Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases

Abstract

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.

Histone clipping: the punctuation in the histone code

“…Third, Marruecos et al investigate a direct interaction with another essential cell signaling molecule, i.e., IkBa. Finally, the findings reported here are coherent with the few common notions about histone clipping (Dhaenens et al , 2015): (i) Histone clipping is strongly associated with developmental transitions (Duncan et al , 2008; De Clerck et al , 2019; Ferrari et al , 2021); (ii) proteins that can cleave histones have all been known for very different functions and are most often not associated with nuclear localization; (iii) hPTMs might regulate the clipping (Duncan et al , 2008; Ferrari et al , 2021); and (iv) very plausibly, clipping is part of a complex regulatory system that has a lot of redundancy (Duncan et al , 2008; Dhaenens et al , 2015; Ferrari et al , 2021). This redundancy is now also confirmed in intestinal differentiation, through either cathepsin L on H3 (Ferrari et al , 2021) or chymotrypsin on H4 (Marruecos et al , 2021).…”

“…Finally, the findings reported here are coherent with the few common notions about histone clipping (Dhaenens et al , 2015): (i) Histone clipping is strongly associated with developmental transitions (Duncan et al , 2008; De Clerck et al , 2019; Ferrari et al , 2021); (ii) proteins that can cleave histones have all been known for very different functions and are most often not associated with nuclear localization; (iii) hPTMs might regulate the clipping (Duncan et al , 2008; Ferrari et al , 2021); and (iv) very plausibly, clipping is part of a complex regulatory system that has a lot of redundancy (Duncan et al , 2008; Dhaenens et al , 2015; Ferrari et al , 2021). This redundancy is now also confirmed in intestinal differentiation, through either cathepsin L on H3 (Ferrari et al , 2021) or chymotrypsin on H4 (Marruecos et al , 2021). However, one aspect of clipping is very different this time: Despite its dramatic nature, inhibition of clipping rarely inhibits cell transition (Duncan et al , 2008), yet in the current work (Marruecos et al , 2021) and in Ferrari et al, (2021), it is shown for the first time that trypsin inhibition can block intestinal differentiation effectively.…”

Retrospective analysis of enhancer activity and transcriptome history

“…In contrast, DCM gene body labelling of canonical PRC1 members Cbx2,4,6,7 and 8 was below background levels, indicating that involvement of PRC1 in ISC maintenance is mediated through ncPRC1 complexes (Fig 5a)(Blackledge et al, 2014). To investigate the role of PRC1 and PRC2 in promoter and enhancer regulation during ISC to enterocyte differentiation, we examined enrichment of H2A119ub or H3K27me3, catalysed by PRC1 and PRC2 respectively, using ISC, crypt and enterocyte ChIP-seq data sets (Chiacchiera et al, 2016a; Ferrari et al, 2021). This analysis revealed a lack of enrichment of both H2Aub119 and H3K27me3 at enhancers at any stage of ISC differentiation (Fig 5c).…”

A Purpose-Built System for Culturing Cells as In Vivo Mimetic 3D Structures

“…Epigenetic marking and histone clipping have been shown to be recovered in spheroids [38] and this has been shown to occur during intestinal differentiation in vivo [50]. Metabolic reprogramming,which may be a key change during cancer development is also demonstrated in 3D spheroids [9].…”