DOCSLIB.ORG

Supplemental Information Posfai Et Al., Polycomb Function During

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Supplemental Information Posfai et al., Polycomb function during oogenesis is required for mouse embryonic development Supplemental Figures and Legends Figure S1. Ring1 and Rnf2 gene deletion strategy during gametogenesis and transcript and protein levels of PRC1 components in wild- type and mutant growing oocytes and early embryos . Figure S2. Reconstitution of endogenous-like levels of PRC1 complex in Ring1m-z+/Rnf2m-z+ early zyotes does not alleviate the developmental arrest at the two-cell stage Figure S3. Ring1 and Rnf2 gene deletion during gametogenesis results in delayed meiotic maturation of oocytes, delayed early embryonic development and arrest before entry into the second cleavage division Figure S4. DNA replication analysis, γ-H2AX patterns and checkpoint activation in control and Ring1m-z+/Rnf2m-z+ embryos Figure S5. Transcriptional shut-down occurs in Ring1/Rnf2 dm GV oocytes Figure S6. Majority of genes misregulated in Ring1/Rnf2 dm GV oocytes are normally expressed in Ring1- or Rnf2 single mutant oocytes Figure S7. Transcripts of developmental regulators de-repressed in Ring1/Rnf2 dm oocytes are only translated after fertilization Figure S8. Ring1/Rnf2 expression during oogenesis defines maternal cytoplasmic and nuclear contributions required for embryonic development. Supplemental Tables Table S1. Gene Ontology (GO) categories of genes up- or down- regulated in Ring1/Rnf2 dm GV oocytes. Table S2. Comparison of Ring1/Rnf2 dm GV oocyte expression profiling data to expression in Rnf2/Eed double mutant mouse ESCs (Leeb et al., 2010). Table S3. Comparison of Ring1/Rnf2 dm GV oocyte expression profiling data to Rnf2 binding profile in wild-type mouse ESCs (Endoh et al., 2008). Table S4. Gene Ontology (GO) categories of genes up- or down- regulated in Ring1/Rnf2 dm GV oocytes, classified according to expression levels of genes during subsequent stages of normal oogenesis. Supplemental Experimental Procedures Mice Antibodies Quantitative real-time RT-PCR primers Comparison to published data sets Comparison to expression patterns during oocyte development and early embryogenesis Defining expression states Defining expression profiles Comparison to expression data of PRC1 or PRC1/PRC2 deficient mouse ESCs and genome wide Rnf2 binding data in mouse ESCs Comparison to H3K27me3 Chip-seq data of mouse ESCs, MEFs and human spermatozoa Statistical analyses A Wild-type spermato spermatid elongating spermatozoon -gonia spermatid +/+ x +/+ early zygote late zygote 2-cell embryo 8-cell blastocyst primordial primary secondary small large GV M-II oocyte oocyte oocyte antral antral oocyte oocyt oocyte oocyte Zygotic transcription Maternal contribution Pan et al., 2005 Zeng et al., 2004 and Zeng et al., 2005 B Conditional mutants spermato spermatid elongating -gonia spermatid spermatozoon F/F Prm1-cre x Zp3-cre Prm1-cre F/F Zp3-cre early zygote late zygote 2-cell embryo 8-cell blastocyst primordial primary secondary small large GV M-II oocyte oocyte oocyte antral antral oocyte oocyt oocyte oocyte Zygotic transcription Maternal contribution C Ring1 Phc1 Rnf2 Phc2 Cbx2 Phc3 relative expression levels primary secondary GV zygote 