NF-kB-induced interplay between histone marks, chromatin topology, and alternative splicing regulation upon HTLV-1 infection – INFLASPLICE

The NF-kB family of transcription factors plays essential roles in multiple physiological and pathological processes. NF-kB activation consists in a rapid and tight coordination of gene transcription between hundred target genes, the majority being involved in cell differentiation, cell proliferation, immune response, and inflammation. Multigene complexes, also called NF-kB factories, have been proposed to spatially and timely coordinate gene transcription of NF-kB-responsive genes. This suggests that perturbation of a single gene within a multigene complex may affect gene expression of other contacted genes. However, the molecular mechanisms underlying NF-kB-induced gene-gene contacts as well as their impacts on other transcription-coupled processes remain unknown.
Infection by Human T-cell Leukemia Virus type 1 (HTLV-1) involves constitutive activation of NF-kB that contributes to the clonal expansion of infected cells, viral persistence, and HTLV-1-associated pathogenesis. We have recently evidenced that, besides transcription, the activation of NF-kB by HTLV-1 TAX protein affects alternative splicing, which is a transcription-coupled process considered as a major actor in transcriptome/proteome diversity and cell phenotype. Upon TAX as well as non-viral NF-kB inducers (TNFa), we demonstrated that the increased RELA occupancy of gene bodies results in recruiting the splicing factor DDX17, which regulates splicing of nearby exons thanks to its RNA helicase activity. Interestingly, RELA regulates alternative splicing in an independent manner of its activity in transcription, raising the question of how these two functions of RELA are coordinated upon NF-kB activation by TAX or non-viral stimuli.
Using integrative analysis of 3D chromatin topology and gene expression profiles, our preliminary data indicate that TAX-induced NF-kB activation promotes gene-gene contacts between genes undergoing transcriptional and alternative splicing regulations. Using epigenetic engineering tools, we demonstrated that the local disruption of gene-gene contacts led to inhibit both DDX17 enrichment of proximal genomic exon target and its corresponding alternative splicing regulation. At the chromatin level, we observed that TAX-induced gene-gene contacts are enriched in RELA, DDX17, and TAX, demonstrating their involvement in multigene complex formation. Furthermore, the analysis of publicly available datasets revealed that RELA:DDX17-regulated genomic exons are enriched in specific histone marks, suggesting that epigenetic changes might coordinate gene-gene contacts, transcription elongation, and alternative splicing upon NF-kB activation. Altogether, these preliminary data propose that alternative splicing does not solely rely on local genomic and epigenetic features of target exon but also depends on its dynamic nuclear localization with other genes coregulated in transcription.
To address these questions, we have set up a very complementary collaboration between our two teams to challenge this model. Team 1 (F. Mortreux) has an internationally recognized experience in studying HTLV-1 and its impacts on post-transcriptional regulations, while Team 2 (R.F. Luco) has a pioneering expertise in studying mechanisms that link epigenetics to alternative splicing regulation. Our objectives are to accurately assess the chromatin occupancy of RELA, DDX17, TAX, and histone marks in gene-gene contacts linked to alternative splicing, to examine the local involvement of histone modifiers and chromatin architecture proteins, and to study the causal relationship that links gene-gene contacts and histone modifications to alternative splicing occurring upon NF-kB activation triggered by TAX and non-viral inducers. These data will depict spatial coordination of gene transcription and alternative splicing upon NF-kB activation, which is a poorly understood aspect of gene regulation upon HTLV-1 infection and in a more general manner in inflammatory diseases.