Genes and Development

Linking H3K79 trimethylation to Wnt signaling through a novel Dot1-containing complex (DotCom).

Publication Date: 2010 Mar 4 PMID: 20203130
Authors: Mohan, M. - Herz, H. M. - Takahashi, Y. H. - Lin, C. - Lai, K. C. - Zhang, Y. - Washburn, M. P. - Florens, L. - Shilatifard, A.
Journal: Genes Dev

Epigenetic modifications of chromatin play an important role in the regulation of gene expression. KMT4/Dot1 is a conserved histone methyltransferase capable of methylating chromatin on Lys79 of histone H3 (H3K79). Here we report the identification of a multisubunit Dot1 complex (DotCom), which includes several of the mixed lineage leukemia (MLL) partners in leukemia such as ENL, AF9/MLLT3, AF17/MLLT6, and AF10/MLLT10, as well as the known Wnt pathway modifiers TRRAP, Skp1, and beta-catenin. We demonstrated that the human DotCom is indeed capable of trimethylating H3K79 and, given the association of beta-catenin, Skp1, and TRRAP, we investigated, and found, a role for Dot1 in Wnt/Wingless signaling in an in vivo model system. Knockdown of Dot1 in Drosophila results in decreased expression of a subset of Wingless target genes. Furthermore, the loss of expression for the Drosophila homologs of the Dot1-associated proteins involved in the regulation of H3K79 shows a similar reduction in expression of these Wingless targets. From yeast to human, specific trimethylation of H3K79 by Dot1 requires the monoubiquitination of histone H2B by the Rad6/Bre1 complex. Here, we demonstrate that depletion of Bre1, the E3 ligase required for H2B monoubiquitination, leads specifically to reduced bulk H3K79 trimethylation levels and a reduction in expression of many Wingless targets. Overall, our study describes for the first time the components of DotCom and links the specific regulation of H3K79 trimethylation by Dot1 and its associated factors to the Wnt/Wingless signaling pathway.

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GEN1/Yen1 and the SLX4 complex: solutions to the problem of Holliday junction resolution.

Publication Date: 2010 Mar 4 PMID: 20203129
Authors: Svendsen, J. M. - Harper, J. W.
Journal: Genes Dev

Chromosomal double-strand breaks (DSBs) are considered to be among the most deleterious DNA lesions found in eukaryotic cells due to their propensity to promote genome instability. DSBs occur as a result of exogenous or endogenous DNA damage, and also occur during meiotic recombination. DSBs are often repaired through a process called homologous recombination (HR), which employs the sister chromatid in mitotic cells or the homologous chromosome in meiotic cells, as a template for repair. HR frequently involves the formation and resolution of four-way DNA structures referred to as the Holliday junction (HJ). Despite extensive study, the machinery and mechanisms used to process these structures in eukaryotes have remained poorly understood. Recent work has identified XPG and UvrC/GIY domain-containing structure-specific endonucleases that can symmetrically cleave HJs in vitro in a manner that allows for religation without additional processing, properties that are reminiscent of the classical RuvC HJ resolvase in bacteria. Genetic studies reveal potential roles for these HJ resolvases in repair after DNA damage and during meiosis. The stage is now set for a more comprehensive understanding of the specific roles these enzymes play in the response of cells to DSBs, collapsed replication forks, telomere dysfunction, and meiotic recombination.

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De novo telomere formation is suppressed by the Mec1-dependent inhibition of Cdc13 accumulation at DNA breaks.

Publication Date: 2010 Mar 1 PMID: 20194442
Authors: Zhang, W. - Durocher, D.
Journal: Genes Dev

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability, since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. Here we report that Mec1, the ATR ortholog, couples the detection of DNA ends with the inhibition of telomerase. Mec1 inhibits telomere healing by phosphorylating Cdc13 on its S306 residue, a phosphorylation event that suppresses Cdc13 accumulation at DSBs. Conversely, telomere addition at accidental breaks is promoted by Pph3, the yeast protein phosphatase 4 (PP4). Pph3 is itself modulated by Rrd1, an activator of PP2A family phosphatases. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DNA breaks. These studies therefore identify a mechanism by which the ATR family of kinases enforces genome integrity, and a process that underscores the contribution of Cdc13 to the fate of DNA ends.

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Differential activation and antagonistic function of HIF-{alpha} isoforms in macrophages are essential for NO homeostasis.

