The Histone H3K79 Methyltransferase Dot1L Is Essential for Mammalian Development and Heterochromatin Structure

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development

The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation.

Histone methylation and DNA methylation cooperatively regulate chromatin structure and gene activity. How these two systems coordinate with each other remains unclear. Here we study the biological function of lysine-specific demethylase 1 (LSD1, also known as KDM1 and AOF2), which has been shown to demethylate histone H3 on lysine 4 (H3K4) and lysine 9 (H3K9). We show that LSD1 is required for gastrulation during mouse embryogenesis. Notably, targeted deletion of the gene encoding LSD1 (namely, Aof2) in embryonic stem (ES) cells induces progressive loss of DNA methylation. This loss correlates with a decrease in DNA methyltransferase 1 (Dnmt1) protein, as a result of reduced Dnmt1 stability. Dnmt1 protein is methylated in vivo, and its methylation is enhanced in the absence of LSD1. Furthermore, Dnmt1 can be methylated by Set7/9 (also known as KMT7) and demethylated by LSD1 in vitro. Our findings suggest that LSD1 demethylates and stabilizes Dnmt1, thus providing a previously unknown mechanistic link between the histone and DNA methylation systems

Pristane-Accelerated Autoimmune Disease in (SWR X NZB) F1 Mice Leads to Prominent Tubulointerstitial Inflammation and Human Lupus Nephritis-Like Fibrosis

<div><p>Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.</p></div

Pristane treatment of SNF1 mice induces human lupus nephritis-like fibrosis.

<p>(A) Gene expression levels of <i>Tnc</i>, <i>Timp1</i>, <i>Col1a2</i>, <i>Acta2</i> in the kidneys of pristane-treated SNF1 mice and Adv-IFNα BWF1 mice (Heatmap shows expression levels in Z-scores). Masson’s Trichrome stain and Tenascin C and alpha smooth muscle actin (ASMA) immunohistochemistry (IHC) were performed on kidney tissue sections from SNF1 with or without pristane treatment (week 14) and BWF1 mice with or without Adv-IFNα injection (week 9). Diseased animals were matched for proteinuria levels. (B) Staining or immunopositive area was quantified in percentage of total tissue. Each dot represents an animal. (* p-value<0.05, **p-value<0.01, unpaired two-tailed Student’s t-test). (C) <i>TNC</i>, <i>TIMP1</i>, <i>COL1A2</i>, <i>ACTA2</i> gene expression levels in normal kidney glomeruli tissues and LN kidney glomeruli tissues from public human microarray dataset. (Heatmap shows expression levels in Z-scores). PAS and tenascin C and ASMA IHC were performed on biopsies from LN patients.</p

Kidney transcriptional response of pristane-SNF1 model and Adv-IFNα BWF1.

<p>(A) Differentially expressed genes were identified using LIMMA package for both mouse models (with fold change difference > |2| and FDR-adjusted p-values <0.05) and using ANOVA test for the human LN kidney dataset (with fold change difference >|1.5| and FDR-adjusted p-values <0.05. Venn Diagram shows the number of differentially expressed genes common between kidneys from pristane-treated SNF1 mice compared to age matched controls, kidneys from Adv-IFNα BWF1 mice compared to age matched controls, and kidney glomeruli tissues from LN patients compared to normal human kidney glomeruli tissues. (B) Gene Set Enrichment Analysis (GSEA) was used to detect whether the upregulated (UP) and downregulated (DN) genes in the human LN were enriched in the top upregulated and top downregulated genes in the kidneys from pristane-treated SNF1 mice respectively. (C) Z-score heatmap showing expression profiles of genes in kidneys from SNF1 mice with and without pristane treatment. All genes shown were differentially expression with a fold change greater than 2 between kidneys from SNF1 at 14 week post-pristane injection vs control (FDR-adjusted p-value<0.05).</p

Serum antibody levels and kidney immune complex deposition in the pristane-SNF1 model and in the Adv-IFNα BWF1 model.

