Fig 1.
MacroH2A expression within the intestinal epithelium.
(A) Analysis of intestinal jejunum crypt or villus tissue fractions for macroH2A variant mRNA levels compared to mouse liver. ΔΔCT method, values normalized to Actb, N = 3 per condition, mean ± SD. (B) MacroH2A isoform mRNA level analysis within Lgr5-eGFPhigh CBCs or Hopx-tdTomato+ reserve ISCs FACS-purified from Lgr5-eGFP-IRES-CreERT2 or Hopx-CreERT2 Rosa26R-LSL-tdTomato mice. ΔΔCT method, values normalized to Actb, N = 3 per condition, mean ± SD. (C) Western blot showing macroH2A1 isoform protein level within FACS-purified populations of CBCs (again, Lgr5-eGFPhigh from Lgr5-eGFP-IRES-CreERT2 mice) or reserve ISCs (Hopx-tdTomato+ from Hopx-CreERT2 Rosa26R-LSL-tdTomato mice). Entire protein lysate from 30,000 CBCs or 20,000 reserve ISCs loaded into each well of gel corresponding to indicated samples on blot. (D) Immunohistochemical straining of pan-macroH2A1 or macroH2A1.1 in macroH2A WT or macroH2A DKO proximal small intestine. 10x objective. Scale bars = 100μm. **p<0.005, ***p<0.0005, ns = not significant, Student’s t-test.
Fig 2.
MacroH2A DKO intestine during homeostasis.
(A) Left: Representative Ki67 immunohistochemistry of macroH2A WT and DKO proximal jejunum. 10x objective. Right: Average Ki67+ crypt height in macroH2A WT vs. DKO proximal jejunum. N = 3 mice per condition, medians, quartiles and ranges of values shown. (B) Average height in microns of crypt-villus axis (distance from base of crypt to tip of villus) of macroH2A WT vs. DKO proximal jejunum (C) Average number of crypts per mm of macroH2A WT vs. DKO proximal jejunum. (D) Representative immunofluorescence and immunohistochemical images of jejunum of macroH2A WT or DKO mice stained for lysozyme (Paneth cells), chromogranin A (enteroendocrine cells), alkaline phosphatase (enterocytes), or alcian blue (goblet cells). Immunofluorescence counterstained with DAPI (blue). Lysozyme, chromogranin A and alkaline phosphatase: 20x objective, alcian blue: 10x objective. Scale bars = 100μm. ns = not significant, Student’s t-test.
Fig 3.
CBC frequency and activity in macroH2A DKO intestine.
(A) Representative phase contrast images of macroH2A WT and DKO crypt-derived organoids, 7 days into culture. Left: 4x objective. Right: 10x objective. (B) Average resulting organoids per well (24-well tissue culture plate) from 100 crypts from macroH2A WT or DKO proximal jejunum from 2-month or 2-year old mice. N = 6 mice per condition, medians, quartiles and ranges of values shown. (C) Aged organoid formation capacity as defined by the average number of organoids that formed as a percent of the number of corresponding organoids that formed from 2-month old crypts per genotype. 10x objective. (D) Left: representative anti-eGFP immunofluorescence of macroH2A WT and DKO jejunum counterstained with DAPI (blue). Right: average Lgr5-eGFP+ cells per crypt. N = 6 mice per condition, medians, quartiles and ranges of values shown. (E) Left: representative flow cytometry plots of EdU content vs. DAPI of within Lgr5-eGFP+ subpopulations of macroH2A WT and DKO proximal jejunal crypt cells. Right: quantitation of Lgr5-eGFP/EdU double positivity as defined by boxed subpopulation on left. N = 4 mice per condition, medians, quartiles and ranges of values shown. *p<0.05, ns = not significant, Student’s t-test. Scale bars = 100μm.
Fig 4.
Reserve ISC frequency and activity in macroH2A DKO intestine.