2-cell Bmi1 oocyte oocyte oocyte wild-type Rnf2 mutant Ring1 mutant Ring1/Rnf2 dm primary secondary GV zygote 2-cell oocyte oocyte oocyte D DAPI Ring1 DAPI Rnf2 DAPI Cbx2 DAPI Bmi1 DAPI Rybp wild-type Rnf2 mutant Ring1 mutant Growing oocyte dm Ring1/Rnf2 E DAPI Ring1DAPI Rnf2 DAPICbx2 DAPI Bmi1 wild-type m-z+ Rnf2 m-z+ 2-cell embryo Ring1 / m-z+ m-z+ Rnf2 Ring1 F Rnf2 Ring1 Ring1/Rnf2 wild-type mutant mutant dm growing mRNA/protein mRNA/protein mRNA/protein mRNA/protein oocyte Ring1 + + + ++ - - - - Rnf2 + + - - + + - - Cbx2 + + + + + + + - Bmi1 + + + + + + + - Ring1m-z+/ wild-type Rnf2m-z+ Ring1m-z+ Rnf2m-z+ 2-cell mRNA/protein mRNA/protein mRNA/protein mRNA/protein embryo Ring1 - - - - - - - - Rnf2 + + - - + + - - Cbx2 + + + - + + + - Bmi1 + + + - + + + - Developmental potential + + + - (beyond the 2-cell stage) Supplemental figure 1 Ring1 and Rnf2 gene deletion strategy during gametogenesis and transcript and protein levels of PRC1 components in wild-type and mutant growing oocytes and early embryos (A) Schematic diagram illustrating different stages of gametogenesis and early embryonic development. Com- parative expression analyses described in Figure 5C and Figure S6D were based on genome-wide transcriptome analyses in wild-type oocytes at five subsequent stages of follicle development (from primordial to large antral follicle stage) (Pan et al., 2005) and in wild-type GV oocytes and early embryos (late zygotes, 2-cell, 8-cell and blastocyst embryos) (Zeng et al., 2004; Zeng and Schultz, 2005). (B) Conditional genetic strategy to generate embryos maternally and zygotically deficient for Rnf2 function. Zp3-cre drives Cre recombinase expression during oogenesis from the primary follicle stage onwards, resulting in loss of Rnf2 expression during the transcriptionally active growth phase of oogenesis. Prm1-cre drives Cre recombinase expression in late round spermatids and during the elongation phase of spermiogenesis during which transcription is largely shut-down in a genome-wide manner. Deletion of Ring1 and Rnf2 in maturing sper- matids did not impair embryonic or postnatal development of double heterozygous offspring (data not shown). The lack of a paternal affect phenotype may be due to the conditional deletion of Rnf2 relatively late during sper- miogenesis, a developmental phase of minor transcriptional activity as compared to Zp3-cre mediated deletion in actively transcribing growing oocytes. (C) Quantitative real-time PCR analysis of gene transcripts of Ring1, Rnf2 and of other PRC1 members Cbx2, Bmi1, Phc1, Phc2 and Phc3 in growing oocytes (from primary and secondary follicles), GV oocytes, zygotes and two-cell embryos, wild-type, single or double deficient for maternal Ring1 and/or Rnf2. Transcript levels were normalized to LnmB1 control. Error bars indicate standard deviation based on 2-3 biological replicates. (D) IF staining for Ring1, Rnf2, Cbx2, Bmi1 and Rybp in wild-type, Rnf2 mutant (Rnf2F/F; Zp3-cre), Ring1 mutant (Ring1-/-) and Ring1/Rnf2 dm (Ring1-/-/Rnf2F/F; Zp3-cre) growing oocytes (from secondary follicles) surrounded by follicle cells in ovary sections from 12-14 day old mice. Ring1 protein level is increased in Rnf2 mutant oocytes. Due to constitutive deletion granulosa cells lack Ring1 protein in Ring1 mutant ovaries. >20 