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Role of RNAi machinery in the formation of heterochromatin Chapter 8 #1 Verdel, A, Jia, S, Gerber, S, Sugiyama, T, Gygi, S, Grewal, SI, Moazed, D (2004) RNAi-mediated targeting of heterochromatin by the RITS complex. Science, 303:672–676. #2 Volpe, TA, Kidner, C, Hall, IM, Teng, G, Grewal, SI, Martienssen, RA (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science, 297:1833–1837. Schizosaccharomyces pombe is different than S. cerevisiae and is more similar to mammals and plants Role of RNAi machinery in the formation of heterochromatin Heterochromatin - condensed chromatin, silenced chromatin Centromeres - include much heterochromatin Centromeres - One does not observe transcription from material adjacent to the centromeres. Transposon repeats - One does not observe transcription. In yeast (S. pombe not S. cerevisiae), mutations in Dicer, Argonaute and RdRp cause such transcripts to appear. meH3lys4 - associated with active genes meH3lys9 - associated with inactive genes (exception is S. cerevisiae). Normally centromeres would have low meH3lys4 and high meH3lys9. Mutants have the opposite. RdRP found associated with centromere (but called RDRC there). PTGS, TGS, RNAi, miRNA PTGS = post-transcriptional gene silencing. TGS = transcriptional gene silencing. RNAi = RNA interference. <-- Most commonly used term. Can cause PTGS or TGS. miRNA = microRNA. A form of PTGS. First indication that RNAi and heterochromatin are linked. Mutations in RNAi machinery (Ago1, Dcr1 or Rdp1) produce phenotypes that are similar to swi6 mutants - centromere disappears, also see appearance of forward and backward larger transcripts from the centromeric region. Side trip - RNAi What was once considered to be a very specific cellular process is now involved in a great many things. RNAi RNAi discovered in C. elegans (first animal) while attempting to use antisense RNA in vivo Control “sense” RNAs also produced suppression of target gene! Craig Mello Andrew Fire sense (and antisense) RNAs were 2006 Nobel Prize in Physiology & Medicine contaminated with dsRNA. dsRNA was the suppressing agent. Previously detected in plants but the mechanism was not figured out before Mello and Fire. Function of RNAi Antiviral - Double stranded RNA is an intermediate in the replication of some RNAi viruses. Suppress transposon activity Suppress expression of repetitive DNA (~1/2 or more of the genome in many organisms) Gene regulation inhibit translation stimulate translation reduce mRNA stability Transcriptional gene silencing Silencing might mean modulation Genome stability - maintenance of heterochromatin Future? probably stimulation of transcription How to evoke RNAi Inject double stranded RNA Express or inject antisense RNA inside a cell Express a gene that has an inverted repeat. Two promoters that point at one other. Expression of 2 different genes whose mRNAs can base-pair over a short region. Info from Molecular Biology by Weaver 4th edition pg 501-507 dsRNA DICER - RNase III family member ATP Dicer RISC - one of the proteins is SLICER. In ADP+Pi p p Drosophila SLICER is the product of the Argonaute gene. Dicer leaves 2nt 3’ p p overhangs & phosphorylated 5’ ends p RISC - RNA-induced silencing complex p -Dicer Argonaute has a PAZ and a PIWI domain. 21-23 nt siRNP -R2D2 PIWI PAZ p RISC -Armitrage loading The 2 domains p of Argonaute RISC=RNA- complex induced ATP PIWI domain forms a shape like an RNase H. silencing ADP+Pi complex. RISC p In mice there are 4 Ago genes but only Ago2 Argonaute mRNA Target recognition appears to be SLICER. Dicer participates in selecting the guide RNA p p Target that is passed on to Argonaute. cleavage mRNA Roles of R2D2 and Armitrage are not clear. p p Amplification dsRNA mRNA ATP p Dicer NTPs ADP+Pi RdRp (RNA directed RNA polymerase) p p PPi Dicer leaves 2nt 3’ p p overhangs & phosphorylated 5’ p ends p ATP Dicer p ADP+Pi -Dicer 21-23 nt siRNP p p p -R2D2 p p p p RISC -Armitrage loading p RISC=RNA- complex induced ATP silencing complex. ADP+Pi p RISC Argonaute mRNA Target recognition p p Target mRNA cleavage Info from pg508 4th ed Molecular