<p>(A) Titers of serum anti-nucleic acid antibodies (ANA) and anti-dsDNA antibodies (Total IgM, IgA, IgG in μg equivalents/ml) in pristane-treated, untreated age matched controls, and 36-week old SNF1 mice with spontaneous disease. (B) Frozen kidney sections from pristane-treated SNF1 mice on weeks 4, 8, 12, and 14, age match control and 36-week old SNF1 mice with spontaneous disease were stained with FITC conjugated anti-IgG Ab to detect immune complex deposition. Staining is representative of 4 mice examined in each group. (C) Frozen kidney sections from Adv-IFNα BWF1 mice on weeks 0, 3, 5, and 9 were stained with FITC conjugated anti-IgG Ab to detect immune complex deposition. Staining is representative of 4 mice examined in each group. (D) Titers of serum total IgG antibodies, anti-nucleic acid antibodies (ANA) were compared between diseased SNF1 pristane mice (14 weeks after treatment) and Adv-IFNα BWF1 mice (9 weeks after treatment). Statistical significance was assessed using unpaired two-tailed Student’s t-test (*** p<0.001, **p<0.01).</p

Pristane treatment accelerates disease in SNF1 lupus-prone mice.

<p>(A) Percentage of non-moribond mice in weeks after birth (SNF1 n = 15) or in weeks after pristane injection (pristane-SNF1 n = 15). Shown is a representative plot of 2 independent experiments. (B) Progression of proteinuria in pristane-treated, untreated age matched controls, and 36-week old SNF1 mice with spontaneous disease. Each symbol indicates an individual mouse. (C) Serum IFNα concentration, and progression of proteinuria and serum cholesterol elevation in Adv-IFNα BWF1 model and untreated age matched controls. Each symbol indicates an individual mouse. (D) Effect of CD40L and BAFF blockade on proteinuria and serum cholesterol in pristane-SNF1 mice (14 weeks after treatment) or Adv-IFNα BWF1 (9 weeks after treatment) either treated with anti-CD40L (MR1), or the isotype control (Ha 4/8) and compared with PBS treated mice. Each symbol indicates an individual mouse. This is a representative experiment of 2 independent experiments. Statistical significance was assessed using One-way Anova test.</p

Innate immune sensors <i>Aim2</i> and <i>Sting</i> are upregulated in pristane-SNF1 model.

<p>(A) Gene expression changes of <i>Aim2</i> and <i>Sting</i> in the kidneys of pristane-treated SNF1 mice and Adv-IFNα BWF1 mice. Data are shown as expression fold change compared to matched control untreated mice. (B) Aim2 and Sting transcripts were detected by RNAscope in kidney tissue sections from SNF1 with or without pristane treatment (week 14) and BWF1 mice with or without Adv-IFNα injection (week 9). Diseased animals were matched for proteinuria levels. (C) Results from image quantification from RNAscope hybridization described in (B) with Aim2, Sting and Ifhi1 RNAscope staining area normalized to the mean of the staining area from the control groups. Each dot represents an animal. (* p-value<0.05, **p-value<0.01, unpaired two-tailed Student’s t-test).</p

Kidney morphology in pristane-SNF1 model and Adv-IFNα BWF1 model.

<p>(<i>A)</i> Glomerular and tubulointerstitial changes in diseased pristane-SNF1 (14 weeks after treatment) and Adv-IFNα-BWF1 models (9 week after treatment) observed using H&E stained kidneys sections. Animals were matched for proteinuria (B) H&E sections of kidneys collected on the weeks indicated from pristane-treated SNF1 and Adv-IFNα injected BWF1 mice were scored for glomerulopathy, tubular dilation, fibrosis and inflammatory infiltrates. The lines represent the average scores, vertical lines represent SEM. Statistical significance was assessed using unpaired two-tailed Student’s t-test (*** p<0.001, **p<0.01). At least 8 animals were analyzed for scoring for each group, except pristane-SNF1 groups at week 4 and week 6 (5 animals per group).</p