Publication Date: 2010 Mar 1 PMID: 20194441
Authors: Takeda, N. - O'Dea, E. L. - Doedens, A. - Kim, J. W. - Weidemann, A. - Stockmann, C. - Asagiri, M. - Simon, M. C. - Hoffmann, A. - Johnson, R. S.
Journal: Genes Dev

Hypoxic response and inflammation both involve the action of the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha. Previous studies have revealed that both HIF-alpha proteins are in a number of aspects similarly regulated post-translationally. However, the functional interrelationship of these two isoforms remains largely unclear. The polarization of macrophages controls functionally divergent processes; one of these is nitric oxide (NO) production, which in turn is controlled in part by HIF factors. We show here that the HIF-alpha isoforms can be differentially activated: HIF-1alpha is induced by Th1 cytokines in M1 macrophage polarization, whereas HIF-2alpha is induced by Th2 cytokines during an M2 response. This differential response was most evident in polarized macrophages through HIF-alpha isoform-specific regulation of the inducible NO synthase gene by HIF-1alpha, and the arginase1 gene by HIF-2alpha. In silico modeling predicted that regulation of overall NO availability is due to differential regulation of HIF-1alpha versus HIF-2alpha, acting to, respectively, either increase or suppress NO synthesis. An in vivo model of endotoxin challenge confirmed this; thus, these studies reveal that the two homologous transcription factors, HIF-1alpha and HIF-2alpha, can have physiologically antagonistic functions, but that their antiphase regulation allows them to coordinately regulate NO production in a cytokine-induced and transcription-dependent fashion.

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miR-125b-2 is a potential oncomiR on human chromosome 21 in megakaryoblastic leukemia.

Publication Date: 2010 Mar 1 PMID: 20194440
Authors: Klusmann, J. H. - Li, Z. - Bohmer, K. - Maroz, A. - Koch, M. L. - Emmrich, S. - Godinho, F. J. - Orkin, S. H. - Reinhardt, D.
Journal: Genes Dev

Children with trisomy 21/Down syndrome (DS) are at high risk to develop acute megakaryoblastic leukemia (DS-AMKL) and the related transient leukemia (DS-TL). The factors on human chromosome 21 (Hsa21) that confer this predisposing effect, especially in synergy with consistently mutated transcription factor GATA1 (GATA1s), remain poorly understood. Here, we investigated the role of Hsa21-encoded miR-125b-2, a microRNA (miRNA) overexpressed in DS-AMKL/TL, in hematopoiesis and leukemogenesis. We identified a function of miR-125b-2 in increasing proliferation and self-renewal of human and mouse megakaryocytic progenitors (MPs) and megakaryocytic/erythroid progenitors (MEPs). miR-125b-2 overexpression did not affect megakaryocytic and erythroid differentiation, but severely perturbed myeloid differentiation. The proproliferative effect of miR-125b-2 on MEPs accentuated the Gata1s mutation, whereas growth of DS-AMKL/TL cells was impaired upon miR-125b repression, suggesting synergism during leukemic transformation in GATA1s-mutated DS-AMKL/TL. Integrative transcriptome analysis of hematopoietic cells upon modulation of miR-125b expression levels uncovered a set of miR-125b target genes, including DICER1 and ST18 as direct targets. Gene Set Enrichment Analysis revealed that this target gene set is down-regulated in DS-AMKL patients highly expressing miR-125b. Thus, we propose miR-125b-2 as a positive regulator of megakaryopoiesis and an oncomiR involved in the pathogenesis of trisomy 21-associated megakaryoblastic leukemia.

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Spermatogenesis rescue in a mouse deficient for the ubiquitin ligase SCF{beta}-TrCP by single substrate depletion.

Publication Date: 2010 Mar 1 PMID: 20194439
Authors: Kanarek, N. - Horwitz, E. - Mayan, I. - Leshets, M. - Cojocaru, G. - Davis, M. - Tsuberi, B. Z. - Pikarsky, E. - Pagano, M. - Ben-Neriah, Y.
Journal: Genes Dev

beta-TrCP, the substrate recognition subunit of a Skp1-Cul1-F-box (SCF) ubiquitin ligase, is ubiquitously expressed from two distinct paralogs, targeting many regulatory proteins for proteasomal degradation. We generated inducible beta-TrCP hypomorphic mice and found that they are surprisingly healthy, yet have a severe testicular defect. We show that the two beta-TrCP paralogs have a nonredundant role in spermatogenesis. The testicular defect is tightly associated with cell adhesion failure within the seminiferous tubules and is fully reversible upon beta-TrCP restoration. Remarkably, testicular depletion of a single beta-TrCP substrate, Snail1, rescued the adhesion defect and restored spermatogenesis. Our studies highlight an unexpected functional reserve of this central E3, as well as a bottleneck in a specific tissue: a single substrate whose stabilization is incompatible with testicular differentiation.

