Stage-dependent and locus-specific role of histone demethylase Jumonji D3 (JMJD3) in the embryonic stages of lung development

Abstract

Histone demethylases have emerged as important players in developmental processes. Jumonji domain containing-3 (Jmjd3) has been identified as a key histone demethylase that plays a critical role in the regulation of gene expression; however, the in vivo function of Jmjd3 in embryonic development remains largely unknown. To this end, we generated Jmjd3 global and conditional knockout mice. Global deletion of Jmjd3 induces perinatal lethality associated with defective lung development. Tissue and stage-specific deletion revealed that Jmjd3 is dispensable in the later stage of embryonic lung development. Jmjd3 ablation downregulates the expression of genes critical for lung development and function, including AQP-5 and SP-B. Jmjd3-mediated alterations in gene expression are associated with locus-specific changes in the methylation status of H3K27 and H3K4. Furthermore, Jmjd3 is recruited to the SP-B promoter through interactions with the transcription factor Nkx2.1 and the epigenetic protein Brg1. Taken together, these findings demonstrate that Jmjd3 plays a stage-dependent and locus-specific role in the mouse lung development. Our study provides molecular insights into the mechanisms by which Jmjd3 regulates target gene expression in the embryonic stages of lung development.

Figure 1. Postnatal lethality and multiple organ defects of Jmjd3 ^−/− embryos.

(A) Postnatal lethality of Jmjd3 ^−/− mice. The skin color of Jmjd3 ^−/− mice became cyanotic minutes after birth. Arrowhead indicates defective lordosis in Jmjd3 ^−/− newborns. (B) The lungs of Jmjd3 ^−/− mice were smaller and not expanded at P0 compared with Jmjd^+/+ mice. (C) H&E staining of P0 lung sections from Jmjd3 ^−/− mice indicates arrested tissue maturation. (D) Less developed yolk sac vascular plexus of Jmjd3 ^−/− embryos at E14.5. (E) Subcutaneous edema on the dorsal region of Jmjd3 ^−/− embryos at E14.5. (F) H&E staining of sagittal sections of Jmjd3 ^−/− embryos at E11.5 indicating defective spinal cord and somite development. S, spinal cord; So, somite; Drg, dorsal root ganglion; H, heart; Lu, lung; Li, liver; C3, centrum of C3; TV, third ventricle; FV, fourth ventricle; MV, mesencephalic vesicle. (G) Failure of umbilical cord resorption (arrowhead) in E18.5 Jmjd3 ^−/− embryos. (H) The spleens of Jmjd3-deficient embryos at E17.5 were smaller than those of WT embryos. (I) H&E staining showing multiple hyperemic areas in the spleen of Jmjd3 ^−/− embryos. (J) Body weight of Jmjd3 ^−/− embryos was significantly lower than that of WT and Jmjd3 ^+/− controls. Data are presented as the mean ± SD. Differences in body weight were analyzed using the Student's t-test.

Figure 2. Effect of tissue-specific Jmjd3 deletion on mouse survival and lung development.

(A) Schematic drawing showing the effects of tissue-specific Cre expression on mouse survival. (B) H&E staining of lung tissues from Jmjd3^f/f:CCSP-Cre and WT embryos at E17.5. (C) H&E staining of lung tissues from Jmjd3^f/f:SPC-Cre and WT embryos at E18.5. (D) Postnatal lethality (within 8 h) was observed in Jmjd3^f/f:Wnt1-Cre embryos. (E) H&E staining of lung tissues from Jmjd3^f/f:Wnt1-Cre and WT embryos at P0 and E17.5.

Figure 3. Stage-specific deletion reveals that Jmjd3 is dispensable for lung development in later E9.5 embryonic stage.

(A) Jmjd3^f/f mice were crossed with CAG-Cre/ESR mice in which Cre expression is globally induced with TM. The survival of Jmjd3^f/f:Cre/ESR pups from maternal mice treated with TM at E4.5, E9.5, and E14.5 was determined. (B) PCR analysis of Jmjd3 deletion efficiency in Jmjd3^f/f:Cre/ESR pups from maternal mice treated with TM at E4.5. (C) PCR analysis of Jmjd3 deletion efficiency in Jmjd3^f/f:Cre/ESR pups from maternal mice treated with TM at E9.5. (D) H&E staining showing the stage-dependent effects of Jmjd3 deletion on lung architecture and its correlation with embryo viability. Bar = 500 µm.

Figure 4. Jmjd3 regulates the expression of genetic markers of lung development.

