Biomedical subjects

En Li

Publications and source records attributed to En Li.

At least 19 recordsLinked to original sources

PubMed2007

Novel pycnodysostosis mouse model uncovers cathepsin K function as a potential regulator of osteoclast apoptosis and senescence.

Pycnodysostosis is a genetic bone disease featuring the unique bone homeostasis disorders of osteolysis and osteopetrosis in the same organism. The pathomechanism for pycnodysostosis has been largely unknown due to the unavailability of a pycnodysostosis mouse model with all the traits of the disease. We generated cathepsin K(-/-) mouse strains in the 129/Sv and C57BL/6J backgrounds and found that, only in the 129/Sv background, cathepsin K(-/-) mice exhibit many characteristics of the human pycnodysostosis-like phenotype. Our data indicated that 129/Sv cathepsin K(-/-) osteoclasts (OCs) lacked normal apoptosis and senescence and exhibited over-growth both in vitro and in vivo. These abnormalities resulted in an unusually high OC number, which is consistent with a recent case study of human pycnodysostosis. Our results show that cathepsin K function has different effects around the skeleton due to site-specific variations in bone homeostasis, such as phenotypes of osteopetrosis in tibiae and osteolysis in calvariae as a result of cathepsin K mutation. Our data demonstrated that the expression levels of p19, p53 and p21 were significantly reduced in 129/Sv cathepsin K(-/-) OCs and forced expression of cathepsin K in pre-OCs induced premature senescence and increased expression of p19, p53 and p21. This is the first evidence that cathepsin K plays a key role in OC apoptosis and senescence, revealing the importance of OC senescence in bone homeostasis. The finding of this novel cathepsin K function provides insight into the pathomechanism of pycnodysostosis and may provide new drug targets for diseases involved in OC-related abnormal bone homeostasis.

Animals↗

PubMed2006

DNA methylation regulates long-range gene silencing of an X-linked homeobox gene cluster in a lineage-specific manner.

DNA methylation is a major epigenetic mechanism that has been suggested to control developmental gene regulation during embryogenesis, but its regulatory mechanisms remain unclear. In this report, we show that CpG islands associated with the X-linked homeobox gene cluster Rhox, which is highly expressed in the extraembryonic trophectoderm, are differentially methylated in a stage- and lineage-specific manner during the post-implantation development of mice. Inactivation of both Dnmt3a and Dnmt3b, DNA methyltransferases essential for the initiation of de novo DNA methylation, abolished the establishment of DNA methylation and the silencing of Rhox cluster genes in the embryo proper. The Dnmt3-dependent CpG-island methylation at the Rhox locus extended for a large genomic region ( approximately 1 Mb) containing the Rhox cluster and surrounding genes. Complementation experiments using embryonic stem (ES) cells deficient in the DNA methyltransferases suggested that the CpG-island methylation by Dnmt3a and Dnmt3b was restricted within this large genomic region, and did not affect the neighboring genes outside it, implicating the existence of region-specific boundaries. Our results suggest that DNA methylation plays important roles in both long-range gene silencing and lineage-specific silencing in embryogenesis.

Animals↗

PubMed2006

DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages.

DNA methylation is necessary for the silencing of endogenous retrotransposons and the maintenance of monoallelic gene expression at imprinted loci and on the X chromosome. Dynamic changes in DNA methylation occur during the initial stages of primordial germ cell development; however, all consequences of this epigenetic reprogramming are not understood. DNA demethylation in postmigratory primordial germ cells coincides with erasure of genomic imprints and reactivation of the inactive X chromosome, as well as ongoing germ cell differentiation events. To investigate a possible role for DNA methylation changes in germ cell differentiation, we have studied several marker genes that initiate expression at this time. Here, we show that the postmigratory germ cell-specific genes Mvh, Dazl and Scp3 are demethylated in germ cells, but not in somatic cells. Premature loss of genomic methylation in Dnmt1 mutant embryos leads to early expression of these genes as well as GCNA1, a widely used germ cell marker. In addition, GCNA1 is ectopically expressed by somatic cells in Dnmt1 mutants. These results provide in vivo evidence that postmigratory germ cell-specific genes are silenced by DNA methylation in both premigratory germ cells and somatic cells. This is the first example of ectopic gene activation in Dnmt1 mutant mice and suggests that dynamic changes in DNA methylation regulate tissue-specific gene expression of a set of primordial germ cell-specific genes.

