Publications by authors named "Charles R Bradshaw"

21 Publications

  • Page 1 of 1

Epigenetic homogeneity in histone methylation underlies sperm programming for embryonic transcription.

Nat Commun 2020 07 13;11(1):3491. Epub 2020 Jul 13.

Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.

Sperm contributes genetic and epigenetic information to the embryo to efficiently support development. However, the mechanism underlying such developmental competence remains elusive. Here, we investigated whether all sperm cells have a common epigenetic configuration that primes transcriptional program for embryonic development. Using calibrated ChIP-seq, we show that remodelling of histones during spermiogenesis results in the retention of methylated histone H3 at the same genomic location in most sperm cell. This homogeneously methylated fraction of histone H3 in the sperm genome is maintained during early embryonic replication. Such methylated histone fraction resisting post-fertilisation reprogramming marks developmental genes whose expression is perturbed upon experimental reduction of histone methylation. A similar homogeneously methylated histone H3 fraction is detected in human sperm. Altogether, we uncover a conserved mechanism of paternal epigenetic information transmission to the embryo through the homogeneous retention of methylated histone in a sperm cells population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-17238-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359334PMC
July 2020

A direct role for SNX9 in the biogenesis of filopodia.

J Cell Biol 2020 04;219(4)

Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201909178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147113PMC
April 2020

Characteristics and homogeneity of N6-methylation in human genomes.

Sci Rep 2019 03 26;9(1):5185. Epub 2019 Mar 26.

Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, CB2 1QN, UK.

A novel DNA modification, N-6 methylated deoxyadenosine (m6dA), has recently been discovered in eukaryotic genomes. Despite its low abundance in eukaryotes, m6dA is implicated in human diseases such as cancer. It is therefore important to precisely identify and characterize m6dA in the human genome. Here, we identify m6dA sites at nucleotide level, in different human cells, genome wide. We compare m6dA features between distinct human cells and identify m6dA characteristics in human genomes. Our data demonstrates for the first time that despite low m6dA abundance, the m6dA mark does often occur consistently at the same genomic location within a given human cell type, demonstrating m6dA homogeneity. We further show, for the first time, higher levels of m6dA homogeneity within one chromosome. Most m6dA are found on a single chromosome from a diploid sample, suggesting inheritance. Our transcriptome analysis not only indicates that human genes with m6dA are associated with higher RNA transcript levels but identifies allele-specific gene transcripts showing haplotype-specific m6dA methylation, which are implicated in different biological functions. Our analyses demonstrate the precision and consistency by which the m6dA mark occurs within the human genome, suggesting that m6dA marks are precisely inherited in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-41601-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435722PMC
March 2019

Chromatin Accessibility Impacts Transcriptional Reprogramming in Oocytes.

Cell Rep 2018 07;24(2):304-311

Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, United Kingdom. Electronic address:

Oocytes have a remarkable ability to reactivate silenced genes in somatic cells. However, it is not clear how the chromatin architecture of somatic cells affects this transcriptional reprogramming. Here, we investigated the relationship between the chromatin opening and transcriptional activation. We reveal changes in chromatin accessibility and their relevance to transcriptional reprogramming after transplantation of somatic nuclei into Xenopus oocytes. Genes that are silenced, but have pre-existing open transcription start sites in donor cells, are prone to be activated after nuclear transfer, suggesting that the chromatin signature of somatic nuclei influences transcriptional reprogramming. There are also activated genes associated with new open chromatin sites, and transcription factors in oocytes play an important role in transcriptional reprogramming from such genes. Finally, we show that genes resistant to reprogramming are associated with closed chromatin configurations. We conclude that chromatin accessibility is a central factor for successful transcriptional reprogramming in oocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.06.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057489PMC
July 2018

Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.

Nat Med 2017 Dec 13;23(12):1424-1435. Epub 2017 Nov 13.

The Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, UK.

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm.4438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722201PMC
December 2017

Reprogramming towards totipotency is greatly facilitated by synergistic effects of small molecules.

Biol Open 2017 Apr 15;6(4):415-424. Epub 2017 Apr 15.

Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/bio.023473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399555PMC
April 2017

H3K4 Methylation-Dependent Memory of Somatic Cell Identity Inhibits Reprogramming and Development of Nuclear Transfer Embryos.

Cell Stem Cell 2017 07 30;21(1):135-143.e6. Epub 2017 Mar 30.

Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK. Electronic address:

Vertebrate eggs can induce the nuclear reprogramming of somatic cells to enable production of cloned animals. Nuclear reprogramming is relatively inefficient, and the development of the resultant embryos is frequently compromised, in part due to the inappropriate expression of genes previously active in the donor nucleus. Here, we identify H3K4 methylation as a major epigenetic roadblock that limits transcriptional reprogramming and efficient nuclear transfer (NT). Widespread expression of donor-cell-specific genes was observed in inappropriate cell types in NT embryos, limiting their developmental capacity. The expression of these genes in reprogrammed embryos arises from epigenetic memories of a previously active transcriptional state in donor cells that is characterized by high H3K4 methylation. Reducing H3K4 methylation had little effect on gene expression in donor cells, but it substantially improved transcriptional reprogramming and development of NT embryos. These results show that H3K4 methylation imposes a barrier to efficient nuclear reprogramming and suggest approaches for improving reprogramming strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2017.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505866PMC
July 2017

Identification of Methylated Deoxyadenosines in Genomic DNA by dA DNA Immunoprecipitation.