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Histone deacetylases 1 and 2 act in concert to promote the G1-to-S progression.

Publication Date: 2010 Mar 1 PMID: 20194438
Authors: Yamaguchi, T. - Cubizolles, F. - Zhang, Y. - Reichert, N. - Kohler, H. - Seiser, C. - Matthias, P.
Journal: Genes Dev

Histone deacetylases (HDACs) regulate gene expression by deacetylating histones and also modulate the acetylation of a number of nonhistone proteins, thus impinging on various cellular processes. Here, we analyzed the major class I enzymes HDAC1 and HDAC2 in primary mouse fibroblasts and in the B-cell lineage. Fibroblasts lacking both enzymes fail to proliferate in culture and exhibit a strong cell cycle block in the G1 phase that is associated with up-regulation of the CDK inhibitors p21(WAF1/CIP1) and p57(Kip2) and of the corresponding mRNAs. This regulation is direct, as in wild-type cells HDAC1 and HDAC2 are bound to the promoter regions of the p21 and p57 genes. Furthermore, analysis of the transcriptome and of histone modifications in mutant cells demonstrated that HDAC1 and HDAC2 have only partly overlapping roles. Next, we eliminated HDAC1 and HDAC2 in the B cells of conditionally targeted mice. We found that B-cell development strictly requires the presence of at least one of these enzymes: When both enzymes are ablated, B-cell development is blocked at an early stage, and the rare remaining pre-B cells show a block in G1 accompanied by the induction of apoptosis. In contrast, elimination of HDAC1 and HDAC2 in mature resting B cells has no negative impact, unless these cells are induced to proliferate. These results indicate that HDAC1 and HDAC2, by normally repressing the expression of p21 and p57, regulate the G1-to-S-phase transition of the cell cycle.

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A degron created by SMN2 exon 7 skipping is a principal contributor to spinal muscular atrophy severity.

Publication Date: 2010 Mar 1 PMID: 20194437
Authors: Cho, S. - Dreyfuss, G.
Journal: Genes Dev

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletions, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, most of the mRNA produced from SMN2 pre-mRNA is exon 7-skipped ( approximately 80%), resulting in a highly unstable and almost undetectable protein (SMNDelta7). We show that this splicing defect creates a potent degradation signal (degron; SMNDelta7-DEG) at SMNDelta7's C-terminal 15 amino acids. The S270A mutation inactivates SMNDelta7-DEG, generating a stable SMNDelta7 that rescues viability of SMN-deleted cells. These findings explain a key aspect of the SMA disease mechanism, and suggest new treatment approaches based on interference with SMNDelta7-DEG activity.

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KDM7 is a dual demethylase for histone H3 Lys 9 and Lys 27 and functions in brain development.

Publication Date: 2010 Mar 1 PMID: 20194436
Authors: Tsukada, Y. - Ishitani, T. - Nakayama, K. I.
Journal: Genes Dev

Methylation of histone H3 Lys 9 and Lys 27 (H3K9 and H3K27) is associated with transcriptional silencing. Here we show that KDM7, a JmjC domain-containing protein, catalyzes demethylation of both mono- or dimethylated H3K9 and H3K27. Inhibition of KDM7 orthologs in zebrafish resulted in developmental brain defects. KDM7 interacts with the follistatin gene locus, and KDM7 depletion in mammalian neuronal cells suppressed follistatin gene transcription in association with increased levels of dimethylated H3K9 and H3K27. Our findings identify KDM7 as a dual demethylase for H3K9 and H3K27 that functions as an eraser of silencing marks on chromatin during brain development.

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Personal genome sequencing: current approaches and challenges.

