Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation

Abstract

Lgr5+ stem cells are crucial to gut epithelium homeostasis; however, how these cells are maintained is not fully understood. Zinc finger HIT-type containing 1 (Znhit1) is an evolutionarily conserved subunit of the SRCAP chromosome remodeling complex. Currently, the function of Znhit1 in vivo and its working mechanism in the SRCAP complex are unknown. Here we show that deletion of Znhit1 in intestinal epithelium depletes Lgr5+ stem cells thus disrupts intestinal homeostasis postnatal establishment and maintenance. Mechanistically, Znhit1 incorporates histone variant H2A.Z into TSS region of genes involved in Lgr5+ stem cell fate determination, including Lgr5, Tgfb1 and Tgfbr2, for subsequent transcriptional regulation. Importantly, Znhit1 promotes the interaction between H2A.Z and YL1 (H2A.Z chaperone) by controlling YL1 phosphorylation. These results demonstrate that Znhit1/H2A.Z is essential for Lgr5+ stem cell maintenance and intestinal homeostasis. Our findings identified a dominant role of Znhit1/H2A.Z in controlling mammalian organ development and tissue homeostasis in vivo.

Fig. 1

Znhit1 deletion disrupts postnatal generation of Lgr5+ ISC. a Znhit1 in situ was performed in intestinal section of 8-week-old C57BL/6 mouse. Scale bar, 50 μm. b Body weight comparison between Znhit1^fl/+; Villin-cre and Znhit1^fl/fl; Villin-cre mice at indicated time. The data represent mean ± s.d. (n = 5 mice per group). Wilcoxon’s rank sum test: **P < 0.01. *P < 0.05. c Kaplan–Meier survival curves of Znhit1^fl/+; Villin-cre and Znhit1^fl/fl; Villin-cre mice (n = 19 mice per genotype) and body size comparison between survived mice at P30. Scale bar, 2 cm. d Paraffin-embedded intestine tissues were stained with hematoxylin and eosin. e Ki67 and Krt20 staining of intestinal sections from Znhit1^fl/+; Villin-cre and Znhit1^fl/fl; Villin-cre mice at P9. f Intestinal crypts were isolated from Znhit1^fl/+; Villin-cre and Znhit1^fl/fl; Villin-cre mice at P9, embedded in Matrigel (100 crypts per well) and cultured for 3 days. The statistical analysis of organoid numbers (n = 5 mice per genotype) was shown as mean ± s.d. Student’s t-test: ***P < 0.001. g GFP staining of intestinal sections from Znhit1^fl/+; Villin-cre; Lgr5-EGFP-IRES-creERT2 and Znhit1^fl/fl; Villin-cre; Lgr5-EGFP-IRES-creERT2 mice at P9. Arrows: Lgr5+ ISCs. h Lgr5 and Olfm4 in situ were performed in intestinal sections at P9. i Intestine was harvested from Znhit1^fl/+; Villin-cre (fl/+) and Znhit1^fl/fl; Villin-cre (fl/fl) mice at P0 to examine the expression of Znhit1, Lgr5, Ascl2, and Olfm4 using qRT-PCR. j Intestine was harvested from Znhit1^fl/+; Villin-cre (fl/+) and Znhit1^fl/fl; Villin-cre (fl/fl) mice at indicated time to examine Lgr5 expression using qRT-PCR. For qRT-PCR, histone H3 was used as an internal control. The statistical data represent mean ± s.d. (n = 3 mice per genotype). Student’s t-test: ***P < 0.001. *P < 0.05. All images are representative of n = 3 mice per genotype. Scale bar, 50 μm

Fig. 2

Znhit1 is essential for Lgr5+ ISC maintenance thus intestinal homeostasis. a Eight-week-old Villin-creERT and Znhit1^fl/fl; Villin-creERT mice were daily injected with tamoxifen for 4 days followed by 7-day waiting period. Top: Scheme of Cre induction strategy. Bottom: Body weight comparison between Villin-creERT and Znhit1^fl/fl; Villin-creERT mice at indicated time following tamoxifen treatment. (n = 5 mice per genotype). b Kaplan–Meier survival curves of Villin-creERT and Znhit1^fl/fl; Villin-creERT mice post tamoxifen administration (n = 9 mice per genotype). c Olfm4 in situ was performed in intestinal sections from Villin-creERT and Znhit1^fl/fl; Villin-creERT mice following tamoxifen treatment. d Intestinal crypts were isolated from Villin-creERT and Znhit1^fl/fl; Villin-creERT mice following tamoxifen treatment, embedded in Matrigel (100 crypts per well) and cultured for 2 days. The statistical analysis of organoid numbers (n = 3 mice per genotype) was shown. e Eight-week-old Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 mice were daily injected with tamoxifen for 3 days followed by 4-day waiting period, then Lgr5+ ISCs were examined by confocal cross-sectioning. The GFP+ cells were quantified for statistical analysis (n = 3 mice per genotype). f Intestinal crypts were isolated from Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice following tamoxifen treatment for immunoblotting with the indicated antibodies. GAPDH served as a loading control. The statistical data represent mean+ s.d. (n = 3 mice per genotype). g Intestinal crypts were isolated from Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice following tamoxifen treatment, embedded in Matrigel (100 crypts per well) and cultured for 7 days. The organoid buddings were quantified for statistical analysis (n = 5 mice per genotype). h Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 mice were daily injected with tamoxifen for 4 days followed by 7-day waiting period. Body weight comparison was shown (n = 3 mice per genotype). The statistical data represent mean ± s.d. Student’s t-test: ***P < 0.001. All images are representative of n = 3 mice per genotype. Scale bar, 50 μm