Bio Protoc 2016 Nov;6(21)

Medical Research Council Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge, UK.

dA DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N-methyldeoxyadenosine (dA). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol , 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol , 2015). This protocol describes the method to perform dA DNA immunoprecipitation (DIP), as was applied to characterize the first dA methylome analysis in higher eukaryotes (Koziol , 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA is pulled down with an antibody that recognizes dA in genomic DNA. After subsequent washes, DNA fragments that do not contain dA are eliminated, and the dA containing fragments are eluted from the antibody in order to be processed further for subsequent analyses.

Background: This protocol was developed in order to identify regions in the genome that contain dA. It can be used to detect dA in different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissini 2013). We developed this protocol and adapted it for the detection of dA in DNA, rather than detecting adenosine methylation RNA. This was required, as no protocol was available at that time to allow the genome-wide identification of dA in eukaryotic DNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.1990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293167PMC
November 2016

Mechanical cell competition kills cells via induction of lethal p53 levels.

Nat Commun 2016 04 25;7:11373. Epub 2016 Apr 25.

The Wellcome Trust/Cancer Research UK Gurdon Institute and Zoology Department, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

Cell competition is a quality control mechanism that eliminates unfit cells. How cells compete is poorly understood, but it is generally accepted that molecular exchange between cells signals elimination of unfit cells. Here we report an orthogonal mechanism of cell competition, whereby cells compete through mechanical insults. We show that MDCK cells silenced for the polarity gene scribble (scrib(KD)) are hypersensitive to compaction, that interaction with wild-type cells causes their compaction and that crowding is sufficient for scrib(KD) cell elimination. Importantly, we show that elevation of the tumour suppressor p53 is necessary and sufficient for crowding hypersensitivity. Compaction, via activation of Rho-associated kinase (ROCK) and the stress kinase p38, leads to further p53 elevation, causing cell death. Thus, in addition to molecules, cells use mechanical means to compete. Given the involvement of p53, compaction hypersensitivity may be widespread among damaged cells and offers an additional route to eliminate unfit cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms11373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848481PMC
April 2016

Sperm is epigenetically programmed to regulate gene transcription in embryos.

Genome Res 2016 08 31;26(8):1034-46. Epub 2016 Mar 31.

Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, CB2 1QN, United Kingdom; Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, United Kingdom;

For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.201541.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971762PMC
August 2016

Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications.

Nat Struct Mol Biol 2016 Jan 21;23(1):24-30. Epub 2015 Dec 21.

Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge.

Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m(5)dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N(6)-methyldeoxyadenosine (m(6)dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m(6)dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.3145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941928PMC
January 2016

Histone H3 lysine 9 trimethylation is required for suppressing the expression of an embryonically activated retrotransposon in Xenopus laevis.

Sci Rep 2015 Sep 21;5:14236. Epub 2015 Sep 21.

Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

Transposable elements in the genome are generally silenced in differentiated somatic cells. However, increasing evidence indicates that some of them are actively transcribed in early embryos and the proper regulation of retrotransposon expression is essential for normal development. Although their developmentally regulated expression has been shown, the mechanisms controlling retrotransposon expression in early embryos are still not well understood. Here, we observe a dynamic expression pattern of retrotransposons with three out of ten examined retrotransposons (1a11, λ-olt 2-1 and xretpos(L)) being transcribed solely during early embryonic development. We also identified a transcript that contains the long terminal repeat (LTR) of λ-olt 2-1 and shows a similar expression pattern to λ-olt 2-1 in early Xenopus embryos. All three retrotransposons are transcribed by RNA polymerase II. Although their expression levels decline during development, the LTRs are marked by histone H3 lysine 4 trimethylation. Furthermore, retrotransposons, especially λ-olt 2-1, are enriched with histone H3 lysine 9 trimethylation (H3K9me3) when their expression is repressed. Overexpression of lysine-specific demethylase 4d removes H3K9me3 marks from Xenopus embryos and inhibits the repression of λ-olt 2-1 after gastrulation. Thus, our study shows that H3K9me3 is important for silencing the developmentally regulated retrotransposon in Xenopus laevis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep14236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585706PMC
September 2015

A Unique Gene Regulatory Network Resets the Human Germline Epigenome for Development.

Cell 2015 Jun;161(6):1453-67

Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK. Electronic address:

Resetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5-7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2015.04.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459712PMC
June 2015

Hierarchical molecular events driven by oocyte-specific factors lead to rapid and extensive reprogramming.

Mol Cell 2014 Aug 24;55(4):524-36. Epub 2014 Jul 24.

Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, UK; Department of Zoology, University of Cambridge, Cambridge CB2 1QN, UK. Electronic address:

Nuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner. We demonstrate that an extensive exchange of somatic- for oocyte-specific factors mediates reprogramming and leads to robust oocyte RNA polymerase II binding and phosphorylation on transplanted chromatin. Moreover, genome-wide binding of oocyte-specific linker histone B4 supports its role in transcriptional reprogramming. Thus, our study reveals the rapid, abundant, and stepwise loading of oocyte-specific factors onto somatic chromatin as important determinants for successful reprogramming.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2014.06.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156308PMC
August 2014

CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans.

J Cell Biol 2014 Feb;204(4):507-22

Wellcome Trust/Cancer Research UK Gurdon Institute, 2 Department of Genetics, and 3 Department of Zoology, University of Cambridge, Cambridge CB2 1QN, England, UK.

Cyclin-dependent kinase (CDK) plays a vital role in proliferation control across eukaryotes. Despite this, how CDK mediates cell cycle and developmental transitions in metazoa is poorly understood. In this paper, we identify orthologues of Sld2, a CDK target that is important for DNA replication in yeast, and characterize SLD-2 in the nematode worm Caenorhabditis elegans. We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos. SLD-2 is a CDK target in vivo, and phosphorylation regulates the interaction with another replication factor, MUS-101. By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans. Finally, using a phosphomimicking sld-2 mutant, we demonstrate that timely CDK phosphorylation of SLD-2 is an important control mechanism to allow normal proliferation in the germline. These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201310083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926958PMC
February 2014

Nuclear Wave1 is required for reprogramming transcription in oocytes and for normal development.

Science 2013 Aug;341(6149):1002-5

Wellcome Trust/Cancer Research UK Gurdon Institute, The Henry Wellcome Building of Cancer and Developmental Biology, Cambridge, UK.

Eggs and oocytes have a remarkable ability to induce transcription of sperm after normal fertilization and in somatic nuclei after somatic cell nuclear transfer. This ability of eggs and oocytes is essential for normal development. Nuclear actin and actin-binding proteins have been shown to contribute to transcription, although their mode of action is elusive. Here, we find that Xenopus Wave1, previously characterized as a protein involved in actin cytoskeleton organization, is present in the oocyte nucleus and is required for efficient transcriptional reprogramming. Moreover, Wave1 knockdown in embryos results in abnormal development and defective hox gene activation. Nuclear Wave1 binds by its WHD domain to active transcription components, and this binding contributes to the action of RNA polymerase II. We identify Wave1 as a maternal reprogramming factor that also has a necessary role in gene activation in development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1240376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3824084PMC
August 2013

Titration of four replication factors is essential for the Xenopus laevis midblastula transition.

Science 2013 Aug 1;341(6148):893-6. Epub 2013 Aug 1.

Wellcome Trust/Cancer Research UK Gurdon Institute, The Henry Wellcome Building of Cancer and Developmental Biology, University of Cambridge, Cambridge CB2 1QN, UK.

The rapid, reductive early divisions of many metazoan embryos are followed by the midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors--Cut5, RecQ4, Treslin, and Drf1--are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1241530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898016PMC
August 2013

Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and apicobasal cell polarity.

Nat Cell Biol 2012 May 27;14(7):666-76. Epub 2012 May 27.

Max Planck Institute of Molecular Cell Biology and Genetics, MPI-CBG, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PAR proteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncb2508DOI Listing
May 2012

Small-molecule-induced DNA damage identifies alternative DNA structures in human genes.

Nat Chem Biol 2012 Feb 5;8(3):301-10. Epub 2012 Feb 5.

Department of Chemistry, University of Cambridge, Cambridge, UK.

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433707PMC
February 2012

Replication stress induces 53BP1-containing OPT domains in G1 cells.

J Cell Biol 2011 Apr 28;193(1):97-108. Epub 2011 Mar 28.

The Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, England, UK.

Chromosomal deletions and rearrangements in tumors are often associated with common fragile sites, which are specific genomic loci prone to gaps and breaks in metaphase chromosomes. Common fragile sites appear to arise through incomplete DNA replication because they are induced after partial replication inhibition by agents such as aphidicolin. Here, we show that in G1 cells, large nuclear bodies arise that contain p53 binding protein 1 (53BP1), phosphorylated H2AX (γH2AX), and mediator of DNA damage checkpoint 1 (MDC1), as well as components of previously characterized OPT (Oct-1, PTF, transcription) domains. Notably, we find that incubating cells with low aphidicolin doses increases the incidence and number of 53BP1-OPT domains in G1 cells, and by chromatin immunoprecipitation and massively parallel sequencing analysis of γH2AX, we demonstrate that OPT domains are enriched at common fragile sites. These findings invoke a model wherein incomplete DNA synthesis during S phase leads to a DNA damage response and formation of 53BP1-OPT domains in the subsequent G1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201011083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082192PMC
April 2011

Systems survey of endocytosis by multiparametric image analysis.

Nature 2010 Mar 28;464(7286):243-9. Epub 2010 Feb 28.

Max Planck Institute for Molecular Cell Biology and Genetics, Germany.

Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-beta and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature08779DOI Listing
March 2010