Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M||WB, ICC, DB, PIA, ChIP||Rb||Affinity Purified||Polyclonal Antibody|
|Presentation||Purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at 2-8°C from date of receipt.|
|Material Size||100 µL|
References | 28 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Chd1 is essential for the high transcriptional output and rapid growth of the mouse epiblast. |
Guzman-Ayala, M; Sachs, M; Koh, FM; Onodera, C; Bulut-Karslioglu, A; Lin, CJ; Wong, P; Nitta, R; Song, JS; Ramalho-Santos, M
Development (Cambridge, England) 142 118-27 2015
The pluripotent mammalian epiblast undergoes unusually fast cell proliferation. This rapid growth is expected to generate a high transcriptional demand, but the underlying mechanisms remain unknown. We show here that the chromatin remodeler Chd1 is required for transcriptional output and development of the mouse epiblast. Chd1(-/-) embryos exhibit proliferation defects and increased apoptosis, are smaller than controls by E5.5 and fail to grow, to become patterned or to gastrulate. Removal of p53 allows progression of Chd1(-/-) mutants only to E7.0-8.0, highlighting the crucial requirement for Chd1 during early post-implantation development. Chd1(-/-) embryonic stem cells (ESCs) have a self-renewal defect and a genome-wide reduction in transcriptional output at both known mRNAs and intergenic transcripts. These transcriptional defects were only uncovered when cell number-normalized approaches were used, and correlate with a lower engagement of RNAP II with transcribed genes in Chd1(-/-) ESCs. We further show that Chd1 directly binds to ribosomal DNA, and that both Chd1(-/-) epiblast cells in vivo and ESCs in vitro express significantly lower levels of ribosomal RNA. In agreement with these findings, mutant cells in vivo and in vitro exhibit smaller and more elongated nucleoli. Thus, the RNA output by both Pol I and II is reduced in Chd1(-/-) cells. Our data indicate that Chd1 promotes a globally elevated transcriptional output required to sustain the distinctly rapid growth of the mouse epiblast.
|Dopamine signaling leads to loss of Polycomb repression and aberrant gene activation in experimental parkinsonism. |
Södersten, E; Feyder, M; Lerdrup, M; Gomes, AL; Kryh, H; Spigolon, G; Caboche, J; Fisone, G; Hansen, K
PLoS genetics 10 e1004574 2014
Polycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq) in combination with expression analysis by RNA-sequencing (RNA-seq) showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease.
|Alternative meiotic chromatid segregation in the holocentric plant Luzula elegans. |
Heckmann, S; Jankowska, M; Schubert, V; Kumke, K; Ma, W; Houben, A
Nature communications 5 4979 2014
Holocentric chromosomes occur in a number of independent eukaryotic lineages. They form holokinetic kinetochores along the entire poleward chromatid surfaces, and owing to this alternative chromosome structure, species with holocentric chromosomes cannot use the two-step loss of cohesion during meiosis typical for monocentric chromosomes. Here we show that the plant Luzula elegans maintains a holocentric chromosome architecture and behaviour throughout meiosis, and in contrast to monopolar sister centromere orientation, the unfused holokinetic sister centromeres behave as two distinct functional units during meiosis I, resulting in sister chromatid separation. Homologous non-sister chromatids remain terminally linked after metaphase I, by satellite DNA-enriched chromatin threads, until metaphase II. They then separate at anaphase II. Thus, an inverted sequence of meiotic sister chromatid segregation occurs. This alternative meiotic process is most likely one possible adaptation to handle a holocentric chromosome architecture and behaviour during meiosis.
|Genome-wide phosphoacetylation of histone H3 at Drosophila enhancers and promoters. |
Kellner, WA; Ramos, E; Van Bortle, K; Takenaka, N; Corces, VG
Genome research 22 1081-8 2012
Transcription regulation is mediated by enhancers that bind sequence-specific transcription factors, which in turn interact with the promoters of the genes they control. Here, we show that the JIL-1 kinase is present at both enhancers and promoters of ecdysone-induced Drosophila genes, where it phosphorylates the Ser10 and Ser28 residues of histone H3. JIL-1 is also required for CREB binding protein (CBP)-mediated acetylation of Lys27, a well-characterized mark of active enhancers. The presence of these proteins at enhancers and promoters of ecdysone-induced genes results in the establishment of the H3K9acS10ph and H3K27acS28ph marks at both regulatory sequences. These modifications are necessary for the recruitment of 14-3-3, a scaffolding protein capable of facilitating interactions between two simultaneously bound proteins. Chromatin conformation capture assays indicate that interaction between the enhancer and the promoter is dependent on the presence of JIL-1, 14-3-3, and CBP. Genome-wide analyses extend these conclusions to most Drosophila genes, showing that the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph is a general feature of enhancers and promoters in this organism.