Publication Date: 2010 Mar 1 PMID: 20194435
Authors: Snyder, M. - Du, J. - Gerstein, M.
Journal: Genes Dev

The revolution in DNA sequencing technologies has now made it feasible to determine the genome sequences of many individuals; i.e., "personal genomes." Genome sequences of cells and tissues from both normal and disease states have been determined. Using current approaches, whole human genome sequences are not typically assembled and determined de novo, but, instead, variations relative to a reference sequence are identified. We discuss the current state of personal genome sequencing, the main steps involved in determining a genome sequence (i.e., identifying single-nucleotide polymorphisms [SNPs] and structural variations [SVs], assembling new sequences, and phasing haplotypes), and the challenges and performance metrics for evaluating the accuracy of the reconstruction. Finally, we consider the possible individual and societal benefits of personal genome sequences.

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Isoform-specific p73 knockout mice reveal a novel role for {Delta}Np73 in the DNA damage response pathway.

Publication Date: 2010 Mar 1 PMID: 20194434
Authors: Wilhelm, M. T. - Rufini, A. - Wetzel, M. K. - Tsuchihara, K. - Inoue, S. - Tomasini, R. - Itie-Youten, A. - Wakeham, A. - Arsenian-Henriksson, M. - Melino, G. - Kaplan, D. R. - Miller, F. D. - Mak, T. W.
Journal: Genes Dev

Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and DeltaNp73 isoforms. As part of our ongoing program to distinguish the biological functions of these isoforms, we generated mice that are selectively deficient for the DeltaNp73 isoform. Mice lacking DeltaNp73 (DeltaNp73(-/-) mice) are viable and fertile but display signs of neurodegeneration. Cells from DeltaNp73(-/-) mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in DeltaNp73(-/-) cells, we discovered a completely new role for DeltaNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. We found that DeltaNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of DeltaNp73 expression show enhanced resistance to chemotherapy.

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The homeobox protein Prox1 is a negative modulator of ERR{alpha}/PGC-1{alpha} bioenergetic functions.

Publication Date: 2010 Mar 1 PMID: 20194433
Authors: Charest-Marcotte, A. - Dufour, C. R. - Wilson, B. J. - Tremblay, A. M. - Eichner, L. J. - Arlow, D. H. - Mootha, V. K. - Giguere, V.
Journal: Genes Dev

Estrogen-related receptor alpha (ERRalpha) and proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) play central roles in the transcriptional control of energy homeostasis, but little is known about factors regulating their activity. Here we identified the homeobox protein prospero-related homeobox 1 (Prox1) as one such factor. Prox1 interacts with ERRalpha and PGC-1alpha, occupies promoters of metabolic genes on a genome-wide scale, and inhibits the activity of the ERRalpha/PGC-1alpha complex. DNA motif analysis suggests that Prox1 interacts with the genome through tethering to ERRalpha and other factors. Importantly, ablation of Prox1 and ERRalpha have opposite effects on the respiratory capacity of liver cells, revealing an unexpected role for Prox1 in the control of energy homeostasis.

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A highly coordinated cell wall degradation machine governs spore morphogenesis in Bacillus subtilis.

Publication Date: 2010 Feb 15 PMID: 20159959
Authors: Morlot, C. - Uehara, T. - Marquis, K. A. - Bernhardt, T. G. - Rudner, D. Z.
Journal: Genes Dev

How proteins catalyze morphogenesis is an outstanding question in developmental biology. In bacteria, morphogenesis is intimately linked to remodeling the cell wall exoskeleton. Here, we investigate the mechanisms by which the mother cell engulfs the prospective spore during sporulation in Bacillus subtilis. A membrane-anchored protein complex containing two cell wall hydrolases plays a central role in this morphological process. We demonstrate that one of the proteins (SpoIIP) has both amidase and endopeptidase activities, such that it removes the stem peptides from the cell wall and cleaves the cross-links between them. We further show that the other protein (SpoIID) is the founding member of a new family of lytic transglycosylases that degrades the glycan strands of the peptidoglycan into disaccharide units. Importantly, we show that SpoIID binds the cell wall, but will only cleave the glycan strands after the stem peptides have been removed. Finally, we demonstrate that SpoIID also functions as an enhancer of SpoIIP activity. Thus, this membrane-anchored enzyme complex is endowed with complementary, sequential, and stimulatory activities. These activities provide a mechanism for processive cell wall degradation, supporting a model in which circumferentially distributed degradation machines function as motors pulling the mother cell membranes around the forespore.