(A) Left: representative flow cytometry plots of SSC-A vs. Hopx-tdTomato+ signal in proximal small intestine crypt cells from macroH2A WT or DKO mice. Right: quantitation of Hopx-tdTomato+ population as a percentage of crypt epithelial cells. N = 5 mice per condition, mean ± SD. (B) Top: homeostatic lineage-tracing scheme: macroH2A WT and DKO Hopx-CreERT2::Rosa26-LSL-tdTomato mice were injected with 2mg tamoxifen for 2 consecutive days followed by a 2-week chase. Bottom: representative anti-tdTomato immunofluorescence (red) counterstained with DAPI (blue) of macroH2A WT and DKO proximal jejunum 2-weeks after induction of Hopx-tdTomato lineage tracing. 4x objective. (C) Left: quantitation of percentage of villi with tracing events after 2 week chase, N = 3 mice per condition, mean ± SD. Right: percentage of villi with tracing events normalized to percentage of Hopx-tdTomato+ ISCs during homeostasis (values in Fig 4A). N = 3 mice per condition, mean ± SD. (D) Left: representative flow cytometry plots of EdU content vs. DAPI of within Hopx-tdTomato+ subpopulations of macroH2A WT and DKO proximal jejunal crypt cells. Right: quantitation of Hopx-tdTomato/EdU double positivity as defined by boxed subpopulation on left. N = 7 mice per condition, medians, quartiles and ranges of values shown. *p<0.05, ns = not significant, Student’s t-test. Scale bar = 100μm.
Fig 5.
Regeneration and DNA damage response in macroH2A DKO intestine.
(A) Left: representative images of Ki-67 immmunohistochemistry within macroH2A WT and DKO proximal jejunum 3 days after exposure of mice to 12 Gy whole body γ-irradiation. 10x objective. Right: quantitation of Ki67+ nascent crypt foci per mm. N = 3 mice per condition, mean ± SD. (B) Top: post-IR lineage tracing scheme: macroH2A WT or DKO Hopx-CreERT2::Rosa26-LSL-tdTomato mice were injected with 2mg tamoxifen 48h and 24h prior to treatment with 12 Gy whole-body gamma irradiation, and 72h later sacrificed for analysis. Bottom: representative immunofluorescence of tdTomato lineage tracing (red) counterstained with DAPI (blue) within macroH2A WT and DKO crypts 72 hours after γ-irradiation. 30x objective (C) Left: quantitation of tdTomato tracing events per 500μm, N = 3 mice per condition, mean ± SD. Right: quantitation of tdTomato tracing events per 500μm normalized to percentage of Hopx-tdTomato+ ISCs during homeostasis (values in Fig 4A), N = 3 mice per condition, mean ± SD. (D) Experimental scheme highlighting the timing of DNA damage and apoptosis analysis (24h post IR) and regeneration and lineage tracing analysis (72h post IR) (E) Left: flow cytometry plots of SSC-A vs. cleaved caspase-3 content within total crypt epithelium or Hopx-tdTomato+ subpopulations of macroH2A WT and DKO proximal jejunal crypt cells 24 hours after γ-irradiation. Right: quantitation of total crypt epithelium CC3 positivity and Hopx-tdTomato+/CC3 double positivity as defined by boxed subpopulation on left. N = 3 mice per condition, mean ± SD. *p<0.05, ***p<0.0005, ns = not significant, Student’s t-test. Scale bars = 100μm.
Fig 6.
MacroH2A’s influence of intestinal tumorigenesis.
(A) MacroH2A mRNA level analysis of healthy human intestinal crypt epithelium and human CRC cell lines. ΔΔCT method, values normalized to GAPD. N = 3 per condition, mean ± SD. (B) Graphical depiction of the H2AFY gene and its exons, including the mutually-exclusive exons of the macroH2A1.1 and macroH2A1.2 splice variants. (C) MacroH2A siRNA knockdown validation in RKO CRC cell line. ΔΔCT method, values normalized to GAPD independently per macroH2A primer relative to luciferace knockdown control. N = 3 per condition, mean ± SD. (D) MTT cell proliferation assay of RKO cell line during macroH2A1.1, 1.2, H2AFY, or control luciferace RNAi knockdown. N = 3 per condition, mean ± SD. (E) Left: representative H&E images of macroH2A WT and DKO Apcmin-derived tumors within the small intestine. 4x objective. Right: quantitation of average total tumors within entire small intestine of macroH2A WT and DKO. N = 8 mice per condition, mean ± SD. *p<0.05, **p<0.005, ***p<0.0005, ns = not significant, Student’s t-test. Scale bar = 100μm.