|Role of anti-angiogenic factor endostatin in the pathogenesis of experimental ulcerative colitis. |
Tolstanova G, Deng X, Khomenko T, Garg P, Paunovic B, Chen L, Sitaraman SV, Shiloach J, Szabo S, Sandor Z.
Life sciences 88 74-81 2011
Vascular endothelial growth factor (VEGF) and pathologic angiogenesis have been demonstrated to play a pathogenic role in the development and progression of inflammatory bowel disease. Thus, we hypothesized that the potent anti-angiogenic factor endostatin might play a beneficial role in experimental ulcerative colitis (UC).
|Calpain-1 cleaves Rad21 to promote sister chromatid separation. |
Panigrahi, AK; Zhang, N; Mao, Q; Pati, D
Molecular and cellular biology 31 4335-47 2011
Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans.
|Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells. |
Stojic, L; Jasencakova, Z; Prezioso, C; Stützer, A; Bodega, B; Pasini, D; Klingberg, R; Mozzetta, C; Margueron, R; Puri, PL; Schwarzer, D; Helin, K; Fischle, W; Orlando, V
Epigenetics & chromatin 4 16 2011
Polycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation.We show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation.Our data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.
|PcG complexes set the stage for epigenetic inheritance of gene silencing in early S phase before replication. |
Lanzuolo, C; Lo Sardo, F; Diamantini, A; Orlando, V
PLoS genetics 7 e1002370 2011
Polycomb group (PcG) proteins are part of a conserved cell memory system that conveys epigenetic inheritance of silenced transcriptional states through cell division. Despite the considerable amount of information about PcG mechanisms controlling gene silencing, how PcG proteins maintain repressive chromatin during epigenome duplication is still unclear. Here we identified a specific time window, the early S phase, in which PcG proteins are recruited at BX-C PRE target sites in concomitance with H3K27me3 repressive mark deposition. Notably, these events precede and are uncoupled from PRE replication timing, which occurs in late S phase when most epigenetic signatures are reduced. These findings shed light on one of the key mechanisms for PcG-mediated epigenetic inheritance during S phase, suggesting a conserved model in which the PcG-dependent H3K27me3 mark is inherited by dilution and not by de novo methylation occurring at the time of replication.
|Coe genes are expressed in differentiating neurons in the central nervous system of protostomes. |
Demilly, A; Simionato, E; Ohayon, D; Kerner, P; Garcès, A; Vervoort, M
PloS one 6 e21213 2011
Genes of the coe (collier/olfactory/early B-cell factor) family encode Helix-Loop-Helix transcription factors that are widely conserved in metazoans and involved in many developmental processes, neurogenesis in particular. Whereas their functions during vertebrate neural tube formation have been well documented, very little is known about their expression and role during central nervous system (CNS) development in protostomes. Here we characterized the CNS expression of coe genes in the insect Drosophila melanogaster and the polychaete annelid Platynereis dumerilii, which belong to different subgroups of protostomes and show strikingly different modes of development. In the Drosophila ventral nerve cord, we found that the Collier-expressing cells form a subpopulation of interneurons with diverse molecular identities and neurotransmitter phenotypes. We also demonstrate that collier is required for the proper differentiation of some interneurons belonging to the Eve-Lateral cluster. In Platynereis dumerilii, we cloned a single coe gene, Pdu-coe, and found that it is exclusively expressed in post mitotic neural cells. Using an original technique of in silico 3D registration, we show that Pdu-coe is co-expressed with many different neuronal markers and therefore that, like in Drosophila, its expression defines a heterogeneous population of neurons with diverse molecular identities. Our detailed characterization and comparison of coe gene expression in the CNS of two distantly-related protostomes suggest conserved roles of coe genes in neuronal differentiation in this clade. As similar roles have also been observed in vertebrates, this function was probably already established in the last common ancestor of all bilaterians.