MeSH Categories: Amidohydrolases/metabolism, Amino Acid Sequence, Bacillus subtilis/enzymology/*physiology, Bacterial Proteins/chemistry/metabolism, Cell Wall/*metabolism, DNA-Binding Proteins/chemistry/metabolism, Endopeptidases/metabolism, Molecular Sequence Data, Polysaccharides/metabolism, Sequence Alignment, Spores, Bacterial/physiology, Transcription Factors/chemistry/metabolism

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A midline switch of receptor processing regulates commissural axon guidance in vertebrates.

Publication Date: 2010 Feb 15 PMID: 20159958
Authors: Nawabi, H. - Briancon-Marjollet, A. - Clark, C. - Sanyas, I. - Takamatsu, H. - Okuno, T. - Kumanogoh, A. - Bozon, M. - Takeshima, K. - Yoshida, Y. - Moret, F. - Abouzid, K. - Castellani, V.
Journal: Genes Dev

Commissural axon guidance requires complex modulations of growth cone sensitivity to midline-derived cues, but underlying mechanisms in vertebrates remain largely unknown. By using combinations of ex vivo and in vivo approaches, we uncovered a molecular pathway controlling the gain of response to a midline repellent, Semaphorin3B (Sema3B). First, we provide evidence that Semaphorin3B/Plexin-A1 signaling participates in the guidance of commissural projections at the vertebrate ventral midline. Second, we show that, at the precrossing stage, commissural neurons synthesize the Neuropilin-2 and Plexin-A1 Semaphorin3B receptor subunits, but Plexin-A1 expression is prevented by a calpain1-mediated processing, resulting in silencing commissural responsiveness. Third, we report that, during floor plate (FP) in-growth, calpain1 activity is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone and sensitization to Sema3B. Finally, we show that the FP cue NrCAM mediates the switch of Plexin-A1 processing underlying growth cone sensitization to Sema3B. This reveals pathway-dependent modulation of guidance receptor processing as a novel mechanism for regulating guidance decisions at intermediate targets.

MeSH Categories: Animals, Axons/metabolism/*physiology, Calpain/metabolism, Cell Adhesion Molecules/metabolism, Chick Embryo, Embryo, Mammalian, Gene Expression Regulation, Developmental, Mice, Nerve Tissue Proteins/metabolism, Neurons/*cytology/metabolism, Neuropilin-2/metabolism, Semaphorins/metabolism, *Signal Transduction

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GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRbeta in the hepatic acute phase response.

Publication Date: 2010 Feb 15 PMID: 20159957
Authors: Venteclef, N. - Jakobsson, T. - Ehrlund, A. - Damdimopoulos, A. - Mikkonen, L. - Ellis, E. - Nilsson, L. M. - Parini, P. - Janne, O. A. - Gustafsson, J. A. - Steffensen, K. R. - Treuter, E.
Journal: Genes Dev

The orphan receptor LRH-1 and the oxysterol receptors LXRalpha and LXRbeta are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRbeta subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor-corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.

MeSH Categories: Acute-Phase Reaction/*immunology, Animals, Anti-Inflammatory Agents/immunology, COS Cells, Cercopithecus aethiops, Female, Gene Expression Regulation, Hela Cells, Humans, Intracellular Signaling Peptides and Proteins/*immunology, Liver/*immunology, Mice, Mice, Inbred C57BL, Mice, Knockout, Orphan Nuclear Receptors/*immunology, Receptors, Cytoplasmic and Nuclear/*immunology, Small Ubiquitin-Related Modifier Proteins/*immunology

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Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA.

Publication Date: 2010 Feb 15 PMID: 20159956
Authors: Menet, J. S. - Abruzzi, K. C. - Desrochers, J. - Rodriguez, J. - Rosbash, M.
Journal: Genes Dev

Transcriptional feedback loops are central to the generation and maintenance of circadian rhythms. In animal systems as well as Neurospora, transcriptional repression is believed to occur by catalytic post-translational events. We report here in the Drosophila model two different mechanisms by which the circadian repressor PERIOD (PER) inhibits CLOCK/CYCLE (CLK/CYC)-mediated transcription. First, PER is recruited to circadian promoters, which leads to the nighttime decrease of CLK/CYC activity. This decrease is proportional to PER levels on DNA, and PER recruitment probably occurs via CLK. Then CLK is released from DNA and sequestered in a strong, approximately 1:1 PER-CLK off-DNA complex. The data indicate that the PER levels bound to CLK change dynamically and are important for repression, first on-DNA and then off-DNA. They also suggest that these mechanisms occur upstream of post-translational events, and that elements of this two-step mechanism likely apply to mammals.

