HDAC1 and HDAC2 independently regulate common and specific intrinsic responses in murine enteroids

www.nature.com/scientificreports OPEN Received: 2 January 2019 Accepted: 19 March 2019 Published: xx xx xxxx HDAC1 and HDAC2 independently regulate common and specific intrinsic responses in murine enteroids Alexis Gonneaud, Naomie Turgeon, Christine Jones, Cassandra Couture, Dominique Lévesque, François-Michel Boisvert, François Boudreau & Claude Asselin Both HDAC1 and HDAC2 are class I deacetylases acting as erasers of lysine-acetyl marks on histones and non-histone proteins. Several histone deacetylase inhibitors, either endogenous to the cell, such as the ketogenic β-hydroxybutyrate metabolite, or exogenous, such as butyrate, a microbialderived metabolite, regulate HDAC activity. Different combinations of intestinal epithelial cell (IEC)-specific Hdac1 and/or Hdac2 deletion differentially alter mucosal homeostasis in mice. Thus, HDAC1 and HDAC2 could act as sensors and transmitters of environmental signals to the mucosa. In this study, enteroid culture models deleted for Hdac1 or Hdac2 were established to determine IEC-specific function as assessed by global transcriptomic and proteomic approaches. Results show that Hdac1 or Hdac2 deficiency altered differentiation of Paneth and goblet secretory cells, which sustain physical and chemical protection barriers, and increased intermediate secretory cell precursor numbers. Furthermore, IEC Hdac1- and Hdac2-dependent common and specific biological processes were identified, including oxidation-reduction, inflammatory responses, and lipid-related metabolic processes, as well as canonical pathways and upstream regulators related to environment-dependent signaling through steroid receptor pathways, among others. These findings uncover unrecognized regulatory similarities and differences between Hdac1 and Hdac2 in IEC, and demonstrate how HDAC1 and HDAC2 may complement each other to regulate the intrinsic IEC phenotype. The small intestinal epithelium is composed of a single row of epithelial cells divided in proliferative crypt and differentiated villus compartments1. Crypt-located reserve intestinal stem cells sustain epithelial renewal by dividing in columnar stem cells generating transit-amplifying cells. These cells further segregate in absorptive enterocytes and secretory progenitor cells, precursors of Paneth, goblet and enteroendocrine cells2. Each differentiated cell lineage contributes to small intestinal functions, notably by establishing physical and chemical barriers between the host and the luminal diet and microbial content, and by providing a sensing and transmitting interface between the lumen and the mucosal immune system3. Indeed, in addition to absorptive and digestive functions, enterocytes, the most abundant intestinal epithelial cells (IEC), achieve selective barrier permeability through tight junction interactions between intestinal epithelial cells4–6. Enterocytes also participate in the chemical barrier by expressing transmembrane mucins as well as cytokines and antimicrobial proteins, in response to the microbial environment7. Goblet cells produce the mucus layer preventing bacterial adhesion to the epithelium as well as various antimicrobial proteins, and deliver luminal antigens to dendritic cells6,8. Crypt-located Paneth cells support the stem cell niche and produce different constitutive or inducible antimicrobial proteins to insure epithelial protection9,10. Many signaling pathways, including the Wnt and Notch pathways, regulate intestinal stem cell maintenance, renewal and differentiation11,12. Intestinal homeostasis is secured by interdependent communication signals between the intestinal mucosal system along with IEC, the luminal environment with diet-derived and microbial products, as well as the microbiota. However, alterations in the intestinal environment or the immune system, in conjunction with genetic susceptibilities, may lead to intestinal defects, including inappropriate inflammatory responses3. Département d’anatomie et biologie cellulaire, Faculté de médecine et des sciences de la santé, Pavillon de recherche appliquée sur le cancer, Université de Sherbrooke, Sherbrooke, Québec, J1E 4K8, Canada. Correspondence and requests for materials should be addressed to C.A. (email: ) Scientific Reports | (2019) 9:5363 | https://doi.org/10.1038/s41598-019-41842-6 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Environmental changes are transmitted to the cell through epigenetic modifications of histones13. Acetylation is one epigenetic signal implicated as environmental sensor. Acetyltransferase readers add an acetyl group to histones on lysines, leading to alterations in DNA-histone interactions or to the production of acetyl marks recognized by bromodomain-containing regulators14. Histone acetylation efficiency is regulated in part by variations in mitochondrial and nucleo-cytosolic acetyl-CoA levels as a result of the cellular metabolic state15,16. Lysine acetylation is also controlled by histone deacetylase (HDAC) erasers that remove acetyl groups from histones and non-histone proteins. Endogenous HDAC activity is inhibited by metabolites including β-hydroxybutyrate17,18, L-carnitine19 and sphingosine-1-phosphate20, as well as diet- and bacterially-derived metabolites, such as butyrate21–24. Among HDACs, HDAC1 and HDAC2 are zinc-dependent class I deacetylases associated with Sin3A, CoREST and NuRD protein complexes regulating transcription, DNA replication and DNA repair, among others25–27. Hdac1 deletion in mice leads to embryonic lethality28 while Hdac2 deficiency results in perinatal lethality stemming from heart defects29. In contrast to no apparent defects or subtle phenotypic alterations in many Hdac1 or Hdac2 tissue-specific deletion models, dual Hdac1 and Hdac2 deficiency triggers extensive differentiation and proliferation alterations in most tissues25. Of note, gene-dosage experiments in mice have indicated different sensitivities to Hdac1 or Hdac2 expression levels. For example, murine brain is altered in mice with one allele of Hdac1 without Hdac2 in neural cells, as opposed to neural cells with one allele of Hdac2 without Hdac130. Similarly, differentiation is altered in mice with one allele of Hdac2 without Hdac1 in epidermal cells, as opposed to epidermal cells with one allele of Hdac1 without Hdac231. In the intestine, IEC-specific Hdac1 and Hdac2 villin-Cre-induced deletion results in increased proliferation, goblet and Paneth cell loss, polarity disruption, activation of Notch, Stat3 and mTOR pathways, as well as increased susceptibility to DSS-induced colitis32,33. While Hdac2 IEC-specific deletion does not alter intestinal homeostasis, Hdac2 deficiency protects against DSS-induced colitis33. In addition, short-term deletion of both Hdac1 and Hdac2 in IEC with the Ah-Cre model leads to proliferation arrest34,35, accompanied by DNA damage responses35. Finally, in contrast to mice with one Hdac1 allele without Hdac2, villin-Cre mice with (...truncated)