Fig 1.
(A) Giemsa-stained sections from the femur of Mll1FC/+; RC/+ and Mll1FC/FC; RC/+ mice. Two weeks after the last tamoxifen gavage mice were sacrificed and the femurs were dissected, decalcified, sectioned and stained. Bone marrow cellularity was severely decreased in Mll1FC/FC; RC/+ mice. Scale bar 100 μm. (B) Kaplan-Meier survival curve. The first day of tamoxifen gavage was day zero. All Mll1FC/+; RC/+ mice (n = 25) survived whereas all Mll1FC/FC; RC/+ mice (n = 33) died within 33 days after tamoxifen induction with a median survival of 11 days. (C) Scheme of the experimental setup for bone marrow transplantation. Donor bone marrow from B6.SJL (CD45.1+) mice was transplanted into lethally irradiated Mll1F/+; RC/+ and Mll1F/F; RC/+ recipients (CD45.2+). Blood chimerism (BC) was measured three times. After 30 weeks Mll1 deletion was achieved by administrating tamoxifen (TAM). FACS analysis for KSL-Slam enriched HSCs (Kit+ Sca1+ Lin- CD48- CD150+ CD34- CD135-) showed comparable numbers in BMTx Mll1FC/+; RC/+ and Mll1FC/FC; RC/+ mice. Dot plots show Lin- CD48- CD150+ CD34- CD135- gated bone marrow (BM) cells of indicated genotypes resolved for the expression of c-Kit and Sca-1. Donor and host cells are distinguished by surface markers CD45.1 and CD45.2. (D) Kaplan-Meier analysis for the onset of diarrhea. Tamoxifen was given by gavage for 6 days to Mll1F/+; RC/+ (n = 6) and Mll1F/F; RC/+ (n = 8). The first day of tamoxifen gavage was day zero. While all BMTx mice with the genotype Mll1FC/+; RC/+ remained healthy, all BMTx Mll1FC/FC; RC/+ mice developed diarrhea with a median of 16.5 days. (E) Antibody staining (brown) showed that MLL1 is expressed in crypts of the small and large intestine but absent in the villus (hematoxylin, purple). Scale bars are 50 μm.
Fig 2.
Mll1 deletion in BMTx mice leads to loss of stem and proliferating cells and increased goblet cells.
(A) Antibody stainings showed reduced MLL1, OLFM4 and SOX9 expression in BMTx Mll1FC/FC; RC/+ intestine compared to controls. Hematoxylin was used as a counterstain for MLL1 and OLFM4 immunohistochemistry (IHC). Expression of Ki67, a marker for proliferating cells was reduced in BMTx Mll1FC/FC; RC/+ mutant intestinal sections. Arrowheads point toward proliferating ISCs. Alcian blue was used as a counterstain for SOX9 and Ki67 IHC, which also revealed enlarged goblet cells (turquoise) in the crypts of BMTx Mll1FC/FC; RC/+ sections. Scale bars are 50 μm. (B) PAS stain to examine goblet cells in villi and crypts of BMTx intestine. Black arrowheads point towards vacuolar structures. Yellow arrowhead points to a mislocalized goblet cell in BMTx Mll1FC/FC; RC/+ crypt. Left panels scale bar 100 μm; middle panels scale bar 50 μm. GOB5 antibody staining of BMTx Mll1FC/FC; RC/+ intestinal sections. Right panels scale bar 100 μm. (C) Left panels; lysozyme antibody staining reveals that Paneth cell numbers remain unchanged. Arrowheads point at Paneth cells. Alcian blue was used as a counterstain and marks goblet cells (turquoise). Middle panels; chromogranin A antibody stain, arrowheads point to the sparse enteroendocrine cells (dark brown) in the villi. Right panels; red enterocytes (arrowheads) covering the villi were visualized by alkaline phosphatase staining. Hematoxylin was used as a counterstain for chromogranin A IHC and alkaline phosphatase histochemical staining. Scale bars are 50 μm.
Fig 3.
Decreased ISCs and increased goblet cells after intestinal specific loss of MLL1.
(A) Kaplan-Meier survival curve. Tamoxifen was given by gavage for 6 days to mice with the genotype Mll1FC/+; Vil-Cre-ERT2/+ (n = 12) and Mll1FC/FC; RC/+ (n = 18). The first day of tamoxifen gavage was day zero. The median survival of mutants is 24 days after tamoxifen. (B) Percent of weight loss of Mll1FC/+; Vil-Cre-ERT2/+ (n = 6) and of Mll1FC/FC; Vil-Cre-ERT2/+ (n = 10) mice. Mean ± s.d. is shown (p = 0.007, Student’s t-test). (C) Decrease in ISC markers, OLFM4 and SOX9 in Mll1FC/FC; Vil-Cre-ERT2/+ intestinal sections. SOX9+ ISCs in Mll1FC/+; Vil-Cre-ERT2/+ intestinal sections are marked with red arrowheads. Proliferating cells in the TA compartment as well as proliferative ISCs (arrowhead) are reduced in Mll1FC/FC; Vil-Cre-ERT2/+ sections. Alcian blue was used as a counterstain after staining for SOX9 and Ki67 and marks goblet cells (turquoise). Scale bars are 100 μm for OLFM4 and 50 μm for SOX9 and Ki67. (D) Left panels; alcian blue staining of goblet cells with nuclear fast red (NFR) to stain nuclei. Middle panels; Paneth cells visualized by staining of granules containing lysozyme (arrowheads). Right panels; enteroendocrine cells stained with chromogranin A antibody (brown; arrowheads) in the villi. Scale bars are 100 μm. (E) Left and middle panels; antibody stainings showed reduced OLFM4 and Ki67 expression in Mll1FC/FC; Vil-Cre-ERT2/+ intestine compared to controls, one week after the first gavage. Hematoxylin was used as a counterstain for OLFM4 and alcian blue was used for Ki67 IHC. Right panel; alcian blue staining of goblet cells with NFR to stain nuclei, one week after the first gavage. Scale bars are 100 μm.