MeSH Categories: Animals, CLOCK Proteins/metabolism, Cells, Cultured, Circadian Rhythm/genetics/*physiology, DNA/*metabolism, DNA Polymerase II/metabolism, Drosophila Proteins/metabolism, Drosophila melanogaster/*genetics/*metabolism, *Gene Expression Regulation, Period Circadian Proteins/*metabolism, Promoter Regions, Genetic, Protein Binding

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The mammalian clock component PERIOD2 coordinates circadian output by interaction with nuclear receptors.

Publication Date: 2010 Feb 15 PMID: 20159955
Authors: Schmutz, I. - Ripperger, J. A. - Baeriswyl-Aebischer, S. - Albrecht, U.
Journal: Genes Dev

Mammalian circadian clocks provide a temporal framework to synchronize biological functions. To obtain robust rhythms with a periodicity of about a day, these clocks use molecular oscillators consisting of two interlocked feedback loops. The core loop generates rhythms by transcriptional repression via the Period (PER) and Cryptochrome (CRY) proteins, whereas the stabilizing loop establishes roughly antiphasic rhythms via nuclear receptors. Nuclear receptors also govern many pathways that affect metabolism and physiology. Here we show that the core loop component PER2 can coordinate circadian output with the circadian oscillator. PER2 interacts with nuclear receptors including PPARalpha and REV-ERBalpha and serves as a coregulator of nuclear receptor-mediated transcription. Consequently, PER2 is rhythmically bound at the promoters of nuclear receptor target genes in vivo. In this way, the circadian oscillator can modulate the expression of nuclear receptor target genes like Bmal1, Hnf1alpha, and Glucose-6-phosphatase. The concept that PER2 may propagate clock information to metabolic pathways via nuclear receptors adds an important facet to the clock-dependent regulation of biological networks.

MeSH Categories: ARNTL Transcription Factors/metabolism, Amino Acid Motifs, Animals, Circadian Rhythm/genetics/*physiology, *Gene Expression Regulation, Glucose/metabolism, Liver/metabolism, Mice, Mice, Knockout, NIH 3T3 Cells, Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism, PPAR alpha/metabolism, Period Circadian Proteins/genetics/*metabolism, Promoter Regions, Genetic, Protein Binding, Receptors, Cytoplasmic and Nuclear/*metabolism

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A divalent switch drives H-NS/DNA-binding conformations between stiffening and bridging modes.

Publication Date: 2010 Feb 15 PMID: 20159954
Authors: Liu, Y. - Chen, H. - Kenney, L. J. - Yan, J.
Journal: Genes Dev

Heat-stable nucleoid structuring protein (H-NS) is an abundant prokaryotic protein that plays important roles in organizing chromosomal DNA and gene silencing. Two controversial binding modes were identified. H-NS binding stimulating DNA bridging has become the accepted mechanism, whereas H-NS binding causing DNA stiffening has been largely ignored. Here, we report that both modes exist, and that changes in divalent cations drive a switch between them. The stiffening form is present under physiological conditions, and directly responds to pH and temperature in vitro. Our findings have broad implications and require a reinterpretation of the mechanism by which H-NS regulates genes.

MeSH Categories: Bacterial Proteins/*metabolism, *Cations, Divalent, DNA, Bacterial/*chemistry/*metabolism, DNA-Binding Proteins/*metabolism, *Gene Expression Regulation, Bacterial, Gene Silencing, Hydrogen-Ion Concentration, Magnesium Chloride/pharmacology, Nucleic Acid Conformation, Polymers/metabolism, Protein Binding/drug effects, Salmonella/*genetics/*metabolism, Temperature

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Essential role of Tip60-dependent recruitment of ribonucleotide reductase at DNA damage sites in DNA repair during G1 phase.