Animals↗

PubMed2006

Loss of the maternal imprint in Dnmt3Lmat-/- mice leads to a differentiation defect in the extraembryonic tissue.

DNA methylation of the genome is essential for mammalian development and plays crucial roles in a variety of biological processes including genomic imprinting. Although the DNA methyltransferase 3-like (Dnmt3L) protein lacks DNA methylase activity, it is thought to establish the maternal imprint in combination with the functional DNA methyltransferases. Oogenesis apparently proceeds normally in female mice homozygous for a targeted deletion of Dnmt3L, but their heterozygous offspring (Dnmt3L(mat-/-)) die before midgestation due to an imprinting defect. In this study, we show that Dnmt3L is required for the establishment of maternal methylation imprints both in the embryos and the placentae and that the placentae of these embryos develop abnormally. There is a defect in the formation of the labyrinth, reduced formation of the spongiotrophoblast layer, excess trophoblast giant cells and insufficient attachment between the chorion layer and the ectoplacental cone. In addition, we demonstrate arrest of proliferation of the extraembryonic tissue without apoptosis in vivo and a disturbance of the cell fate of Dnmt3L(mat-/-) trophoblastic stem cells in vitro. Furthermore, we report that DNA methylation during oogenesis is essential for the establishment of imprinting Mash2. These findings provide evidence that not only is DNA methylation required for the appropriate maternal imprint in the placenta but that the appropriate imprint is absolutely required for vertebrate placentation.

Animals↗

PubMed2006

Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases.

Histone methylation regulates chromatin structure, transcription, and epigenetic state of the cell. Histone methylation is dynamically regulated by histone methylases and demethylases such as LSD1 and JHDM1, which mediate demethylation of di- and monomethylated histones. It has been unclear whether demethylases exist that reverse lysine trimethylation. We show the JmjC domain-containing protein JMJD2A reversed trimethylated H3-K9/K36 to di- but not mono- or unmethylated products. Overexpression of JMJD2A but not a catalytically inactive mutant reduced H3-K9/K36 trimethylation levels in cultured cells. In contrast, RNAi depletion of the C. elegans JMJD2A homolog resulted in an increase in general H3-K9Me3 and localized H3-K36Me3 levels on meiotic chromosomes and triggered p53-dependent germline apoptosis. Additionally, other human JMJD2 subfamily members also functioned as trimethylation-specific demethylases, converting H3-K9Me3 to H3-K9Me2 and H3-K9Me1, respectively. Our finding that this family of demethylases generates different methylated states at the same lysine residue provides a mechanism for fine-tuning histone methylation.

Animals↗

PubMed2006

Targeted ablation of Par-4 reveals a cell type-specific susceptibility to apoptosis-inducing agents.

The prostate apoptosis response-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli, and down-regulation of this protein has been suggested to be a key event during tumorigenesis. Several studies suggest an essential function for the COOH-terminal leucine repeats/death domain of Par-4 in mediating apoptosis. We investigated the biological role of this domain in vivo by generating knock-out mice expressing a Par-4 mutant protein lacking the COOH terminus domain. We found that the Par-4 mutant mice are viable and fertile with no overt phenotype, thus excluding an essential role for the COOH terminus domain of Par-4 in embryogenesis and developmental apoptosis. To determine the requirement of Par-4 for apoptosis, we treated primary fibroblasts with various stimuli that trigger mitochondria and membrane receptor cell death pathways. Fibroblasts isolated from Par-4 mutant mice are as sensitive as the wild-type cells to these apoptosis-inducing agents. Similar effects were observed following RNA interference (RNAi)-mediated knockdown of Par-4 in these cells. In contrast, RNAi-mediated depletion of Par-4 in HeLa cells resulted in a significant inhibition of apoptosis induced by various proapoptotic agents. Taken together, our findings provide strong genetic evidence that the proapoptotic function of Par-4 is dependent on the cellular context and raise the possibility that alterations of Par-4 function may occur during carcinogenesis.