|Epigenetic changes in the brain: measuring global histone modifications. |
Rumbaugh G, Miller CA
Methods Mol Biol 670 263-74. 2011
Recent years have witnessed an explosion of research on the role of epigenetic modifications, such as DNA methylation and histone protein acetylation and phosphorylation, in neuroscience. These changes exert control over gene expression and have been shown to play important roles in a variety of neural processes, including learning and memory. We and others have also recently shown that epigenetic changes may contribute to neurodegenerative disorders, such as Alzheimer's disease. Western blot analysis with antibodies raised against specific histone modifications is a relatively simple technique able to reveal the type, location, and degree of histone posttranslational modifications produced by an experimental manipulation. Here we provide a step-by-step protocol for isolating histone proteins from tissue and measuring these posttranslational modifications.
|Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina. |
E C Lelièvre,M Lek,H Boije,L Houille-Vernes,V Brajeul,A Slembrouck,J E Roger,J A Sahel,J M Matter,F Sennlaub,F Hallböök,O Goureau,X Guillonneau
Developmental biology 358 2011
During development, progenitor cells of the retina give rise to six principal classes of neurons and the Müller glial cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-helix-loop-helix transcription factor necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Both GABAergic H1 and non-GABAergic H3 horizontal cells were induced following the forced expression of Ptf1a. We describe Ptf1a as a strong, negative regulator of Atoh7 expression. Furthermore, the Rbpj-interacting domains of Ptf1a protein were required for its effects on cell fate specification. Together, these data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina.
|Histone H3 Phosphorylation (S10, S28) and Phosphoacetylation (K9S10) are Differentially Associated With Gene Expression In Liver of Rats Treated In Vivo With Acute Ethanol. |
James TT, Aroor AR, Lim RW, Shukla SD
The Journal of pharmacology and experimental therapeutics 2011
The epigenetic histone modification by ethanol is emerging as one of the mechanisms for its deleterious effects in the liver. In this context, we have investigated the role of histone H3 phosphorylation at serine-10 (P-H3-S10), and serine 28 (P-H3-S28) in liver after acute ethanol treatment in vivo. Ethanol was administered intraperitoneally (IP) in male Sprague-Dawley rats. Ethanol dose response (1-5g/ Kg body weight) and time course (1-4 h) experiments were conducted and various parameters were monitored. Steatosis, and necrosis (serum ALT) of the liver increased in 4h suggesting liver injury. There were differences between P-H3-S10 and P-H3-S28 at 1h, the latter was more sensitive to lower ethanol doses. Interestingly, phosphorylation of both serines disappeared at the highest dose used (5g/Kg). We also examined phosphoacetylation of histone H3 at K9/S10 and observed a dramatic increase. The changes in histone H3 phosphorylation, and phosphoacetylation were also accompanied with expression of early response genes (c-Fos, c-Jun, MKP-1). Chromatin immunoprecipitation (ChIP) assays in samples from 1.5h and 4h of ethanol administration indicated that increased histone H3 phosphorylation at serine 28 was associated with the promoters of c-Jun and PAI-1. In conclusion, this study demonstrates for the first time that in vivo exposure of liver to acute ethanol induced phosphorylation, and phosphoacetylation of histone H3, and these modifications are differentially involved in the mRNA expression of genes.
|Coordination of cell proliferation and anterior-posterior axis establishment in the mouse embryo. |
Stuckey, DW; Clements, M; Di-Gregorio, A; Senner, CE; Le Tissier, P; Srinivas, S; Rodriguez, TA
Development (Cambridge, England) 138 1521-30 2011
During development, the growth of the embryo must be coupled to its patterning to ensure correct and timely morphogenesis. In the mouse embryo, migration of the anterior visceral endoderm (AVE) to the prospective anterior establishes the anterior-posterior (A-P) axis. By analysing the distribution of cells in S phase, M phase and G2 from the time just prior to the migration of the AVE until 18 hours after its movement, we show that there is no evidence for differential proliferation along the A-P axis of the mouse embryo. Rather, we have identified that as AVE movements are being initiated, the epiblast proliferates at a much higher rate than the visceral endoderm. We show that these high levels of proliferation in the epiblast are dependent on Nodal signalling and are required for A-P establishment, as blocking cell division in the epiblast inhibits AVE migration. Interestingly, inhibition of migration by blocking proliferation can be rescued by Dkk1. This suggests that the high levels of epiblast proliferation function to move the prospective AVE away from signals that are inhibitory to its migration. The finding that initiation of AVE movements requires a certain level of proliferation in the epiblast provides a mechanism whereby A-P axis development is coordinated with embryonic growth.
