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

Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells.

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase-mediated disruption of this allele results in hemimethylation of approximately 20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival

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

Defective heart development in hypomorphic Lsd1 mice

Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates several target proteins, including histones, DNMT1 and p53. Lsd1 can act as either a transcriptional activator or repressor, depending on the histone lysine it demethylates; its specificity for either H3K4 or H3K9 appears to be determined by its binding partners. We have previously demonstrated that a complete loss of this protein results in early embryonic lethality. However, it was also noted that no adult mice homozygous for the floxed allele survived, even though this allele should be indistinguishable from wild-type Lsd1 after splicing removes the LoxP sites. Homozygous pups die perinatally, with most animals showing major heart development defects. The Aof22lox allele contains two point mutations; the resulting protein shows reduced interaction with known binding partners and decreased enzymatic activity. The expression of a very limited subset of genes is altered in the hearts. This includes an increase in CK2beta expression, the regulatory subunit of the CK2 kinase, which results in E-cadherin hyperphosphorylation. These results identify a previously unknown role for Lsd1 in heart development, through the control of E-cadherin phosphorylation

Lsd1 expression in the developing heart.

<p>Wild-type embryos at the indicated development stages (E8.5 to E13.5, and E18.5) were dissected and processed for immunohistochemistry. H&E staining was performed, along with staining with anti-Lsd1 to visual the Lsd1 expression pattern during heart development. Note that in all panels, the black bar represents 100 µm.</p

Figure 5

<p><b>Changes in gene expression in the hypomorphic hearts.</b> Expression of gene products, examined by qRT-PCR using RNA isolated from E18.5 embryonic hearts. Data represents mean +/− SD from 3 to 5 animals of each genotype, and was normalized such that the expression of the mRNA in wild-type animals was equal to 1 (*: p<0.05). (A) Lsd1 mRNA levels were decreased by approximately 50%, confirming the microarray data. Nkx2-5, a heart marker, Ncam, and β-catenin were not found to be significantly altered in the hypomorphic hearts. The expression of Tescalcin and Fblim1, two proteins with potential roles in heart development that were identified by microarray, was also analyzed. The increase in Tescalcin expression was recapitulated by the qRT-PCR, while no difference in Fblim1 expression between the wild-type and hypomorphic hearts was noted. CamK2β showed minor increases in expression. (B) Expression of Wnt targets in the wild-type and hypomorphic hearts. The Wnt signaling pathway target genes Wnt11, Lrp6, Kit, and Isl1 were examined for changes in mRNA levels in the hypomorphic hearts by qRT-PCR. No statistically significant changes were noted in the expression of any of these genes. (C) mRNA expression of epithelial and mesenchymal markers is identical between wild-type and 2lox/2lox hearts. Expression of the epithelial markers Pecam and VE-Cadherin, and the mesenchymal markers B4galt6 and Fn1 was examined by qRT-PCR using RNA isolated from E18.5 embryonic hearts. No statistically significant difference was noted in the expression of any of these mRNAs.</p