Animals↗

PubMed2006

DNA methyltransferases control telomere length and telomere recombination in mammalian cells.

Here, we describe a role for mammalian DNA methyltransferases (DNMTs) in telomere length control. Mouse embryonic stem (ES) cells genetically deficient for DNMT1, or both DNMT3a and DNMT3b have dramatically elongated telomeres compared with wild-type controls. Mammalian telomere repeats (TTAGGG) lack the canonical CpG methylation site. However, we demonstrate that mouse subtelomeric regions are heavily methylated, and that this modification is decreased in DNMT-deficient cells. We show that other heterochromatic marks, such as histone 3 Lys 9 (H3K9) and histone 4 Lys 20 (H4K20) trimethylation, remain at both subtelomeric and telomeric regions in these cells. Lack of DNMTs also resulted in increased telomeric recombination as indicated by sister-chromatid exchanges involving telomeric sequences, and by the presence of 'alternative lengthening of telomeres' (ALT)-associated promyelocytic leukaemia (PML) bodies (APBs). This increased telomeric recombination may lead to telomere-length changes, although our results do not exclude a potential involvement of telomerase and telomere-binding proteins in the aberrant telomere elongation observed in DNMT-deficient cells. Together, these results demonstrate a previously unappreciated role for DNA methylation in maintaining telomere integrity.

Animals↗

PubMed2006

G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis.

Oct-3/4 is a POU domain homeobox gene that is expressed during gametogenesis and in early embryonic cells, where it has been shown to be important for maintaining pluripotency. Following implantation, this gene undergoes a novel multi-step programme of inactivation. Transcriptional repression is followed by a pronounced increase in histone H3 methylation on Lys 9 that is mediated by the SET-containing protein, G9a. This step sets the stage for local heterochromatinization via the binding of HP1 and is required for subsequent de novo methylation at the promoter by the enzymes Dnmt3a/3b. Genetic studies show that these epigenetic changes actually have an important role in the inhibition of Oct-3/4 re-expression, thereby preventing reprogramming.

Acetylation↗

PubMed2006

Stochastic imprinting in the progeny of Dnmt3L-/- females.

The cis-acting regulatory sequences of imprinted genes are subject to germline-specific epigenetic modifications, the imprints, so that this class of genes is exclusively expressed from either the paternal or maternal allele in offspring. How genes are differentially marked in the germlines remains largely to be elucidated. Although the exact nature of the mark is not fully known, DNA methylation [at differentially methylated regions (DMRs)] appears to be a major, functional component. Recent data in mice indicate that Dnmt3a, an enzyme with de novo DNA methyltransferase activity, and the related protein Dnmt3L are required for methylation of imprinted loci in germ cells. Maternal methylation imprints, in particular, are strictly dependent on the presence of Dnmt3L. Here, we show that, unexpectedly, methylation imprints can be present in some progeny of Dnmt3L(-/-) females. This incomplete penetrance of the effect of Dnmt3L deficiency in oocytes is neither embryo nor locus specific, but stochastic. We establish that, when it occurs, methylation is present in both embryo and extra-embryonic tissues and results in a functional imprint. This suggests that this maternal methylation is inherited, directly or indirectly, from the gamete. Our results indicate that in the absence of Dnmt3L, factors such as Dnmt3a and possibly others can act alone to mark individual DMRs. However, establishment of appropriate maternal imprints at all loci does require a combination of all factors. This observation can provide a basis to understand mechanisms involved in some sporadic cases of imprinting-related diseases and polymorphic imprinting in human.