Publication Date: 2010 Feb 15 PMID: 20159953
Authors: Niida, H. - Katsuno, Y. - Sengoku, M. - Shimada, M. - Yukawa, M. - Ikura, M. - Ikura, T. - Kohno, K. - Shima, H. - Suzuki, H. - Tashiro, S. - Nakanishi, M.
Journal: Genes Dev

A balanced deoxyribonucleotide (dNTP) supply is essential for DNA repair. Here, we found that ribonucleotide reductase (RNR) subunits RRM1 and RRM2 accumulated very rapidly at damage sites. RRM1 bound physically to Tip60. Chromatin immunoprecipitation analyses of cells with an I-SceI cassette revealed that RRM1 bound to a damage site in a Tip60-dependent manner. Active RRM1 mutants lacking Tip60 binding failed to rescue an impaired DNA repair in RRM1-depleted G1-phase cells. Inhibition of RNR recruitment by an RRM1 C-terminal fragment sensitized cells to DNA damage. We propose that Tip60-dependent recruitment of RNR plays an essential role in dNTP supply for DNA repair.

MeSH Categories: Animals, DNA Damage/*physiology, G1 Phase/*physiology, Gene Knockdown Techniques, Hela Cells, Histone Acetyltransferases/genetics/*metabolism, Humans, Mice, Ribonucleotide Reductases/*metabolism

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The DMM complex prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa.

Publication Date: 2010 Mar 1 PMID: 20139222
Authors: Honda, S. - Lewis, Z. A. - Huarte, M. - Cho, L. Y. - David, L. L. - Shi, Y. - Selker, E. U.
Journal: Genes Dev

Transposable elements are common in genomes and must be controlled. Many organisms use DNA methylation to silence such selfish DNA, but the mechanisms that restrict the methylation to appropriate regions are largely unknown. We identified a JmjC domain protein in Neurospora, DNA METHYLATION MODULATOR-1 (DMM-1), that prevents aberrant spreading of DNA and histone H3K9 methylation from inactivated transposons into nearby genes. Mutation of a conserved residue within the JmjC Fe(II)-binding site abolished dmm-1 function, as did mutations in conserved cysteine-rich domains. Mutants defective only in dmm-1 mutants grow poorly, but growth is restored by reduction or elimination of DNA methylation using the drug 5-azacytosine or by mutation of the DNA methyltransferase gene dim-2. DMM-1 relies on an associated protein, DMM-2, which bears a DNA-binding motif, for localization and proper function. HP1 is required to recruit the DMM complex to the edges of methylated regions.

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The histone demethylase UTX enables RB-dependent cell fate control.

Publication Date: 2010 Feb 15 PMID: 20123895
Authors: Wang, J. K. - Tsai, M. C. - Poulin, G. - Adler, A. S. - Chen, S. - Liu, H. - Shi, Y. - Chang, H. Y.
Journal: Genes Dev

Trimethylation of histone H3 on Lys 27 (H3K27me3) is key for cell fate regulation. The H3K27me3 demethylase UTX functions in development and tumor suppression with undefined mechanisms. Here, genome-wide chromatin occupancy analysis of UTX and associated histone modifications reveals distinct classes of UTX target genes, including genes encoding Retinoblastoma (RB)-binding proteins. UTX removes H3K27me3 and maintains expression of several RB-binding proteins, enabling cell cycle arrest. Genetic interactions in mammalian cells and Caenorhabditis elegans show that UTX regulates cell fates via RB-dependent pathways. Thus, UTX defines an evolutionarily conserved mechanism to enable coordinate transcription of a RB network in cell fate control.

MeSH Categories: Animals, Caenorhabditis elegans/metabolism, Cell Differentiation/*physiology, Cell Line, Tumor, Cell Proliferation, Cells, Cultured, Chromatin/metabolism, *Gene Expression Regulation, Genome/genetics, Humans, Jumonji Domain-Containing Histone Demethylases/genetics/*metabolism, Methylation, Mice, Neoplasms/metabolism, Retinoblastoma Binding Proteins/genetics/*metabolism

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Jarid2 and PRC2, partners in regulating gene expression.

Publication Date: 2010 Feb 15 PMID: 20123894
Authors: Li, G. - Margueron, R. - Ku, M. - Chambon, P. - Bernstein, B. E. - Reinberg, D.
Journal: Genes Dev

The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing.

MeSH Categories: Animals, Cell Differentiation, Embryonic Stem Cells/cytology/metabolism, *Gene Expression Regulation, Developmental, Histone-Lysine N-Methyltransferase/metabolism, Mice, Nerve Tissue Proteins/genetics/*metabolism, Protein Binding, Repressor Proteins/*metabolism

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