follicles were analyzed from 6 mice for each IF staining. Scale bar = 10 μm. (E) IF staining for Ring1, Rnf2, Cbx2 and Bmi1 in two-cell embryos, wild-type, single or double deficient for maternal Ring1 and/or Rnf2. Scale bar = 20 μm. (F) Summary of mRNA and protein expression of PRC1 components in growing oocytes and two-cell embryos, wild-type, single or double deficient for maternal Ring1 and/or Rnf2. A m-z+ m-z+ C Ring1 /Rnf2 control control Rnf2 mRNA water water DAPI Rnf2 merge injected injected injected O 2 H DAPI Ring1m+z+; Rnf2m-z+ DAPI Rnf2 merge Bmi1 O 2 H B Rnf2 50 ng Rnf2 mRNA 10 ng relative mRNA expression levels Rnf2 mRNA H2OH2O 50ng 10ng1ng 0.1ng m+z+ m-z+ control Ring1 ; Rnf2 1 ng D Rnf2 mRNA Rnf2 0.1 ng Rnf2 mRNA relative protein expression levels H2O H2O 50ng 10ng 1ng 0.1ng (9) (4) (5) (8) (8) (5) control Ring1m+z+; Rnf2m-z+ E 50 ng Rnf2 mRNA injected 2 ng Rnf2 mRNA injected control Ring1m-z+; Rnf2m-z+ control Ring1m-z+; Rnf2m-z+ 100 100 80 80 60 60 40 40 % of embryos 20 % of embryos 20 0 0 H2O Rnf2 mRNA H2O Rnf2 mRNA H2O Rnf2 mRNA H2O Rnf2 mRNA (45) (30) (26) (30) (35) (27) (21) (30) fragmented 2-cell 4-cell morula zygote 3-cell 8-cell blastocyst Supplemental figure 2 Reconstitution of endogenous-like levels of PRC1 complex in Ring1m-z+/Rnf2m-z+ early zyotes does not alleviate the developmental arrest at the two-cell stage (A) Microinjection of myc-tagged Rnf2 mRNA into Ring1m-z+/Rnf2m-z+ zygotes leads to de-novo PRC1 com- plex formation. IF analyses of control and Ring1m-z+/Rnf2m-z+ zygotes microinjected with water or with myc- tagged Rnf2 mRNA. Embryos were stained with anti-Bmi1 antibodies to visualize reconstitution of chromatin bound PRC1. Scale bar = 20 μm. (B) Rnf2 mRNA levels measured by quantitative real-time PCR in water injected control zygotes and Ring1m- z+/Rnf2m-z+ zygotes microinjected with 0 (water), 50, 10, 1, 0.1 ng of myc-tagged Rnf2 mRNA. Early IVF zygotes were injected 3-5 hpf and harvested 12 hpf. Transcript levels were normalized to LnmB1 control. Error bars indicate standard deviation based on 2-3 biological replicates. (C) IF staining with anti-Rnf2 antibody of water injected control zygotes and Ring1m-z+/Rnf2m-z+ zygotes microinjected with 0 (water), 50, 10, 1, 0.1 ng of myc-tagged Rnf2 mRNA. Early IVF zygotes were injected 3-5 hpf and harvested 12 hpf. Scale bar = 20 μm. (D) Quantification of Rnf2 protein levels by measuring total fluorescent signal (Y axis) of IF staining with anti- Rnf2 antibody in panel (B) embryos. Total fluorescent signal was measured from both PN together. Error bars indicate standard deviation based on the number of embryos analyzed (indicated in brackets). (E) Microinjection of 50 (left panel) or 2 (right panel) ng of myc-tagged Rnf2 mRNA into early zyotes does not alleviate the developmental arrest at the two-cell stage. Diagram showing developmental potential of control and Ring1m-z+/Rnf2m-z+ embryos, microinjected at the early zygote stage with water or Rnf2 mRNA. Developmen- tal potential was scored at embryonic day 3.5. A 21 day ovary control Ring1-/-; Rnf2F/F; Zp3-cre B C 100 80 2.5-3.5
Recommended publications
  • Down-Regulation of Stem Cell Genes, Including Those in a 200-Kb Gene Cluster at 12P13.31, Is Associated with in Vivo Differentiation of Human Male Germ Cell Tumors

