The histone methyltransferase Dot1 is required for DNA damage repair and proper development in Dictyostelium

Posttranslational histone modifications play an important role in modulating gene expression and chromatin structure. Here we report the identification of histone H3K79 dimethylation in the simple eukaryote Dictyostelium discoideum. We have deleted the D. discoideum Dot1/KMT4 homologue and demonstrate that it is the sole enzyme responsible for histone H3K79me2. Cells lacking Dot1 are reduced in growth and delayed in development, but do not show apparent changes in cell cycle regulation. Furthermore, our results indicate that Dot1 contributes to UV damage resistance and DNA repair in D. discoideum. In summary, the data support the view that the machinery controlling the setting of histone marks is evolutionary highly conserved and provide evidence that D. discoideum is a suitable model system to analyze these modifications and their functions during development and differentiation.     

H3K56me3 Is a Novel,Conserved Heterochromatic Mark That Largely but Not Completely Overlaps with H3K9me3 in Both Regulation and Localization

Histone lysine (K) methylation has been shown to play a fundamental role in modulating chromatin architecture and regulation of gene expression. Here we report on the identification of histone H3K56, located at the pivotal, nucleosome DNA entry/exit point, as a novel methylation site that is evolutionary conserved. We identify trimethylation of H3K56 (H3K56me3) as a modification that is present during all cell cycle phases, with the exception of S-phase, where it is underrepresented on chromatin. H3K56me3 is a novel heterochromatin mark, since it is enriched at pericentromeres but not telomeres and is thereby similar, but not identical, to the localization of H3K9me3 and H4K20me3. Possibly due to H3 sequence similarities, Suv39h enzymes, responsible for trimethylation of H3K9, also affect methylation of H3K56. Similarly, we demonstrate that trimethylation of H3K56 is removed by members of the JMJD2 family of demethylases that also target H3K9me3. Furthermore, we identify and characterize mouse mJmjd2E and its human homolog hKDM4L as novel, functionally active enzymes that catalyze the removal of two methyl groups from trimethylated H3K9 and K56. H3K56me3 is also found in C. elegans, where it co-localizes with H3K9me3 in most, but not all, tissues. Taken together, our findings raise interesting questions regarding how methylation of H3K9 and H3K56 is regulated in different organisms and their functional roles in heterochromatin formation and/or maintenance.     

Specificity determinants of a novel Nck interaction with the juxtamembrane domain of the epidermal growth factor receptor

Nck is a ubiquitously expressed adaptor protein containing Src homology 2 (SH2) and Src homology 3 (SH3) domains. It integrates downstream effector proteins with cell membrane receptors, such as the epidermal growth factor receptor (EGFR). EGFR plays a critical role in cellular proliferation and differentiation. The 45-residue juxtamembrane domain of EGFR (JM), located between the transmembrane and kinase domains, regulates receptor activation and trafficking to the basolateral membrane of polarized epithelia through a proline-rich motif that resembles a consensus SH3 domain binding site. We demonstrate here that the JM region can bind to Nck, showing a notable binding preference for the second SH3 domain. To elucidate the structural determinants for this interaction, we have determined the NMR solution structures of both the first and second Nck SH3 domains (Nck1-1 and Nck1-2). These domains adopt a canonical SH3 beta-barrel-like fold, containing five antiparallel strands separated by three loop regions and one 3 10-helical turn. Chemical shift perturbation studies have identified the residues that form the binding cleft of Nck1-2, which are primarily located in the RT and n-Src loops. JM binds to Nck1-2 with an affinity of approximately 80 microM through a positively charged sequence near the N-terminus, as opposed to the polyproline sequence. The two Nck SH3 domains exhibit both steric and electrostatic differences in their RT-Src and n-Src loops, and a model of the Nck1-2 domain complexed with the JM highlights the factors that define the putative binding mode for this ligand.     

