Dual function of PHF16 in reinstating homeostasis of murine intestinal epithelium after crypt regeneration.

Intestinal stem cells (ISCs) are highly vulnerable to damage, being in a constant state of proliferation. Reserve stem cells repair the intestinal epithelium following damage-induced ablation of ISCs. Here, we report that the epigenetic regulator plant homology domain (PHD) finger protein 16 (PHF16) restores homeostasis of the intestinal epithelium after initial damage-induced repair. In Phf16-/Y mice, revival stem cells (revSCs) showed defects in exiting the regenerative state, and intestinal crypt regeneration failed even though revSCs were still induced in response to tissue damage, as observed by single-cell RNA sequencing (scRNA-seq). Analysis of Phf16-/Y intestinal organoids by RNA sequencing (RNA-seq) and ATAC sequencing identified that PHF16 restores homeostasis of the intestinal epithelium by inducing retinoic acid receptor (RAR)/retinoic X receptor (RXR) target genes through HBO1-mediated histone H3K14 acetylation, while at the same time counteracting YAP/TAZ activity by ubiquitination of CDC73. Together, our findings demonstrate the importance of timely suppression of regenerative activity by PHF16 for the restoration of gut homeostasis after acute tissue injury.

Isoforms of GPR35 have distinct extracellular N-termini that allosterically modify receptor-transducer coupling and mediate intracellular pathway bias.

Within the intestine, the human G protein-coupled receptor (GPCR) GPR35 is involved in oncogenic signaling, bacterial infections, and inflammatory bowel disease. GPR35 is known to be expressed as two distinct isoforms that differ only in the length of their extracellular N-termini by 31 amino acids, but detailed insights into their functional differences are lacking. Through gene expression analysis in immune and gastrointestinal cells, we show that these isoforms emerge from distinct promoter usage and alternative splicing. Additionally, we employed optical assays in living cells to thoroughly profile both GPR35 isoforms for constitutive and ligand-induced activation and signaling of 10 different heterotrimeric G proteins, ligand-induced arrestin recruitment, and receptor internalization. Our results reveal that the extended N-terminus of the long isoform limits G protein activation yet elevates receptor-ß-arrestin interaction. To better understand the structural basis for this bias, we examined structural models of GPR35 and conducted experiments with mutants of both isoforms. We found that a proposed disulfide bridge between the N-terminus and extracellular loop 3, present in both isoforms, is crucial for constitutive G13 activation, while an additional cysteine contributed by the extended N-terminus of the long GPR35 isoform limits the extent of agonist-induced receptor-ß-arrestin2 interaction. The pharmacological profiles and mechanistic insights of our study provide clues for the future design of isoform-specific GPR35 ligands that selectively modulate GPR35-transducer interactions and allow for mechanism-based therapies against, for example, inflammatory bowel disease or bacterial infections of the gastrointestinal system.