Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.
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 (∼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.[Keywords: DNA methylation; Dnmt3a; Dnmt3b; Rhox] Supplemental material is available at http://www.genesdev.org.
DNA methylation regulates development and many epigenetic processes in mammals, and it is required for somatic cell growth and survival. In contrast, embryonic stem (ES) cells can self-renew without DNA methylation. It remains unclear whether any lineage-committed cells can survive without DNA-methylation machineries. Unlike in somatic cells, DNA methylation is dispensable for imprinting and X-inactivation in the extraembryonic lineages. In ES cells, DNA methylation prevents differentiation into the trophectodermal fate. Here, we created triple-knockout (TKO) mouse embryos deficient for the active DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) by nuclear transfer (NT), and we examined their development. In chimeric TKO-NT and WT embryos, few TKO cells were found in the embryo proper, but they contributed to extraembryonic tissues. TKO ES cells showed increasing cell death during their differentiation into epiblast lineages, but not during differentiation into extraembryonic lineages. Furthermore, we successfully established trophoblastic stem cells (ntTS cells) from TKO-NT blastocysts. These TKO ntTS cells could self-renew, and they retained the fundamental gene expression patterns of stem cells. Our findings indicated that extraembryonic-lineage cells can survive and proliferate in the absence of DNA methyltransferases and that a cell's response to the stress of epigenomic damage is cell type dependent.
PKNbeta is a novel isoform of PKNalpha, which is one of the target protein kinases for the small GTPase Rho. By yeast two-hybrid screening of a human embryonic kidney 293 cell cDNA library with the PKNbeta linker region containing proline-rich motifs as a bait, clones encoding Graf (GAP for Rho Associated with Focal adhesion kinase) and a novel Graf-related protein, termed Graf2, were isolated. The full length of Graf2 contains a putative PH domain, a RhoGAP domain, and an SH3 domain as well as Graf. Northern and Western blot analyses demonstrated that Graf2 is expressed in several tissues, with the highest expression in skeletal muscle. Recombinant Graf2 exhibited GTPase-activating activity toward the small GTPase RhoA and Cdc42Hs, but not toward Rac1, in vitro. The SH3 domains of Graf and Graf2 purified from Escherichia coli bound directly to PKNbeta. Graf or Graf2 was co-immunoprecipitated with PKNbeta in COS-7 cells transiently transfected with Graf or Graf2 and PKNbeta expression constructs. The catalytically active form of PKNbeta phosphorylated Graf and Graf2 in vitro. The interplay of PKNbeta and the GTPase-activating proteins, Graf and Graf2, may offer a novel mechanism regulating the Rho-mediated signaling.
DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.
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