    Down-Regulation of Stem Cell Genes, Including Those in a 200-Kb Gene Cluster at 12P13.31, Is Associated with in Vivo Differentiation of Human Male Germ Cell Tumors

    Research Article Down-Regulation of Stem Cell Genes, Including Those in a 200-kb Gene Cluster at 12p13.31, Is Associated with In vivo Differentiation of Human Male Germ Cell Tumors James E. Korkola,1 Jane Houldsworth,1,2 Rajendrakumar S.V. Chadalavada,1 Adam B. Olshen,3 Debbie Dobrzynski,2 Victor E. Reuter,4 George J. Bosl,2 and R.S.K. Chaganti1,2 1Cell Biology Program and Departments of 2Medicine, 3Epidemiology and Biostatistics, and 4Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York Abstract on the degree and type of differentiation (i.e., seminomas, which Adult male germ cell tumors (GCTs) comprise distinct groups: resemble undifferentiated primitive germ cells, and nonseminomas, seminomas and nonseminomas, which include pluripotent which show varying degrees of embryonic and extraembryonic embryonal carcinomas as well as other histologic subtypes patterns of differentiation; refs. 2, 3). Nonseminomatous GCTs are exhibiting various stages of differentiation. Almost all GCTs further subdivided into embryonal carcinomas, which show early show 12p gain, but the target genes have not been clearly zygotic or embryonal-like differentiation, yolk sac tumors and defined. To identify 12p target genes, we examined Affymetrix choriocarcinomas, which exhibit extraembryonal forms of differ- (Santa Clara, CA) U133A+B microarray (f83% coverage of 12p entiation, and teratomas, which show somatic differentiation along genes) expression profiles of 17 seminomas, 84 nonseminoma multiple lineages (3). Both seminomas and embryonal carcinoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p are known to express stem cell markers, such as POU5F1 (4) and were significantly overexpressed, including GLUT3 and REA NANOG (5).
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
  • RING1 Antibody (N-Term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP14560A

    RING1 Antibody (N-Term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP14560A

    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 RING1 Antibody (N-term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP14560A Specification RING1 Antibody (N-term) - Product Information Application WB,E Primary Accession Q06587 Other Accession Q6MGB6, O35730, NP_002922.2 Reactivity Human Predicted Mouse, Rat Host Rabbit Clonality Polyclonal Isotype Rabbit Ig Antigen Region 95-123 RING1 Antibody (N-term) - Additional Information RING1 Antibody (N-term) (Cat. #AP14560a) Gene ID 6015 western blot analysis in MDA-MB453 cell line lysates (35ug/lane).This demonstrates the Other Names RING1 antibody detected the RING1 protein E3 ubiquitin-protein ligase RING1, 632-, (arrow). Polycomb complex protein RING1, RING finger protein 1, Really interesting new gene 1 protein, RING1, RNF1 RING1 Antibody (N-term) - Background Target/Specificity This gene belongs to the RING finger family, This RING1 antibody is generated from members of rabbits immunized with a KLH conjugated synthetic peptide between 95-123 amino which encode proteins characterized by a RING acids from the N-terminal region of human domain, a RING1. zinc-binding motif related to the zinc finger domain. The gene Dilution product can bind DNA and can act as a WB~~1:1000 transcriptional repressor. It is associated with the multimeric polycomb Format group protein complex. Purified polyclonal antibody supplied in PBS The gene product interacts with the polycomb with 0.09% (W/V) sodium azide. This group proteins BMI1, antibody is purified through a protein A EDR1, and CBX4, and colocalizes with these column, followed by peptide affinity proteins in large purification.
  • Phase Separation by the Polyhomeotic Sterile Alpha Motif Compartmentalizes Polycomb Group Proteins and Enhances Their Activity

    Phase Separation by the Polyhomeotic Sterile Alpha Motif Compartmentalizes Polycomb Group Proteins and Enhances Their Activity

