HMGA1/SMAD3 Promoter Complex Mediates PD-L1-Dependent Transcriptional Regulation of GAS6, EGR1 and PD-L1

Programmed death-ligand 1 (PD-L1) partners with specificity Protein 1 (SP1) or signal transducer and activator of transcription 3 (STAT3) to modulate the transcription of growth arrest-specific 6 (GAS6) and early growth response protein 1 (EGR1), necessitating mediators to avoid feedback. Based on binding and stemness data, high mobility group A1 (HMGA1) and Small Mother Against Decapentaplegic3 (SMAD3) were identified as potential mediators in this context. While the SMAD3-P300-STAT3 complex facilitates SMAD3-STAT3 crosstalk, it remains unclear whether the PD-L1-HMGA1-SP1 or PD-L1-SMAD3-SP1 complexes bind to GAS6 and EGR1 promoters to regulate their transcription.

MG63 osteosarcoma cells and SW620 colon cancer cells with unidentified nuclear PD-L1 function were chosen for our study. Chromatin immunoprecipitation and co-immunoprecipitation assays were performed to evaluate SP1, HMGA1, SMAD3, STAT3, P300 and PD-L1 (also denoted CD274) enrichment at the GAS6 and EGR1 promoters; the existence of the PD-L1-(HMGA1 or SMAD3)-SP1 complexes; whether P300 binds to STAT3; and whether HMGA1 and SMAD3 bind to P300. The alterations in GAS6, EGR1 and PD-L1 mRNA levels after their combined over-expression and/or knockdown were assessed via qPCR. Two representative target genes identified via PD-L1 chromatin immunoprecipitation (ChIP)-seq were examined to determine whether HMGA1 and SMAD3 were enriched at their promoters.

PD-L1, HMGA1, SMAD3, SP1, P300 and STAT3 were enriched at GAS6 and EGR1 promoters in two cell lines. HMGA1 or SMAD3 antibody pulled down PD-L1 and SP1; PD-L1 antibody pulled down HMGA1, SMAD3 and SP1; P300 antibody pulled down STAT3; and, surprisingly, HMGA1 and SMAD3 antibodies pulled down P300. Combined over-expression or knockdown significantly altered GAS6, EGR1 and PD-L1 mRNA levels. PD-L1 ChIP-seq indicated 114 target genes, among which PD-L1 and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) were chosen to verify the promoter enrichment of HMGA1 and SMAD3.

Our study provides initial evidence that PD-L1 might form HMGA1- and SMAD3-dependent complexes to bind the GAS6, EGR1 and CD274 promoters, thus modulating the transcription of GAS6, EGR1 and PD-L1 mRNA in cancer and sarcoma cells.