Animals↗

PubMed2006

Lsh is involved in de novo methylation of DNA.

Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in Lsh-/- embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA.

Animals↗

PubMed2006

Meiotic and epigenetic aberrations in Dnmt3L-deficient male germ cells.

The DNA methyltransferase-like protein Dnmt3L is necessary for the establishment of genomic imprints in oogenesis and for normal spermatogenesis (Bourc'his et al., 2001; Hata et al., 2002). Also, a paternally imprinted gene, H19, loses DNA methylation in Dnmt3L-/- spermatogonia (Bourc'his and Bestor, 2004; Kaneda et al., 2004). To determine the reason for the impaired spermatogenesis in the Dnmt3L-/- testes, we have carried out a series of histological and molecular studies. We show here that Dnmt3L-/- germ cells were arrested and died around the early meiotic stage. A microarray-based gene expression-profiling analysis revealed that various gonad-specific and/or sex-chromosome-linked genes were downregulated in the Dnmt3L-/- testes. In contrast, expression of retrovirus-like intracisternal A-particle (IAP) sequences was upregulated; consistent with this observation, a specific IAP copy showed complete loss of DNA methylation. These findings indicate that Dnmt3L regulates germ cell-specific gene expression and IAP suppression, which are critical for male germ cell proliferation and meiosis.

Animals↗

PubMed2006

Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

Amino Acid Sequence↗

PubMed2006

Dnmt3a2 targets endogenous Dnmt3L to ES cell chromatin and induces regional DNA methylation.

DNA methylation is involved in fundamental cellular processes such as silencing of genes and transposable elements, but the underlying mechanism of regulation of DNA methylation is largely unknown. DNA methyltransferase 3-like protein (Dnmt3L), a member of the Dnmt3 family of proteins, is required during the establishment of DNA methylation patterns in germ cells. Dnmt3L does not possess enzymatic activity. Rather, in vitro analysis indicates that Dnmt3L stimulates DNA methylation by both Dnmt3a and Dnmt3b through direct binding to these proteins. In the current study, we demonstrated that in vivo, Dnmt3L physically and functionally interacted with the Dnmt3 isoform Dnmt3a2. In wild-type embryonic stem (ES) cells, but not in cells lacking Dnmt3a, endogenous Dnmt3L was concentrated in chromatin foci. In ES cells deficient in both Dnmt3a and Dnmt3b, Dnmt3L was distributed diffusely throughout the nucleus and cytoplasm, and ectopic expression of Dnmt3a2, but not Dnmt3a or Dnmt3b, restored wild-type Dnmt3L localization. We showed that endogenous Dnmt3L physically interacted with Dnmt3a2, but not Dnmt3a or Dnmt3b, in ES cells and embryonic testes. We also found that specific CpG sites were demethylated upon depletion of either Dnmt3a or Dnmt3L, but not Dnmt3b, in ES cells. These results provide evidence for a physical and functional interaction between Dnmt3L and Dnmt3a2 in the nucleus. We propose that Dnmt3a2 recruits Dnmt3L to chromatin, and induces regional DNA methylation in germ cells.

Animals↗

PubMed2006

Essential dosage-dependent functions of the transcription factor yin yang 1 in late embryonic development and cell cycle progression.

Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing approximately 75%, approximately 50%, and approximately 25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation.

Animals↗

PubMed2006

Roles for Dnmt3b in mammalian development: a mouse model for the ICF syndrome.