    ARTICLE https://doi.org/10.1038/s41467-020-19435-z OPEN Phase separation by the polyhomeotic sterile alpha motif compartmentalizes Polycomb Group proteins and enhances their activity Elias Seif1, Jin Joo Kang1,2, Charles Sasseville1, Olga Senkovich3, Alexander Kaltashov1, Elodie L. Boulier1, ✉ Ibani Kapur1,2, Chongwoo A. Kim3 & Nicole J. Francis 1,2,4 1234567890():,; Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification. 1 Institut de recherches cliniques de Montréal, 110 Avenue des Pins Ouest, Montréal, QC H2W 1R7, Canada. 2 Division of Experimental Medicine, McGill University, 1001 Decarie Boulevard, Montreal, QC H4A 3J1, Canada. 3 Department of Biochemistry and Molecular Genetics, Midwestern University, 19555N.
  • PHC1 (D-10): Sc-390880

    PHC1 (D-10): Sc-390880

    SAN TA C RUZ BI OTEC HNOL OG Y, INC . PHC1 (D-10): sc-390880 BACKGROUND APPLICATIONS Polycomb group (PcG) proteins assemble into multimeric protein complexes, PHC1 (D-10) is recommended for detection of PHC1 of mouse, rat and human which are involved in maintaining the transcriptional repressive state of genes origin by Western Blotting (starting dilution 1:100, dilution range 1:100- throughout development. PHC1 ( polyhomeotic homolog 1), also known as 1:1000), immunoprecipitation [1-2 µg per 100-500 µg of total protein (1 ml EDR1, HPH1 or RAE28, is a 1,004 amino acid nuclear protein that is a compo - of cell lysate)], immunofluorescence (starting dilution 1:50, dilution range nent of the PcG multiprotein PRC1 complex. Specifically, the PcG PRC1 com - 1:50-1:500) and solid phase ELISA (starting dilution 1:30, dilution range plex modifies histones, remodels chromatin and mediates monoubiquination 1:30- 1:3000). of Histone H2A. Other constituent proteins involved in the PcG PRC1 complex Suitable for use as control antibody for PHC1 siRNA (h): sc-95881, PHC1 are Mel-18, Bmi-1, M33, MPc2, MPc3, RING1, Ring1b, as well as several siRNA (m): sc-152203, PHC1 shRNA Plasmid (h): sc-95881-SH, PHC1 shRNA others. Existing as a homodimer, PHC1 contains one FCS-type zinc finger Plasmid (m): sc-152203-SH, PHC1 shRNA (h) Lentiviral Particles: sc-95881-V and a SAM (sterile motif) domain. PHC1 is encoded by a gene located on α and PHC1 shRNA (m) Lentiviral Particles: sc-152203-V. human chromosome 12, which encodes over 1,100 genes and comprises approximately 4.5% of the human genome.
  • Genome-Wide DNA Methylation Analysis of KRAS Mutant Cell Lines Ben Yi Tew1,5, Joel K

    Genome-Wide DNA Methylation Analysis of KRAS Mutant Cell Lines Ben Yi Tew1,5, Joel K

    www.nature.com/scientificreports OPEN Genome-wide DNA methylation analysis of KRAS mutant cell lines Ben Yi Tew1,5, Joel K. Durand2,5, Kirsten L. Bryant2, Tikvah K. Hayes2, Sen Peng3, Nhan L. Tran4, Gerald C. Gooden1, David N. Buckley1, Channing J. Der2, Albert S. Baldwin2 ✉ & Bodour Salhia1 ✉ Oncogenic RAS mutations are associated with DNA methylation changes that alter gene expression to drive cancer. Recent studies suggest that DNA methylation changes may be stochastic in nature, while other groups propose distinct signaling pathways responsible for aberrant methylation. Better understanding of DNA methylation events associated with oncogenic KRAS expression could enhance therapeutic approaches. Here we analyzed the basal CpG methylation of 11 KRAS-mutant and dependent pancreatic cancer cell lines and observed strikingly similar methylation patterns. KRAS knockdown resulted in unique methylation changes with limited overlap between each cell line. In KRAS-mutant Pa16C pancreatic cancer cells, while KRAS knockdown resulted in over 8,000 diferentially methylated (DM) CpGs, treatment with the ERK1/2-selective inhibitor SCH772984 showed less than 40 DM CpGs, suggesting that ERK is not a broadly active driver of KRAS-associated DNA methylation. KRAS G12V overexpression in an isogenic lung model reveals >50,600 DM CpGs compared to non-transformed controls. In lung and pancreatic cells, gene ontology analyses of DM promoters show an enrichment for genes involved in diferentiation and development. Taken all together, KRAS-mediated DNA methylation are stochastic and independent of canonical downstream efector signaling. These epigenetically altered genes associated with KRAS expression could represent potential therapeutic targets in KRAS-driven cancer. Activating KRAS mutations can be found in nearly 25 percent of all cancers1.
  • RING1 Proteins Contribute to Early Proximal-Distal Specification of The