|PP1/Repo-Man Dephosphorylates Mitotic Histone H3 at T3 and Regulates Chromosomal Aurora B Targeting. |
Qian, Junbin, et al.
Curr. Biol., 21: 766-73 (2011) 2011
The transient mitotic histone H3 phosphorylation by various protein kinases regulates chromosome condensation and segregation, but the counteracting phosphatases have been poorly characterized [1-8]. We show here that PP1γ is the major histone H3 phosphatase acting on the mitotically phosphorylated (ph) residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify Repo-Man, a chromosome-bound interactor of PP1γ , as a selective regulator of H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph dephosphorylation by an indirect mechanism but directly and specifically targets H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the end of mitosis. Consistent with these findings, Repo-Man modulates in a PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase B and its substrate MCAK. Our study defines a novel mechanism by which PP1 counteracts Aurora B.
|Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex. |
Drobic, B; Pérez-Cadahía, B; Yu, J; Kung, SK; Davie, JR
Nucleic acids research 38 3196-208 2010
Upon activation of the ERK and p38 MAPK pathways, the MSK1/2-mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early (IE) gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and sequential chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 the ATPase subunit of the SWI/SNF remodeler, is recruited to the promoter of target genes by transcription factors such as Elk-1 or NF-kappaB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of IE genes enabling the binding of transcription factors like JUN and the onset of transcription.
|Frequent mitotic activity in banal melanocytic nevi uncovered by immunohistochemical analysis. |
Katharina Glatz,Christoph Hartmann,Milos Antic,Heinz Kutzner
The American Journal of dermatopathology 32 2010
The presence and distribution of mitotic figures is an important discriminatory parameter in the assessment of melanocytic lesions. We evaluated the number and distribution of mitotic figures in 353 randomly collected melanocytic nevi of various subtypes by hematoxylin and eosin (H&E) staining and immunohistochemically with the 2 mitotic markers Phospho-Histone H3 Ser28 (PHH3) and MPM2. At least 1 mitotic figure was present in 19.5%, 31.3%, and 42.8% of H&E-, PHH3-, and MPM2-stained lesions, respectively. In common compound nevi, the mean number of dermal mitoses amounted to 0.024/mm dermal surface area in the H&E staining (PHH3: 0.061; MPM2: 0.087) and to 0.175/mm in Spitz nevi (PHH3: 0.325; MPM2: 0.45). Nevi exhibiting mitotic figures were significantly more frequent in the youngest age group (0-20 years) than in patients older than 50 years (P < 0.0001). In the upper half of the dermis, mitotic activity was roughly 3 times as frequent as compared with the lower half. Clusters of mitotic figures within the dermis were not observed. Mitotic activity in obviously benign melanocytic nevi is not rare even in the deep dermal part. More than 2 mitotic figures per lesion can usually be explained either by the nevus subtype, young patient age, traumatization, or inflammation. PHH3 and MPM2 are a valuable diagnostic adjunct in the evaluation of melanocytic tumors allowing more sensitive and faster recognition of mitotic figures and their distribution.