(A) Microarray analysis showing representative genes that were differentially expressed between WT and Jmjd3 ^−/− lung tissues at E17.5. Red, upregulated genes; blue, downregulated genes. (B) qPCR was done to validate the expression of selected genes in WT and Jmjd3 ^−/− lung tissues. Gene expression levels were normalized to β-actin. Gene expression levels in WT lung tissue were defined as 1. Data are presented as the mean ± SD. *P<0.05 and **P<0.001 (Student's t test). (C) Immunoblot analysis of the protein expression of selected genes in the lung tissues of Jmjd3 ^+/+ and Jmjd3 ^−/− embryos at E19.5. (D) Immunohistocytochemical staining of SP-B, CC10, and AQP-5 protein expression in the lung tissues of Jmjd3 ^+/+ and Jmjd3 ^−/− embryos at E19.5. Data shown are representative of three independent experiments. Bar = 50 µm. (E) Immunohistocytochemical staining of SP-B, CC10, and AQP-5 expression in the lungs of Jmjd3^f/f, Jmjd3^f/f:CreESR/TM, Jmjd3^f/f:SPC-Cre, and Jmjd3^f/f:Wnt1-Cre mice. Data shown are representative of three independent experiments. Bar = 50 µm.

Figure 5. Jmjd3 ablation affects global histone methylation in lung tissues and methylation status of the promoter regions of target genes.

(A) Global gene methylation analysis of Jmjd3 ^+/+ and Jmjd3 ^−/− lung tissues at E17.5 by ChIP-Seq. ↑, methylation increased; ↓, methylation decreased. (B) ChIP-Seq analysis of H3K27me3 and H3K4me3 levels in the promoter and gene body regions of the SP-B gene in Jmjd3 ^+/+ and Jmjd3 ^−/− lung tissues at E17.5. Data shown are representative of three independent experiments. (C) Jmjd3 binding to the SP-B promoter in lung tissues was determined by ChIP-PCR. Chromatin was immunoprecipitated from the lung tissues of E17.5 Jmjd3 ^+/+ and Jmjd3 ^−/− embryos. Primer design for the ChIP-PCR assay of mouse SP-B promoter regions (top panel). The primers sets cover the following regions: A, −2347–−2142; B, −2065–−1835; C, −1451–−1334; D, −1001–−878; E, −516–−383; F, −218–+14. ChIP-PCR assay showing Jmjd3 binding around 2 kb upstream of the TSS of the SP-B promoter region (bottom panel). (D) ChIP-qPCR analysis of histone methylation levels in the SP-B promoter region in the lung tissues of E17.5 Jmjd3 ^+/+ and Jmjd3 ^−/− embryos. (E) Locus-specific demethylation analysis of Jmjd3 by ChIP-Seq. ChIP-Seq was done to determine the H3K27 and H3K4 methylation level of genes located in the region (∼280 kb) containing SP-B on chromosome 6 and the region (∼160 kb) containing AQP-5 on chromosome 15. Arrows indicate the gene expression direction.

Figure 6. Jmjd3 coregulates SP-B expression with Nkx2.1 and Brg1.

(A) The interaction between Jmjd3 and Nkx2.1 was evaluated by coimmunoprecipitation (CoIP) analysis of 293T cells expressing HA-tagged Jmjd3 and FLAG-tagged Nkx2.1. (B) A cell-based luciferase assay was used to evaluate SP-B promoter activity in 293T cells cotransfected with Nkx2.1, Jmjd3, and mouse SP-B promoter-linked episomal luciferase vector (containing Nkx2.1 binding sites). (C) The interaction between Jmjd3 and Brg1 was determined by CoIP analysis of 293T cells expressing HA-tagged Jmjd3 and FLAG-tagged Brg1. (D) Cell-based luciferase assay was used to evaluate SP-B promoter activity in 293T cells cotransfected with Nkx2.1, Jmjd3, Brg1, and mouse SP-B promoter-linked episomal luciferase vector. *P<0.05 (Student's t test). (E) Generation of Jmjd3 deletion constructs. Numbers represent the corresponding amino acid residue position in the Jmjd3 coding region. (F) CoIP analysis of the interaction between Nkx2.1 and HA-tagged truncated Jmjd3 proteins. (G) CoIP analysis of the interaction between Brg1 and truncated Jmjd3 proteins. (H) CoIP analysis of the interaction among endogenous Nkx2.1, Brg1, and Jmjd3 in WT and Jmjd3-deficient lung tissues. (I) ChIP-PCR and ChIP-qPCR analysis of Nkx2.1 and Brg1 binding to SP-B promoter regions in WT and Jmjd3-deficient lung tissues. *P<0.05 (Student's t test).

Figure 7. The demethylase activity of Jmjd3 is required for the regulation of SP-B expression.

(A) WT, but not mutant, Jmjd3 enhanced NKx2.1 and Brg1-mediated SP-B promoter activity. Data are presented as the mean ± SD from three independent experiments. *P<0.05 (Student's t test). (B) qPCR analysis of SP-B expression in Jmjd3-specific shRNA-expressing H441 cells transfected with WT or mutant Jmjd3. Data are presented as the mean ± SD from three independent experiments. (C) Immunoblot analysis of SP-B protein in Jmjd3-specific shRNA-expressing H441 cells transfected with WT or mutant Jmjd3. (D) A proposed model explaining how Jmjd3 specifically upregulates SP-B expression by interacting with Nkx2.1 and Brg1 in the SP-B promoter.