Targeting P-TEFb recruitment and super-enhancers function suppress the development of Th1-mediated EAU disease

Abstract

Purpose: We aimed to determine how T-bet acts through the enhancer elements to drive Th1 differentiation and also to provide further insight into the mechanism of super-enhancer function using human and mice models.

Methods: To identify the lineage-specific genes and groups of genes repressed by JQ1 and/or Flavopiridol in Th1 cells, wild-type C57BL/6 mouse naive CD4+ T cells were cultured under Th1 and Th2 polarizing conditions and run for gene expression profiling using the Mouse GE 8x60K microarrays (Agilent).The libraries were generated for RNA-seq and RNA-Chip and quantified and then sequenced on an Illumina HiSeq (50bp paired-end) and Illumina GAIIx ro HiSeq 2000 sequencer, respectively. To induce EAU, B10RIII mice aged 5-8 weeks were immunized with 300 mg IRBP_161-180 emulsified with Complete Freund’s adjuvant, supplemented with 1.5 mg/ml Mycobacterium tuberculosis complete H37 Ra (1:1 v/v). Each mouse also received 0.4mg Bordetella pertussis toxin intraperitoneally. Flavopiridol and JQ1 were then administered by daily intraperitoneal injection and disease progression scored by retinal funduscopy and histology.

Results: We revealed that T-bet acts through super-enhancers to recruit P-TEFb and activate lineage-specific transcriptional elongation. We showed that recruitment of P-TEFb to Th1 genes depends on T-bet activity and both T-bet and P-TEFb function at super-enhancers to activate transcription of enhancer RNAs. We also demonstrated that P-TEFb activity was required for Ifng eRNA production and treating Th1 cells with JQ1 and Flavopiridol resulted in a marked reduction in eRNA levels. This requirement for P-TEFb function renders lineage-specific genes hyper-sensitive to inhibitors of transcriptional elongation both in vitro and in vivo in a mouse model of uveitis. EAU mice treated with Flavopiridol and JQ1 showed suppression of disease which associated with inhibition of super-enhancer function. Flow cytometry of CD4+ T cells sorted from the retina and lymph node revealed that expression of the super-enhancer-associated gene products Ifng, Tnf, Fasl, Il18r1 and Ctla4 were downregulated by Flavopiridol and JQ1.

Conclusions: Using in vitro and in vivo models, we concluded that P-TEFb binds extensively to the super-enhancers of lineage-specific genes and revealed a molecular mechanism for the selective effect of those drugs on genes involved in mediating inflammation.