Stochastic binding of Ca2+ ions in the dyadic cleft; continuous versus random walk description of diffusion

Ca²⁺ signaling in the dyadic cleft in ventricular myocytes is fundamentally discrete and stochastic. We study the stochastic binding of single Ca²⁺ ions to receptors in the cleft using two different models of diffusion: a stochastic and discrete Random Walk (RW) model, and a deterministic continuous model. We investigate whether the latter model, together with a stochastic receptor model, can reproduce binding events registered in fully stochastic RW simulations. By evaluating the continuous model goodness-of-fit for a large range of parameters, we present evidence that it can. Further, we show that the large fluctuations in binding rate observed at the level of single time-steps are integrated and smoothed at the larger timescale of binding events, which explains the continuous model goodness-of-fit. With these results we demonstrate that the stochasticity and discreteness of the Ca²⁺ signaling in the dyadic cleft, determined by single binding events, can be described using a deterministic model of Ca²⁺ diffusion together with a stochastic model of the binding events, for a specific range of physiological relevant parameters. Time-consuming RW simulations can thus be avoided. We also present a new analytical model of bimolecular binding probabilities, which we use in the RW simulations and the statistical analysis.     

Gnarley1 is a dominant mutation in the knox4 homeobox gene affecting cell shape and identity.

Maize leaves have a stereotypical pattern of cell types organized into discrete domains. These domains are altered by mutations in knotted1 (kn1) and knox (for kn1-like homeobox) genes. Gnarley (Gn1) is a dominant maize mutant that exhibits many of the phenotypic characteristics of the kn1 family of mutants. Gn1 is unique because it changes parameters of cell growth in the basal-most region of the leaf, the sheath, resulting in dramatically altered sheath morphology. The strongly expressive allele Gn1-R also gives rise to a floral phenotype in which ectopic carpels form. Introgression studies showed that the severity of the Gn1-conferred phenotype is strongly influenced by genetic background. Gn1 maps to knox4, and knox4 is ectopically expressed in plants with the Gn1-conferred phenotype. Immunolocalization experiments showed that the KNOX protein accumulates at the base of Gn1 leaves in a pattern that is spatially and temporally correlated with appearance of the mutant phenotype. We further demonstrate that Gn1 is knox4 by correlating loss of the mutant phenotype with insertion of a Mutator transposon into knox4.     

Isolation, characterization, and mapping of a human acid beta-galactosidase cDNA

A lambda gt11 human testicular cDNA library was screened with degenerate oligonucleotide probe mixtures based on amino acid sequence data generated from cyanogen bromide fragments and tryptic fragments of purified human beta-galactosidase. Six positive clones were identified after screening 2 x 10(6) plaques. The sequences of these six clones were determined and found to be derived from two different cDNAs. The sequence of the longest of these cDNAs is nearly identical to that recently determined by Oshima et al. (1988). It codes for a 76-kD protein and all 11 peptides that were generated from the purified enzyme. The second clone is shorter by 393 bp in the central portion of the coding region. Analysis by Northern blotting revealed the presence of a single mRNA species of 2.45 kb in lymphoblasts and testicular tissue. It is deduced from the amino acid sequence data that proteolytic processing of the precursor form of beta-galactosidase must occur by cleavage in the carboxy-terminal portion of the polypeptide perhaps around amino acid 530 at a uniquely hydrophilic sequence. Using a probe generated from the 3' region of the cDNA, we have mapped the locus coding for human beta-galactosidase to chromosome 3p21-3pter.     

Monitoring starvation risk: adjustments of body reserves in greenfinches (Carduelis Chloris L.) during periods of unpredictable foraging success

Foragers can put on fat as an energy reserve to reduce the riskof starvation. Reserves are necessary to survive periods whenenergy intake is impossible, and additional reserves can serveas a buffer against periods of little success when foragingis unpredictable; however, maintaining the maximum possiblebody reserves may be detrimental when measured against a costof carrying fat. Experiments with greenfinches (Carduelis ChlorisL) showed that the birds maintained reserves below the levelpermitted by food availability. Greenfinches reduced body reserveswhen exposed to lower metabolic requirements and predictableforaging success; reserves were increased when ambient temperaturewas lowered or foraging success was made more unpredictable.The response to unpredictability was statedependent. Fatterbirds increased their reserves less. The adjustments of energyreserves according to requirements and environmental predictabilitysuggest that it is costly to carry fat and that this cost isbalanced against the benefits of carrying body reserves as aninsurance against starvation.     

XGef mediates early CPEB phosphorylation during Xenopus oocyte meiotic maturation

Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process.