|The human CDK8 subcomplex is a histone kinase that requires Med12 for activity and can function independently of mediator. |
Knuesel, MT; Meyer, KD; Donner, AJ; Espinosa, JM; Taatjes, DJ
Molecular and cellular biology 29 650-61 2009
The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable "CDK8 subcomplex" in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12--but not Med13--to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex--unlike the recombinant submodule--is unable to phosphorylate the Pol II CTD.Full Text Article
|BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity. |
Rudolph, D; Steegmaier, M; Hoffmann, M; Grauert, M; Baum, A; Quant, J; Haslinger, C; Garin-Chesa, P; Adolf, GR
Clinical cancer research : an official journal of the American Association for Cancer Research 15 3094-102 2009
Antimitotic chemotherapy remains a cornerstone of multimodality treatment for locally advanced and metastatic cancers. To identify novel mitosis-specific agents with higher selectivity than approved tubulin-binding agents (taxanes, Vinca alkaloids), we have generated inhibitors of Polo-like kinase 1, a target that functions predominantly in mitosis.The first compound in this series, suitable for i.v. administration, has entered clinical development. To fully explore the potential of Polo-like kinase 1 inhibition in oncology, we have profiled additional compounds and now describe a novel clinical candidate.BI 6727 is a highly potent (enzyme IC(50) = 0.87 nmol/L, EC(50) = 11-37 nmol/L on a panel of cancer cell lines) and selective dihydropteridinone with distinct properties. First, BI 6727 has a pharmacokinetic profile favoring sustained exposure of tumor tissues with a high volume of distribution and a long terminal half-life in mice (V(ss) = 7.6 L/kg, t(1/2) = 46 h) and rats (V(ss) = 22 L/kg, t(1/2) = 54 h). Second, BI 6727 has physicochemical and pharmacokinetic properties that allow in vivo testing of i.v. as well as oral formulations, adding flexibility to dosing schedules. Finally, BI 6727 shows marked antitumor activity in multiple cancer models, including a model of taxane-resistant colorectal cancer. With oral and i.v. routes of administration, the total weekly dose of BI 6727 is most relevant for efficacy, supporting the use of a variety of well-tolerated dosing schedules.These findings warrant further investigation of BI 6727 as a tailored antimitotic agent; clinical studies have been initiated.
|Global transcription in pluripotent embryonic stem cells. |
Efroni, S; Duttagupta, R; Cheng, J; Dehghani, H; Hoeppner, DJ; Dash, C; Bazett-Jones, DP; Le Grice, S; McKay, RD; Buetow, KH; Gingeras, TR; Misteli, T; Meshorer, E
Cell stem cell 2 437-47 2008
The molecular mechanisms underlying pluripotency and lineage specification from embryonic stem cells (ESCs) are largely unclear. Differentiation pathways may be determined by the targeted activation of lineage-specific genes or by selective silencing of genome regions. Here we show that the ESC genome is transcriptionally globally hyperactive and undergoes large-scale silencing as cells differentiate. Normally silent repeat regions are active in ESCs, and tissue-specific genes are sporadically expressed at low levels. Whole-genome tiling arrays demonstrate widespread transcription in coding and noncoding regions in ESCs, whereas the transcriptional landscape becomes more discrete as differentiation proceeds. The transcriptional hyperactivity in ESCs is accompanied by disproportionate expression of chromatin-remodeling genes and the general transcription machinery. We propose that global transcription is a hallmark of pluripotent ESCs, contributing to their plasticity, and that lineage specification is driven by reduction of the transcribed portion of the genome.
|Cyclin A1-deficient mice lack histone H3 serine 10 phosphorylation and exhibit altered aurora B dynamics in late prophase of male meiosis. |
Nickerson, HD; Joshi, A; Wolgemuth, DJ
Developmental biology 306 725-35 2007
Male mice lacking cyclin A1 protein are sterile. Their sterility results from an arrest in the meiotic cell cycle of spermatocytes, which we now identify as occurring at late diplotene, immediately before diakinesis. The stage of arrest in cyclin A1-deficient mice is distinct from the arrest seen in spermatocytes that are deficient in its putative catalytic partner Cdk2, which occurs much earlier in pachytene. The arrest in cyclin A1-deficient spermatocytes is also accompanied by an unusual clustering of centromeric heterochromatin. Consistent with a possible defect in the centromeric region, immunofluorescent staining of cyclin A1 protein shows localization in the region of the centromere. Phosphorylation of histone H3 at serine 10 in pericentromeric heterochromatin, which normally occurs in late diplotene, is reduced in spermatocytes from heterozygous Ccna1(+/-) testes and completely absent in spermatocytes with no cyclin A1 protein. Concomitantly, the levels of pericentromeric aurora B kinase, known to phosphorylate histone H3 during meiosis, are partially reduced in spermatocytes from testes of heterozygous mice and further reduced in homozygous null spermatocytes. These data suggest a critical and concentration-dependent function for cyclin A1 in the pericentromeric region in late diplotene of meiosis, perhaps in assembly or function of the passenger protein complex.