Fig. 3

Znhit1 controls the transcription of Lgr5+ ISC fate-determining genes. a, b Eight-week-old Villin-creERT (+/+) and Znhit1^fl/fl; Villin-creERT (fl/fl) mice were daily injected with tamoxifen for 4 days followed by 7-day waiting period. Intestinal crypts were harvested for RNA-seq (a) and qRT-PCR (b) to analyze the gene expression changes. Clustered heatmap of log2-transformed RPKMs shows the differentially expressed genes after Znhit1 deletion. Log2-transformed fold changes of indicated genes were marked in right. c Tgfb1 and Tgfbr2 staining of intestinal sections from Villin-creERT (+/+) and Znhit1^fl/fl; Villin-creERT (fl/fl) mice following tamoxifen treatment. d Eight-week-old Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice were daily injected with tamoxifen for 3 days followed by 4-day waiting period. Intestinal crypts were harvested to examine the expression of indicated genes using qRT-PCR. e Phospho-Smad2 staining of intestinal sections from Villin-creERT (+/+) and Znhit1^fl/fl; Villin-creERT (fl/fl) mice following tamoxifen treatment. f Intestinal crypts were isolated from Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice following tamoxifen treatment and subjected to in vitro culture in the presence of 3 μM CHIR99021 and/or 10 μM SB431542 for 4 days. Mock: DMSO. g The cultured organoids were harvested to examine the expression of indicated genes using qRT-PCR. For qRT-PCR, histone H3 was used as an internal control. The statistical data represent mean ± s.d. (n = 3 mice per genotype or treatment). Student’s t-test: ***P < 0.001. **P < 0.01. All images are representative of n = 3 mice per genotype. Scale bar, 50 μm

Fig. 4

Znhit1 incorporates H2A.Z for transcriptional regulation. a Distribution of H2A.Z on genome of intestinal crypts. Eight-week-old Villin-creERT (+/+) and Znhit1^fl/fl; Villin-creERT (fl/fl) mice were daily injected with tamoxifen for 3 days followed by 5-day waiting period. Intestinal crypts were harvested for ChIP-seq. ChIP-seq signals for H2A.Z binding at Ereg and Fbp1 loci were shown as examples. b Venn diagram showing the overlap between TSS H2A.Z binding genes and Znhit1-regulated genes. The significance was evaluated by Fisher’s exact test. c ChIP-seq signals for H2A.Z binding at Lgr5, Clic6, Tgfbr2, and Tgfb1 loci. d Eight-week-old Villin-creERT (+/+) and Znhit1^fl/fl; Villin-creERT (fl/fl) mice were daily injected with tamoxifen for 3 days followed by 5-day waiting period. Intestinal crypts were harvested and ChIP-qPCR was performed to examine the fold enrichment of H2A.Z in TSS region of indicated genes. e Intestinal crypts were harvested from H2afv^+/+; H2afz^+/+; Villin-cre and H2afv^fl/fl; H2afz^fl/fl; Villin-cre mice at P9 to examine the expression of indicated genes using qRT-PCR. Histone H3 was used as an internal control. f Intestinal crypts were harvested from H2afv^+/+; H2afz^+/+; Villin-cre and H2afv^fl/fl; H2afz^fl/fl; Villin-cre mice at P9 and ChIP-qPCR was performed to examine the fold enrichment of H3K4me3 and H3K27me3 in TSS region of indicated genes. The statistical data represent mean ± s.d. (n = 3 mice per genotype). Student’s t-test: ***P < 0.001. **P < 0.01. *P < 0.05

Fig. 5

Znhit1 mediates H2A.Z incorporation by enhancing the interaction between H2A.Z and YL1. a Eight-week-old Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice were daily injected with tamoxifen for 3 days followed by 4-day waiting period, then intestinal crypts were isolated for immunoblotting with the indicated antibodies. GAPDH served as a loading control. b Intestinal crypt (wild-type) were harvested for anti-YL1 immunoprecipitation then anti-phospho-Ser/Thr (p-Akt substrate) immunoblotting. c Intestinal crypt (wild-type) lysis was treated with rSAP for dephosphorylation then subjected to immunoblotting with the indicated antibodies. d Intestinal crypts were harvested from Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice following tamoxifen treatment for anti-YL1 immunoprecipitation then anti-p-Akt immunoblotting. WCL IB: whole cell lysis immunoblotting. e Intestinal crypts (wild-type) were harvested for anti-H2A.Z immunoprecipitation then anti-YL1 immunoblotting. Percentage of H2A.Z-bound phospho-YL1 (p-YL1) and non-phospho-YL1 (non-p-YL1) was quantitated. The statistical data represent mean ± s.d. (n = 3 mice). Student’s t-test: ***P < 0.001. f Intestinal crypt (wild-type) lysis treated with rSAP were subjected to anti-YL1 immunoprecipitation then immunoblotting with the indicated antibodies. g Cultured organoids (wild-type) were treated with 25 μM LY294002 for 12 h then harvested for anti-YL1 immunoprecipitation followed by immunoblotting. h Intestinal crypts were harvested from Znhit1^+/+; Olfm4-IRES-eGFPCreERT2 (+/+) and Znhit1^fl/fl; Olfm4-IRES-eGFPCreERT2 (fl/fl) mice following tamoxifen treatment for anti-YL1 immunoprecipitation then anti-H2A.Z immunoblotting. i Working model of how Znhit1-mediated H2A.Z incorporation regulates the transcription of Lgr5+ ISC fate determiners