE, differentially expressed; qPCR, quantitative real-time PCR; DEU, differential exon usage; Ctrl, control; PARP, poly(ADP-ribose) polymerase; CPT, camptothecin; Mer, merbarone; -lap, -lapachone; CARD, caspase recruitment domain; CTCF, CCCTC-binding factor; Br-dUTP, bromodeoxyuridine triphosphate; DNA-PK, DNA-dependent protein kinase; MHCII, major histocompatibility complex class I
In agreement with Abramson et al [6], that etoposide alone (Etop) was able to increase, albeit to a lesser extent (4, 7, and 3-fold, respectively), the expression of AIRE-dependent S100A8 and IVL and AIRE-independent PSMD4 genes (Fig. 1A). These results suggest a strong synergistic effect of AIRE and low-dose etoposide on target gene expression, which is much higher than etoposide alone
Summary
The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. We applied the RNA-seq approach to systematically analyze the effect of etoposide, which blocks the religation activity of the TOP2 enzymes, in the doxycyclineing; IP, immunoprecipitation; FAIRE-seq, formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing; 3C, chromosome conformation capture; CEACAM, carcinoembryonic antigenlike cellular adhesion molecule; Etop, etoposide; Dox, doxycycline; DE, differentially expressed; qPCR, quantitative real-time PCR; DEU, differential exon usage; Ctrl, control; PARP, poly(ADP-ribose) polymerase; CPT, camptothecin; Mer, merbarone; -lap, -lapachone; CARD, caspase recruitment domain; CTCF, CCCTC-binding factor; Br-dUTP, bromodeoxyuridine triphosphate; DNA-PK, DNA-dependent protein kinase; MHCII, major histocompatibility complex class I We tested whether this overlap implies that the expression of AIRE could entail changes in CTCF-mediated chromatin interactions using the 3C method, and we were able to determine altered chromatin looping in the CEACAM gene cluster
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