PTGER4-dependent epigenetic regulation of intestinal epithelial subtypes during homeostasis and Crohn’s Disease Public

Abstract

Background: Crohn’s Disease (CD) is a relapsing-remitting inflammatory bowel disease (IBD) that can also manifest in the rectum as perianal fistulizing CD. During this time, mucosal inflammation can disrupt epithelial cell differentiation, decreasing the abundance of fully differentiated cell types that mitigate healing. Gastrointestinal organoids lack fully differentiated cell types and thus can serve as a model to mechanistically investigate pathways that promote them, potentially giving rise to new therapeutic targets. We hypothesize that PTGER4, an epithelial receptor encoded by an IBD risk gene, is involved in epigenetic modulation that promotes expression of gene programs involved in secretory lineage formation. Methods: Rectal mucosal biopsy samples from Emory University/Children’s Healthcare of Atlanta (CHOA) were used for crypt extraction and organoid culturing. Localization of HDAC cell type expression was determined by immunofluorescence on surgical tissue sections. Freshly isolated crypts and established organoids were used for biochemical analysis on chemically inhibited HDAC or HES1 samples, including rt-PCR and Western blotting (WB). Inhibition of PTGER4 was also investigated under these conditions. Results: Class I and II HDACs were broadly expressed in the epithelium, stromal, and immune compartments of the mucosa, with relatively high levels of HDAC4 in epithelial cells. We found that PTGER4 stimulation in organoids with prostaglandin E2 (PGE2) decreased histone deacetylases phosphorylation at HDAC4(S246)/5(S259)/7(S155), but not HDAC4(S632)/5(S661)/7(S486), and found that LMK-235, an inhibitor of HDACs 4/5, can increase SPINK4 expression. This contrasted with butyrate stimulation of organoids that increased the phosphorylation of HDAC4(S246)/5(S259)/7(S155), increased levels of HDAC 4 and 5, and depleted levels of HDAC7, but also increased SPINK4 expression. Inhibition of HDACs with butyrate or small molecule inhibitors of HDACs had variable effects on freshly isolated crypts and organoids, likely reflecting patient heterogeneity in these pathways. Conclusion: These results suggest that PTGER4 regulates secretory lineage pathways by activating HDAC4 and 5 in a manner opposite of the signals coming from the microbiome (i.e. butyrate), and these opposing signals, one received apically by the epithelium from the microbiome, the other basolaterally from stromal cells, likely balance one another during homeostasis to promote the appropriate proportions of epithelial subtypes, but the process is disrupted during inflammation and further complicated by patient specific responses.

Table of Contents

Table of Contents

INTRODUCTION...................................................................................................................... ........................ ........................ ........................ ........................ ........................ 1

Figure 1. Intestinal crypt stem cell differentiation is mediated by signaling factors and differential gene expression...............................................................................................2

Figure 2. PCA analysis of scRNA-seq from mucosal epithelium and organoid culture. .................................................................................................................... ........................ ..8

Figure 3. Analysis of scRNA-seq data derived from mucosal and organoid samples from the same patients. ..............................................................................................9

METHODS ..................................................................................................................... ........................ ........................ ........................ ........................ ........................ .12

BIOSPECIMEN COLLECTION AND ORGANOID CULTURING...................................................................................................................... ........................ ........................ ... 13

EXTRACTING ORGANOID CRYPTS AND TREATING WITH DIFFERENT HDAC INHIBITORS...................................................................................................................... 13

TREATING ORGANOID CULTURES WITH DIFFERENT INHIBITORS..................................................................................................................... ........................ ................. 14

WESTERN BLOTTING...................................................................................................................... ........................ ........................ ........................ ........................ ......... 14

RNA EXTRACTION AND QUANTITATIVE PCR.............................................................................................. ........................ ........................ ........................ ........................ 14

IMMUNOFLUORESCENCE.............................................................................................. ........................ ........................ ........................ ........................ ........................ 15

RESULTS.............................................................................................. ........................ ........................ ........................ ........................ ........................ ........................ 15

DETECTION OF HDACS IN THE INTESTINAL EPITHELIUM IN VIVO AND IN VITRO...................................................................................................................... ........................ 15

Figure 4. Class I and II HDACs are expressed in the intestinal epithelium ............................................................................................................................................................. 16

Figure 5. SPINK4 (red) expression in MUC2 positive goblet cells (green) in surgical tissue.........................................................................................................................................17

Figure 6. PGE2 and LMK-235 increases SPINK4 expression levels.............................................................................................................................................................................18

PGE2-PTGER4 AXIS REGULATES HDAC 4, 5, AND 7 PHOSPHORYLATION LEVELS...................................................................................................................................................18

Figure 7. Histone deacetylases HDAC4(S246)/5(S259)/7(S155) phosphorylation, but not HDAC4(S632)/5(S661)/7(S486) change during PGE2 treatment through PTGER4.......................19

PAN-INHIBITION OF HDACS CHANGES PHOSPHORYLATION AND TOTAL HDAC PROTEIN LEVELS...................................................................................................................... 20

Figure 8. Inhibition of HDACs by a pan-inhibitor, sodium butyrate, increases HDAC4(S246)/5(S259)/7(S155) phosphorylation and total HDAC protein levels and influences gene expression of epithelial differentiation marker genes..............................................................................................................................................................................................................21

STEM CELL TECHNOLOGIES HUMAN ORGANOID DIFFERENTIATION MEDIATM IS NOT SUFFICIENT TO INDUCE SECRETORY CELLS.................................................................... 21

Figure 9. No significant increase in secretory cell marker genes in organoids treated with differentiation media versus standard organoid media........................................................ 22

INHIBITION OF HDACS BY HDAC CLASS I AND CLASS II INHIBITORS IN ORGANOIDS............................................................................................................................................22

INHIBITION OF HES1 AND PAN-INHIBITION OF HDACS IN FRESHLY ISOLATED RECTAL CRYPTS....................................................................................................... ....................23

Figure 10. Inhibition of HDACs by HDAC Class I and Class II inhibitors in organoids..................................................................................................................................................24

Figure 11. Inhibition of HES1 and pan-inhibition of HDACs by butyrate in organoid crypts.........................................................................................................................................25

Figure 12. Graphical summary................................................................................................................................................................................................................................26

DISCUSSION.........................................................................................................................................................................................................................................................27

REFERENCES........................................................................................................................................................................................................................................................33

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Biology, General Biology, Cell
Mot-clé	Crohn's Disease Intestinal Epithelial Cell Differentiation Inflammatory Bowel Disease Histone Deacetylases
Committee Chair / Thesis Advisor	Jason Matthews, Emory University Subra Kugathasan, Emory University
Committee Members	Arri Eisen, Emory University Adam Gracz, Emory University Rheinallt Jones, Emory University

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