    RING1 Proteins Contribute to Early Proximal-Distal Specification of The

    © 2016. Published by The Company of Biologists Ltd | Development (2016) 143, 276-285 doi:10.1242/dev.127506 RESEARCH ARTICLE RING1 proteins contribute to early proximal-distal specification of the forelimb bud by restricting Meis2 expression Nayuta Yakushiji-Kaminatsui1,*,‡, Takashi Kondo1,2,3, Takaho A. Endo4, Yoko Koseki1,2, Kaori Kondo1,2,3, Osamu Ohara4, Miguel Vidal5 and Haruhiko Koseki1,2,‡ ABSTRACT subunits to catalyze histone H2A monoubiquitylation at lysine 119 Polycomb group (PcG) proteins play a pivotal role in silencing (H2AK119ub1). PRC1 also targets chromatin compaction by SAM developmental genes and help to maintain various stem and domain polymerization of PHC1 and PHC2, a mammalian PH precursor cells and regulate their differentiation. PcG factors also ortholog, via H3K27me3 recognition by chromodomain proteins regulate dynamic and complex regional specification, particularly in such as CBX7 and CBX2 (Cao et al., 2002; Endoh et al., 2012; mammals, but this activity is mechanistically not well understood. In Isono et al., 2013; Kuzmichev et al., 2002). Recent studies identified this study, we focused on proximal-distal (PD) patterning of the a variant PRC1 complex containing RYBP (Ring1 and YY1 mouse forelimb bud to elucidate how PcG factors contribute to a binding protein)/YAF (YY1-associated factor), RING1A/B and a regional specification process that depends on developmental distinct PCGF subunit (Gao et al., 2012; Tavares et al., 2012), and signals. Depletion of the RING1 proteins RING1A (RING1) and demonstrated that H2AK119ub1 mediated by this variant PRC1 RING1B (RNF2), which are essential components of Polycomb leads to the recruitment of PRC2 and placement of H3K27me3 to repressive complex 1 (PRC1), led to severe defects in forelimb initiate Polycomb repression (Blackledge et al., 2014).
  • Polycomb Group Proteins Ring1a/B Are Functionally Linked to the Core Transcriptional Regulatory Circuitry to Maintain ES Cell Identity Mitsuhiro Endoh1, Takaho A

    Polycomb Group Proteins Ring1a/B Are Functionally Linked to the Core Transcriptional Regulatory Circuitry to Maintain ES Cell Identity Mitsuhiro Endoh1, Takaho A

    Development ePress online publication date 13 March 2008 RESEARCH ARTICLE 1513 Development 135, 1513-1524 (2008) doi:10.1242/dev.014340 Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity Mitsuhiro Endoh1, Takaho A. Endo2, Tamie Endoh1, Yu-ichi Fujimura1, Osamu Ohara1, Tetsuro Toyoda2, Arie P. Otte3, Masaki Okano4, Neil Brockdorff5, Miguel Vidal1,6 and Haruhiko Koseki1,* The Polycomb group (PcG) proteins mediate heritable silencing of developmental regulators in metazoans, participating in one of two distinct multimeric protein complexes, the Polycomb repressive complexes 1 (PRC1) and 2 (PRC2). Although PRC2 has been shown to share target genes with the core transcription network, including Oct3/4, to maintain embryonic stem (ES) cells, it is still unclear whether PcG proteins and the core transcription network are functionally linked. Here, we identify an essential role for the core PRC1 components Ring1A/B in repressing developmental regulators in mouse ES cells and, thereby, in maintaining ES cell identity. A significant proportion of the PRC1 target genes are also repressed by Oct3/4. We demonstrate that engagement of PRC1 at target genes is Oct3/4-dependent, whereas engagement of Oct3/4 is PRC1-independent. Moreover, upon differentiation induced by Gata6 expression, most of the Ring1A/B target genes are derepressed and the binding of Ring1A/B to their target loci is also decreased. Collectively, these results indicate that Ring1A/B-mediated Polycomb
  • Molecular Genetics of Microcephaly Primary Hereditary: an Overview