ICF (Immunodeficiency, Centromeric instability and Facial anomalies) syndrome is a rare autosomal recessive disease caused by mutations in the DNA methyltransferase gene DNMT3B. To investigate the function of Dnmt3b in mouse development and to create animal models for ICF syndrome, we have generated three mutant alleles of Dnmt3b in mice: one carrying a deletion of the catalytic domain (null allele) and two carrying ICF-like missense mutations in the catalytic domain. The Dnmt3b null allele results in embryonic lethality from E14.5 to E16.5 with multiple tissue defects, including liver hypotrophy, ventricular septal defect and haemorrhage. By contrast, mice homozygous for the ICF mutations develop to term and some survive to adulthood. These mice show phenotypes that are reminiscent of ICF patients, including hypomethylation of repetitive sequences, low body weight, distinct cranial facial anomalies and T cell death by apoptosis. These results indicate that Dnmt3b plays an essential role at different stages of mouse development, and that ICF missense mutations cause partial loss of function. These mutant mice will be useful for further elucidation of the pathogenic and molecular mechanisms underlying ICF syndrome.

Animals↗

PubMed2005

Regulation of muscle growth by multiple ligands signaling through activin type II receptors.

Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn(-/-) mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo.

Activin Receptors, Type II↗

PubMed2005

DNA methylation is required for silencing of ant4, an adenine nucleotide translocase selectively expressed in mouse embryonic stem cells and germ cells.

The capacity for cellular differentiation is governed not only by the repertoire of available transcription factors but by the accessibility of cis-regulatory elements. Studying changes in epigenetic modifications during stem cell differentiation will help us understand how cells maintain or lose differentiation potential. We investigated changes in DNA methylation during the transition of pluripotent embryonic stem cells (ESCs) into differentiated cell types. Using a methylation-sensitive restriction fingerprinting method, we identified a novel adenine nucleotide (ADP/ATP) translocase gene, Ant4, that was selectively hypomethylated and expressed in undifferentiated mouse ESCs. In contrast to other pluripotent stem cell-specific genes such as Oct-4 and Nanog, the Ant4 gene was readily derepressed in differentiated cells after 5-aza-2'-deoxycytidine treatment. Moreover, expression of de novo DNA methyltransferases Dnmt3a and Dnmt3b was essential for repression and DNA methylation of the Ant4 gene during ESC differentiation. Although the deduced amino acid sequence of Ant4 is highly homologous to the previously identified Ant isoforms, the expression of Ant4 was uniquely restricted to developing gametes in adult mice, and its promoter hypomethylation was observed only in testis. Additionally, Ant4 was expressed in primordial germ cells. These data indicate that Ant4 is a pluripotent stem cell- and germ cell-specific isoform of adenine nucleotide translocase in mouse and that DNA methylation plays a primary role in its transcriptional silencing in somatic cells.

Animals↗

PubMed2005

Bone morphogenetic protein (BMP) type II receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells.

Bone morphogenetic protein (BMP) ligands signal by binding the BMP type II receptor (BMPR2) or the activin type II receptors (ActRIIa and ActRIIb) in conjunction with type I receptors to activate SMADs 1, 5, and 8, as well as members of the mitogen-activated protein kinase family. Loss-of-function mutations in Bmpr2 have been implicated in tumorigenesis and in the etiology of primary pulmonary hypertension. Because several different type II receptors are known to recognize BMP ligands, the specific contribution of BMPR2 to BMP signaling is not defined. Here we report that the ablation of Bmpr2 in pulmonary artery smooth muscle cells, using an ex vivo conditional knock-out (Cre-lox) approach, as well as small interfering RNA specific for Bmpr2, does not abolish BMP signaling. Disruption of Bmpr2 leads to diminished signaling by BMP2 and BMP4 and augmented signaling by BMP6 and BMP7. Using small interfering RNAs to inhibit the expression of other BMP receptors, we found that wild-type cells transduce BMP signals via BMPR2, whereas BMPR2-deficient cells transduce BMP signals via ActRIIa in conjunction with a set of type I receptors distinct from those utilized by BMPR2. These findings suggest that disruption of Bmpr2 leads to the net gain of signaling by some, but not all, BMP ligands via the activation of ActRIIa.

Activin Receptors, Type II↗