Circ-myh8/KAT7 Affects PANoptosis in Pulmonary Arterial Smooth Muscle Cells: Involvement of Super-Enhancers in FOSL2 Expression

Abstract

Background: Pulmonary hypertension is a pathophysiological condition characterized by multiple forms of regulated cell death. PANoptosis, which is an inflammation-driven mode of regulated cell death, is regulated by the PANoptosome. Super-enhancers (SEs) have been implicated in the pathology of pulmonary hypertension by modulating the transcriptional regulation of target genes. However, it remains unclear whether SEs influence the onset of PANoptosis in pulmonary arterial smooth muscle cells (PASMCs) via the regulation of specific target genes.

Methods: Chromatin immunoprecipitation (ChIP)-sequencing was integrated with RNA sequencing to identify SE-regulated target genes in hypoxic PASMCs. Bioinformatics analysis was used to predict transcription factors with potential binding sites in the promoter or SE regions of these target genes. Coimmunoprecipitation and ChIP-polymerase chain reaction were conducted to validate the epigenetic regulatory mechanisms governing SE-regulated target genes. Immunoblotting was performed to assess the expression of PANoptosis proteins, whereas YP1 (YO-PRO-1; Oxazole yellow) and PI (propidium iodide) fluorescence staining and immunofluorescence were used to evaluate the effects of transcription factors and SE on PANoptosis in PASMCs. Additionally, reverse transcriptase-quantitative polymerase chain reaction, immunoblotting, and ChIP-polymerase chain reaction assays were used to investigate the impact of the SE region of the target gene on PANoptosis in PASMCs following CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9)-mediated knockdown of the SE regions of the target genes.

Results: A combination of ChIP sequencing and RNA sequencing analyses confirmed that FOSL2 (Fos-like antigen 2) is a target gene regulated by SEs. Bioinformatics predictions revealed that the transcription factor MAZ (myc-associated zinc finger protein) has binding sites within both the SE and promoter regions of FOSL2. Coimmunoprecipitation and ChIP-polymerase chain reaction experiments demonstrated that KAT7 (lysine acetyltransferase 7) interacts with H3K27ac (acetylation of lysine 27 of histone H3) and that circ_chr11_67292179-67294612 (circ-myh8) and KAT7 enhances H3K27ac enrichment in the SE region of FOSL2. CRISPR-Cas9-mediated knockdown of the FOSL2 SE resulted in a reduction in PANoptosis in PASMCs. Additionally, ChIP-polymerase chain reaction assays revealed that FOSL2 functions as a transcription factor that binds to the promoters of PANoptosis-related genes.

Conclusions: Circ-myh8 RNA, which functions jointly with KAT7, enhances H3K27ac enrichment, thereby promoting SE activity for FOSL2 transcription. This process ultimately contributes to the induction of PANoptosis in PASMCs. These findings elucidate the role of the circ-myh8/KAT7/SE/FOSL2 axis in the regulation of PANoptosis in PASMCs, thus offering new insights into the pathophysiology of PANoptosis in pulmonary hypertension. These findings may lead to novel therapeutic strategies for the treatment of pulmonary hypertension.

Keywords: FOSL2; PANoptosis; pulmonary hypertension; super‐enhancer.