    Molecular Genetics of Microcephaly Primary Hereditary: an Overview

    brain sciences Review Molecular Genetics of Microcephaly Primary Hereditary: An Overview Nikistratos Siskos † , Electra Stylianopoulou †, Georgios Skavdis and Maria E. Grigoriou * Department of Molecular Biology & Genetics, Democritus University of Thrace, 68100 Alexandroupolis, Greece; [email protected] (N.S.); [email protected] (E.S.); [email protected] (G.S.) * Correspondence: [email protected] † Equal contribution. Abstract: MicroCephaly Primary Hereditary (MCPH) is a rare congenital neurodevelopmental disorder characterized by a significant reduction of the occipitofrontal head circumference and mild to moderate mental disability. Patients have small brains, though with overall normal architecture; therefore, studying MCPH can reveal not only the pathological mechanisms leading to this condition, but also the mechanisms operating during normal development. MCPH is genetically heterogeneous, with 27 genes listed so far in the Online Mendelian Inheritance in Man (OMIM) database. In this review, we discuss the role of MCPH proteins and delineate the molecular mechanisms and common pathways in which they participate. Keywords: microcephaly; MCPH; MCPH1–MCPH27; molecular genetics; cell cycle 1. Introduction Citation: Siskos, N.; Stylianopoulou, Microcephaly, from the Greek word µικρoκεϕαλi´α (mikrokephalia), meaning small E.; Skavdis, G.; Grigoriou, M.E. head, is a term used to describe a cranium with reduction of the occipitofrontal head circum- Molecular Genetics of Microcephaly ference equal, or more that teo standard deviations
  • Vitamin D Genes & Exposure in Relation to Kidney Cancer by Sara

    Vitamin D Genes & Exposure in Relation to Kidney Cancer by Sara

    Vitamin D Genes & Exposure in Relation to Kidney Cancer by Sara Karami B.S., Biology, James Madison University, 2002 M.P.H, Epidemiology, The George Washington University, 2004 A Dissertation submitted to The Faculty of The Columbian College of Arts and Science of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 31, 2009 Dissertation directed by Katherine L. Hunting Professor of Environmental and Occupational Health and of Epidemiology and Biostatistics and Lee E. Moore Epidemiological Investigator, NIH, NCI The Columbian College of Arts and Science of The George Washington University certifies that Sara Karami has passed the Final Examination for the degree of Doctor of Philosophy as of August 12, 2009. This is the final and approved form of the dissertation. Vitamin D Genes & Exposure in Relation to Kidney Cancer Sara Karami Dissertation Research Committee: Katherine L. Hunting, Professor of Environmental and Occupational Health and of Epidemiology and Biostatistics, Dissertation Director Lee E. Moore, Epidemiological Investigator, NIH, NCI, Co-Director Paul H. Levine, Professor of Epidemiology and Biostatistics, Committee Member Yinglei Lai, Assistant Professor of Statistics, Committee Member ii © Copyright 2009 by Sara Karami All rights reserved iii Dedication The author wishes to thank everyone involved in the dissertation process for their guidance and support. Special thanks to Dr. Lee Moore, Dr. Katherine Hunting, Dr. Paul Levine, Dr. Yinglei Lai, Dr. Sean Cleary, and Dr. Donte Verme. iv Acknowledgement The author wishes to acknowledge the National Cancer Institute, the International Agency for Research on Cancer, and the School of Public Health and Health Services of The George Washington University for their assistance.
  • Structural and Spatial Chromatin Features at Developmental Gene Loci in Human Pluripotent Stem Cells