Fig 4.
RNA profiling of Mll1FC/FC; Lgr5-eGFP-CreERT2/+ and control ISCs.
(A) ISCs were sorted from control (Mll1FC/+; Lgr5-eGFP-CreERT2/+) (n = 4) and Mll1FC/FC; Lgr5-eGFP-CreERT2/+ (n = 4) mice 4 days after tamoxifen induction was completed and subjected to RNA profiling. MA plot visualizing the log2-fold change differences according to expression levels. Red dots represent significant DEGs at a 5% FDR. (B) Plots show biological processes (BP) that are enriched in genes up- or downregulated in Mll1FC/FC; Lgr5-eGFP-CreERT2/+ compared to control ISCs. Analysis was performed using the gene ontology (GO)/BP/FAT database of DAVID 6.8. (C) (D) GSEA shows significant negative or positive correlation of genes from the stem (C) and goblet cell (D) signature gene set in Mll1FC/FC; Lgr5-eGFP-CreERT2/+ compared to control ISCs (4 days after tamoxifen). The signature gene sets originate from [87]. NES: normalized enrichment score. (E) DESeq normalized counts for genes coding for transcription factors downregulated in Mll1FC/FC; Lgr5-eGFP-CreERT2/+ compared to control ISCs (4 days after tamoxifen). Mean+s.d. is shown; n = 4; p<0.05, Wald test. (F) DESeq normalized counts for genes coding for ISC markers downregulated in Mll1FC/FC; Lgr5-eGFP-CreERT2/+ compared to control ISCs (4 days after tamoxifen). Mean+s.d. is shown; n = 4; p<0.05, Wald test. (G) Western blot analysis for GATA4 protein on crypts from two pairs of Mll1FC/+; RC/+ and Mll1FC/FC; RC/+ mice. GAPDH serves as loading control. (H) Representative ChIP-qPCR analysis specific for H3K4me3 occupancy at promoter regions. Crypts from Mll1FC/+; RC/+ or Mll1FC/FC; RC/+ mice were crosslinked and chromatin immunoprecipitation was performed with and without (mock) H3K4me3 antibody. Target genes were selected from Table 1. Mean+s.d. is shown; n = 3 *p<0.05, **p<0.01, ***p<0.001, Student’s t test.
Table 1.
Common mRNAs downregulated after removal of MLL1 from ISCs and/or organoids.
Fig 5.
RNA profiling of wt Paneth cells after deletion of Mll1 in the neighboring ISCs.
(A) Wt Paneth cells neighboring either Mll1FC/+; Lgr5-eGFP-CreERT2/+ or Mll1FC/FC; Lgr5-eGFP-CreERT2/+ ISCs were sorted 4 days after tamoxifen induction was completed and were subjected to RNA profiling. MA plot visualizing the log2-fold change differences according to expression levels. Red dots represent significant DEGs at a 5% FDR. (B) Enriched terms of biological processes and pathways down- and upregulated using DAVID GO/BP/FAT and KEGG database. (C) GSEA shows significant negative or positive correlation of genes from the GO ERAD pathway, GO oxidative phosphorylation and Paneth cell signature gene set in wt Paneth cells neighboring either Mll1FC/FC; Lgr5-eGFP-CreERT2/+ or Mll1FC/+; Lgr5-eGFP-CreERT2/+ ISCs. The Paneth cell signature gene set originates from [87]. NES: normalized enrichment score. (D) DESeq normalized counts for selected genes differentially regulated in the ERAD pathway, oxidative phosphorylation and Paneth cell signature gene set. Mean+s.d. is shown; n = 4; p<0.05, Wald test.
Fig 6.
Mll1 deletion in organoids results in loss of budding and formation of spheres.
(A) Differential interference contrast (DIC) images of Mll1FC/+; RC/+ and Mll1FC/FC; RC/+ organoids. Organoids were induced with 4-hydroxy tamoxifen for 24 h. Upon passaging (P), the mutant starts to loose its budding morphology giving rise to an undifferentiated cyst-like appearance. Scale bar 100 μm. (B) Quantification of number of buds per organoid shown in A. Mean+s.d. is shown; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Student’s t test. (C) mRNA profiling of Mll1FC/+; RC/+ and Mll1FC/FC; RC/+ intestinal organoids after passage 1. Volcano plot visualizing the log2-fold change differences according to expression levels. Blue and red dots represent significant down- and upregulated DEGs, respectively, at a 5% FDR. (D, E) Enriched terms of biological processes after passage 1 upregulated (D) and downregulated (E) using DAVID GO/BP/FAT.
Fig 7.
MLL1 is essential for niche homeostasis.
ISCs are anchored in the crypt in close association with Paneth cells. Loss of MLL1 in ISCs causes transcriptional changes with downregulation of intestine specific transcription factors such as Pitx1, Pitx2, Foxa1 and Gata4 as well as chromatin accessory factor Bahcc1. Subsequently loss of stem cell identity perturbs Paneth cell identity accompanied with changes in metabolism and protein folding.