Differential RELA and GR recruitment to the BIRC3/BIRC2 locus: Molecular insight as to combinatorial regulation by proinflammatory cytokines and glucocorticoid

In pulmonary epithelial cells, baculoviral inhibitor of apoptosis repeat-containing (BIRC) gene, BIRC3, and to a lesser extent, BIRC2, mRNAs were upregulated by interleukin (IL)-1β and tumor necrosis factor-α. Glucocorticoids also induced BIRC3 mRNA and the glucocorticoid receptor (GR) was recruited to GR-binding regions (GBRs) proximal to, and within, BIRC3 in A549 and BEAS-2B cells. Four such GBRs drove glucocorticoid-inducible, Organon 34517-antagonized, transcription in A549 cells. IL-1β and tumor necrosis factor-α recruited the nuclear factor (NF)-κB transactivating subunit, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), to RELA-binding regions (RBRs) (R4 and R7) upstream of the BIRC3 and BIRC2 transcription start sites. These RBRs drove IL-1β-inducible transcription that involved NF-κB. Thus, direct regulation of BIRC3 by GR and BIRC3/BIRC2 by NF-κB is indicated. IL-1β-plus-budesonide also recruited RELA to multiple GBRs, whereas GR was recruited to the main IL-1β-induced RBR (R4), effects that correlated with positive IL-1β/glucocorticoid transcriptional cooperativity or additivity. At the R5 GBR, RELA was not recruited and both GR binding and glucocorticoid-dependent transcription reduced (infra-additivity) on cotreatment. Similarly, the R7 RBR barely recruited GR and IL-1β-induced transcription showed infra-additivity with IL-1β-plus-glucocorticoid. R8 recruited GR and RELA primarily with IL-1β-plus-glucocorticoid and revealed transcriptional synergy. Thus, GR/RELA-corecruitment yielded positive cooperative and additive transcriptional effects, whereas recruiting one factor alone associated with infra-additivity. Furthermore, DNA looping revealed how multiple RBRs/GBRs may integrate to control transcription. Because IL-1β- and glucocorticoid-dependent coupregulation of apoptotic/antiapoptotic genes was widespread, the combinatorial recruitment of RELA/GR to regulatory genes, including BIRC3, CFLAR plus others in the NF-κB pathway, may be critical to cell fate determination in inflammation. SIGNIFICANCE STATEMENT: Identification of functional GR- and RELA-binding regions at the BIRC3/BIRC2 locus explains the upregulation of BIRC3 expression by glucocorticoids and inflammatory cytokines. Cytokine-plus-glucocorticoid cotreatment revealed positive cooperative and additive interactions between GR and RELA, whereas DNA regions binding only one factor showed reduced effects on binding and transcription. These region-specific outcomes, combined with DNA looping between regulatory regions, provides insight as to how factors at multiple DNA regions may integrate their outputs to produce combinatorial regulation of apoptotic/antiapoptotic genes.