    Structural and Spatial Chromatin Features at Developmental Gene Loci in Human Pluripotent Stem Cells

    ARTICLE DOI: 10.1038/s41467-017-01679-x OPEN Structural and spatial chromatin features at developmental gene loci in human pluripotent stem cells Hiroki Ikeda1, Masamitsu Sone1,2, Shinya Yamanaka1,3 & Takuya Yamamoto 1,2,4 Higher-order chromatin organization controls transcriptional programs that govern cell properties and functions. In order for pluripotent stem cells (PSCs) to appropriately respond 1234567890 to differentiation signals, developmental gene loci should be structurally and spatially regu- lated to be readily available for immediate transcription, even though these genes are hardly expressed in PSCs. Here, we show that both chromatin interaction profiles and nuclear positions at developmental gene loci differ between human somatic cells and hPSCs, and that changes in the chromatin interactions are closely related to the nuclear repositioning. Moreover, we also demonstrate that developmental gene loci, which have bivalent histone modifications, tend to colocalize in PSCs. Furthermore, this colocalization requires PRC1, PRC2, and TrxG complexes, which are essential regulatory factors for the maintenance of transcriptionally poised developmental genes. Our results indicate that higher-order chro- matin regulation may be an integral part of the differentiation capacity that defines pluripotency. 1 Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. 2 Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. 3 Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA. 4 AMED-CREST, AMED 1-7-1 Otemach, Chiyodaku, Tokyo 100-0004, Japan. Correspondence and requests for materials should be addressed to T.Y.
  • Table S1. Identified Proteins with Exclusive Expression in Cerebellum of Rats of Control, 10Mg F/L and 50Mg F/L Groups

    Table S1. Identified Proteins with Exclusive Expression in Cerebellum of Rats of Control, 10Mg F/L and 50Mg F/L Groups

    Table S1. Identified proteins with exclusive expression in cerebellum of rats of control, 10mg F/L and 50mg F/L groups. Accession PLGS Protein Name Group IDa Score Q3TXS7 26S proteasome non-ATPase regulatory subunit 1 435 Control Q9CQX8 28S ribosomal protein S36_ mitochondrial 197 Control P52760 2-iminobutanoate/2-iminopropanoate deaminase 315 Control Q60597 2-oxoglutarate dehydrogenase_ mitochondrial 67 Control P24815 3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase type 1 84 Control Q99L13 3-hydroxyisobutyrate dehydrogenase_ mitochondrial 114 Control P61922 4-aminobutyrate aminotransferase_ mitochondrial 470 Control P10852 4F2 cell-surface antigen heavy chain 220 Control Q8K010 5-oxoprolinase 197 Control P47955 60S acidic ribosomal protein P1 190 Control P70266 6-phosphofructo-2-kinase/fructose-2_6-bisphosphatase 1 113 Control Q8QZT1 Acetyl-CoA acetyltransferase_ mitochondrial 402 Control Q9R0Y5 Adenylate kinase isoenzyme 1 623 Control Q80TS3 Adhesion G protein-coupled receptor L3 59 Control B7ZCC9 Adhesion G-protein coupled receptor G4 139 Control Q6P5E6 ADP-ribosylation factor-binding protein GGA2 45 Control E9Q394 A-kinase anchor protein 13 60 Control Q80Y20 Alkylated DNA repair protein alkB homolog 8 111 Control P07758 Alpha-1-antitrypsin 1-1 78 Control P22599 Alpha-1-antitrypsin 1-2 78 Control Q00896 Alpha-1-antitrypsin 1-3 78 Control Q00897 Alpha-1-antitrypsin 1-4 78 Control P57780 Alpha-actinin-4 58 Control Q9QYC0 Alpha-adducin 270 Control Q9DB05 Alpha-soluble NSF attachment protein 156 Control Q6PAM1 Alpha-taxilin 161