|Role of apoptosis in low-dose hyper-radiosensitivity. |
S A Krueger,M C Joiner,M Weinfeld,E Piasentin,B Marples
Radiation research 167 2007
Little is known about the mode of cell killing associated with low-dose hyper-radiosensitivity, the radiation response that describes the enhanced sensitivity of cells to small doses of ionizing radiation. Using a technique that measures the activation of caspase 3, we have established a relationship between apoptosis detected 24 h after low-dose radiation exposure and low-dose hyper-radiosensitivity in four mammalian cell lines (T98G, U373, MR4 and 3.7 cells) and two normal human lymphoblastoid cell lines. The existence of low-dose hyper-radiosensitivity in clonogenic survival experiments was found to be associated with an elevated level of apoptosis after low-dose exposures, corroborating earlier observations (Enns et al., Mol. Cancer Res. 2, 557-566, 2004). We also show that enriching populations of MR4 and V79 cells with G(1)-phase cells, to minimize the numbers of G(2)-phase cells, abolished the enhanced low-dose apoptosis. These cell-cycle enrichment experiments strengthen the reported association between low-dose hyper-sensitivity and the radioresponse of G(2)-phase cells. These data are consistent with our current hypothesis to explain low-dose hyper-radiosensitivity, namely that the enhanced sensitivity of cells to low doses of ionizing radiation reflects the failure of ATM-dependent repair processes to fully arrest the progression of damaged G(2)-phase cells harboring unrepaired DNA breaks entering mitosis.
|Progression from mitotic catastrophe to germ cell death in Caenorhabditis elegans lis-1 mutants requires the spindle checkpoint. |
Edgar A Buttner, Aleksandra J Gil-Krzewska, Anandita K Rajpurohit, Craig P Hunter
Developmental biology 305 397-410 2007
Deletion of the lissencephaly disease gene LIS-1 in humans causes an extreme disorganization of the brain associated with significant reduction in cortical neurons. Here we show that deletion or RNA interference (RNAi) of Caenorhabditis elegans lis-1 results in a reduction in germline nuclei and causes a variety of cellular, developmental, and neurological defects throughout development. Our analysis of the germline defects suggests that the reduction in nuclei number stems from dysfunctional mitotic spindles resulting in cell cycle arrest and eventually programmed cell death (apoptosis). Deletion of the spindle checkpoint gene mdf-1 blocks lis-1(lf)-induced cell cycle arrest and germline apoptosis, placing the spindle checkpoint pathway upstream of the programmed cell death pathway. These results suggest that apoptosis may contribute to the cell-sparse pathology of lissencephaly.Full Text Article
|Asymmetry in histone H3 variants and lysine methylation between paternal and maternal chromatin of the early mouse zygote. |
van der Heijden, GW; Dieker, JW; Derijck, AA; Muller, S; Berden, JH; Braat, DD; van der Vlag, J; de Boer, P
Mechanisms of development 122 1008-22 2005
In mammalian fertilization, the paternal genome is delivered to the secondary oocyte by sperm with protamine compacted DNA, while the maternal genome is arrested in meiotic metaphase II. Thus, at the beginning of fertilization, the two gametic chromatin sets are strikingly different. We elaborate on this contrast by reporting asymmetry for histone H3 type in the pre-S-phase zygote when male chromatin is virtually devoid of histone H3.1/3.2. Localization of the histone H3.3/H4 assembly factor Hira with the paternal chromatin indicates the presence of histone H3.3. In conjunction with this, we performed a systematic immunofluorescence analysis of histone N-tail methylations at position H3K4, H3K9, H3K27 and H4K20 up to the young pronucleus stage and show that asymmetries reported earlier are systematic for virtually all di- and tri-methylations but not for mono-methylation of H3K4 and H4K20, the only marks studied present in the early male pronucleus. For H4K20 the expanding male chromatin is rapidly mono-methylated. This coincides with the formation of maternally derived nucleosomes, a process which is observed as early as sperm chromatin decondensation occurs. Absence of tri-methylated H3K9, tri-methylated H4K20 and presence of loosely anchored HP1-beta combined with the homogenous presence of mono-methylated H4K20 suggests the absence of a division of the paternal chromatin in eu- and heterochromatin. In summary the male, in contrast to female G1 chromatin, is uniform and contains predominantly histone H3.3 as histone H3 variant.
