Mylène Blais

HDAC1 and HDAC2 Restrain the Intestinal Inflammatory Response by Regulating Intestinal Epithelial Cell Differentiation

Acetylation and deacetylation of histones and other proteins depends on histone acetyltransferases and histone deacetylases (HDACs) activities, leading to either positive or negative gene expression. HDAC inhibitors have uncovered a role for HDACs in proliferation, apoptosis and inflammation. However, little is known of the roles of specific HDACs in intestinal epithelial cells (IEC). We investigated the consequences of ablating both HDAC1 and HDAC2 in murine IECs. Floxed Hdac1 and Hdac2 homozygous mice were crossed with villin-Cre mice. Mice deficient in both IEC HDAC1 and HDAC2 weighed less and survived more than a year. Colon and small intestinal sections were stained with hematoxylin and eosin, or with Alcian blue and Periodic Acid Schiff for goblet cell identification. Tissue sections from mice injected with BrdU for 2 h, 14 h and 48 h were stained with anti-BrdU. To determine intestinal permeability, 4-kDa FITC-labeled dextran was given by gavage for 3 h. Microarray analysis was performed on total colon RNAs. Inflammatory and IEC-specific gene expression was assessed by Western blot or semi-quantitative RT-PCR and qPCR with respectively total colon protein and total colon RNAs. HDAC1 and HDAC2-deficient mice displayed: 1) increased migration and proliferation, with elevated cyclin D1 expression and phosphorylated S6 ribosomal protein, a downstream mTOR target; 2) tissue architecture defects with cell differentiation alterations, correlating with reduction of secretory Paneth and goblet cells in jejunum and goblet cells in colon, increased expression of enterocytic markers such as sucrase-isomaltase in the colon, increased expression of cleaved Notch1 and augmented intestinal permeability; 3) loss of tissue homeostasis, as evidenced by modifications of claudin 3 expression, caspase-3 cleavage and Stat3 phosphorylation; 4) chronic inflammation, as determined by inflammatory molecular expression signatures and altered inflammatory gene expression. Thus, epithelial HDAC1 and HDAC2 restrain the intestinal inflammatory response, by regulating intestinal epithelial cell proliferation and differentiation.

The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis

Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from dextran sulfate sodium (DSS)-induced murine colitis. Although tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal disease activity index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability, and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation.

The histone H3K27 methylation mark regulates intestinal epithelial cell density‐dependent proliferation and the inflammatory response

Polycomb‐group proteins form multimeric protein complexes involved in transcriptional silencing. The Polycomb Repressive complex 2 (PRC2) contains the Suppressor of Zeste‐12 protein (Suz12) and the histone methyltransferase Enhancer of Zeste protein‐2 (Ezh2). This complex, catalyzing the di‐ and tri‐methylation of histone H3 lysine 27, is essential for embryonic development and stem cell renewal. However, the role of Polycomb‐group protein complexes in the control of the intestinal epithelial cell (IEC) phenotype is not known. We show that Suz12 and Ezh2 were differentially expressed along the intestinal crypt‐villus axis. ShRNA‐mediated Suz12 depletion in the IEC‐6 rat crypt‐derived cell line decreased Ezh2 expression and H3K27 di‐trimethylation. Suz12‐depleted cells achieved higher cell densities after confluence, with increased cyclin D2 and cyclin D3 protein levels, and increased STAT3 activation in post‐confluent cells. Suz12 depletion specifically increased mostly developmental, cell adhesion and immune response gene expression, including neuronal and inflammatory genes. Suz12 depletion directly and indirectly de‐regulated the IL‐1β‐dependent inflammatory response, as demonstrated by decreased MAPK p38 activation as opposed to JNK activation, and altered basal and stimulated expression of inflammatory genes, including transcription factors such as C/EBPβ. Of note, this positive effect on cell proliferation and inflammatory gene expression was revealed in the absence of the cyclin‐dependent kinase inhibitor p16, a main target negatively regulated by PRC2. These results demonstrate that the PRC2 complex, in addition to keeping in check non‐IEC differentiation pathways, insures the proper IEC response to cell density as well as to external growth and inflammatory signals, by controlling specific signaling pathways. J. Cell. Biochem. 114: 1203–1215, 2013.

Regulation of C/EBPδ-dependent transactivation by histone deacetylases in intestinal epithelial cells

The C/EBPδ transcription factor is involved in the positive regulation of the intestinal epithelial cell acute phase response. C/EBPδ regulation by histone deacetylases (HDACs) during the course of inflammation remains to be determined. Our aim was to examine the effect of HDACs on C/EBPδ‐dependent regulation of haptoglobin, an acute phase protein induced in intestinal epithelial cells in response to pro‐inflammatory cytokines. HDAC1, HDAC3, and HDAC4 were expressed in intestinal epithelial cells, as determined by Western blot. GST pull‐down assays showed specific HDAC1 interactions with the transcriptional activation and the b‐ZIP C/EBPδ domains, while the co‐repressor mSin3A interacts with the C‐terminal domain. Immunoprecipitation assays confirmed the interaction between HDAC1 and the N‐terminal C/EBPδ amino acid 36–164 domain. HDAC1 overexpression decreased C/EBPδ transcriptional activity of the haptoglobin promoter, as assessed by transient transfection and luciferase assays. Chromatin immunoprecipitation analysis showed a displacement of HDAC1 from the haptoglobin promoter in response to inflammatory stimuli and an increased acetylation of histone H3 and H4. HDAC1 silencing by shRNA expression increased both basal and IL‐1β‐induced haptoglobin mRNA levels in epithelial intestinal cells. Our results suggest that interactions between C/EBPs and HDAC1 negatively regulate C/EBPδ‐dependent haptoglobin expression in intestinal epithelial cells. J. Cell. Biochem. 103: 1573–1583, 2008.