|G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex. |
Lukaszewicz, A; Savatier, P; Cortay, V; Giroud, P; Huissoud, C; Berland, M; Kennedy, H; Dehay, C
Neuron 47 353-64 2005
We have investigated the cell cycle-related mechanisms that lead to the emergence of primate areas 17 and 18. These areas are characterized by striking differences in cytoarchitectonics and neuron number. We show in vivo that (1) area 17 precursors of supragranular neurons exhibit a shorter cell cycle duration, a reduced G1 phase, and a higher rate of cell cycle reentry than area 18 precursors; (2) area 17 and area 18 precursors show contrasting and specific levels of expression of cyclin E (high in area 17, low in area 18) and p27Kip1 (low in area 17, high in area 18); (3) ex vivo up- and downmodulation of cyclin E and p27Kip1 show that both regulators influence cell cycle kinetics by modifying rates of cell cycle progression and cell cycle reentry; (4) modeling the areal differences in cell cycle parameters suggests that they contribute to areal differences in numbers of precursors and neuron production.
|Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice. |
Lin, Y; Page, DC
Developmental biology 288 309-16 2005
Genes of the DAZ family play critical roles in germ cell development in mammals and other animals. In mice, Dazl mRNA is first observed at embryonic day 11.5 (E11.5), but previous studies using Dazl-deficient mice of mixed genetic background have largely emphasized postnatal spermatogenic defects. Using an inbred C57BL/6 background, we show that Dazl is required for embryonic development and survival of XY germ cells. By E14.5, expression of germ cell markers (Mvh, Oct4, Dppa3/Stella, GCNA and MVH protein) was reduced in XY Dazl-/- gonads. By E15.5, most remaining germ cells in XY Dazl-/- embryos exhibited apoptotic morphology, and XY Dazl-/- gonads contained increased numbers of TUNEL-positive cells. The rare XY Dazl-/- germ cells that persisted until birth maintained a nuclear morphology that resembled that of wildtype germ cells at E12.5-E13.5, a critical developmental period when XY germ cells lose pluripotency and commit to a spermatogonial fate. We propose that Dazl is required as early as E12.5-E13.5, shortly after its expression is first detected, and that inbred Dazl-/- mice of C57BL/6 background provide a reproducible standard for exploring Dazl's roles in embryonic germ cell development.
|Topoisomerase inhibitor induced dephosphorylation of H1 and H3 histones as a consequence of cell cycle arrest. |
Nicole Happel, Arne Sommer, Kristina Hänecke, Werner Albig, Detlef Doenecke
Journal of cellular biochemistry 95 1235-47 2005
Posttranslational modifications of histones have an integral function in the structural and functional organization of chromatin. Several changes in the modification state of histones could be observed after induction of apoptosis with topoisomerase inhibitors and other inducers. Most of these studies include the analysis of the state of phosphorylation of histones, and the results are to some extent controversial, depending on cell lines and agents used. In the present study we compared the kinetics of the dephosphorylation of H1 and H3 histones with apoptosis markers after treatment of leukemic cell lines with topoisomerase inhibitors. In parallel, we determined cell cycle parameters in detail. Dephosphorylation of both histone classes started within 1 h of induction, and no direct correlation with timing and intensity of the investigated apoptotic features could be observed. In contrast, we show that the effect of topoisomerase inhibitors on the state of H1 and H3 phosphorylation is not directly related to apoptosis, but reflects the changes in the cell cycle distribution of cells treated with these inducers.
|Barrier-to-Autointegration Factor influences specific histone modifications. |
Montes de Oca, R; Andreassen, PR; Wilson, KL
Nucleus (Austin, Tex.) 2 580-90 2001
Defects in the nuclear envelope or nuclear 'lamina' networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.
|Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation. |
Goto, H, et al.
J. Biol. Chem., 274: 25543-9 (1999) 1999
Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.