Biology 4th ed. Robert F. Weaver. p p McGraw Hill Publishers Source of special genes micro- special genes RNAs special genes introns or 3’ UTRs 12 One interrelated gene silencing system siRNA 21-27 n (sizes vary) with a 2 n overhang at the 3’ end. miRNAs 21-24 n Some people say that miRNA differs because it is expressed from native genes and used to regulate endogenous genes whereas RNAi is artificial. This distinction is artificial. Why is this new? If the siRNA’s are so common why were they undetected for so long? We have too many acronyms :( PTGS - Post-transcriptional Gene Silencing RISC=RNA-induced Silencing Complex, targets mRNA RITS= RNA-induced Transcriptional Silencing RDRC = RNA-dependent RNA polymerase complex Heterochromatin formation Figure 2 pg 105 Epigenetics by Allis, Jenuwein and Reinberg. 2007 CSH press Clr4 is a H3K9 methylase CLR4 CLR4 Swi6 can bind Clr4 Both can bind H3K9me through their chromodomain Fission yeast centromere Central domain is fundamentally different from other regions. Uses CENP-A (cenH3) This is a histone-like protein. It replaces H3. Outer heterochromatin is called Clr4-dependent heterochromatin. Outer domain promotes heterochromatin also find homologies between Mat loci and at telomere associated regions. At Mat this homology is called cenH. Promotes Clr4-dependent heterochromatin. Some centromeres have genes. Some tRNA genes are between outer repeat and central kinetochore domain. These prevent the heterochromatin from moving into the central domain. CLR4 Just like all heterochromatin it tries to spread. Propagation Saccharomyces cerevisiae <--- RECAP OF LAST TOPIC!!! pg 69 Allis et al Rap1 recruits Sir4 ORC recruits Sir1 Abf1 recruits Sir3 ORC = origin recognition complex pg 72 Common feature of heterochromatin is its spreading from a nucleation site Telomeric repeat is recog by Rap1 and maybe yKu These two proteins can bind Sir4 proteins Sir4 binds Sir2 and Sir3 Sir2 is a HDAC that deacetylates H4K16ac & H3k9ac !!!!! This can then spread through region w/o Rap1 because Sir3 and 4 can bind the Looping can be important for spreading deacetylated H4 & H3 tails which helps the see figure 6 spreading.Saccharomycescerevisiae sir2 produces O-acetyl-ADP-ribose NAD --> O-acetyl-ADP-ribose which stimulates Sir3 multimerization and Sir3 binding to Sir4 and Sir2 letters to nature cells, corresponding to silent chromatin domains at the nuclear histone H3 only when Lys 9 has been methylated by SUVAR39H1. periphery11, whereas the Clr4-G341D strain shows loss of localiza- In S. pombe, Lys 9 methylase activity is required for transcriptional tion from the nuclear periphery and accumulation of more diffuse repression and HP1 localization. Thus, these data make a direct staining over the nucleolus (Fig. 4c). The Clr4-G341D strain shows correlation between methylated, HP1-bound histones and tran- a very similar pattern of Swi6 distribution to that of the Clr4D strain scriptionally silent heterochromatin. lacking Clr4 (ref. 5). The speci®city of the Swi6 antibody is The fact that HP1 is associated with the enzyme that methylates demonstrated using a Swi6D strain (Fig. 4c). and `marks' histones for HP1 binding suggests a self-maintenance To con®rm that Swi6 association with silent chromatin is depen- model for how HP1 spreads over chromatin to form heterochro- dent on SET domain function, we performed chromatin immuno- matically repressed regions (Fig. 5). An extension of this model precipitation with anti-Swi6 antibodies (Fig. 4d). Centromeric could also explain how silenced heterochromatin could be passed chromatin, known to be associated with Swi6, was enriched relative on during DNA replication. The SUVAR39H1 methylase, bound to to the control (euchromatin) locus in the immunoprecipitated methylated histones via HP1, could direct the methylation of newly sample compared with the total extract12. In both the clr4-G341D deposited histones. This self-maintaining mechanism would then and clr4¢ strains, enrichment of this centromeric sequence was lost. Thus, a mutation in the Clr4 SET domain that abolishesClr4 (yeast) histone is H3 methylase activity disrupts recruitment of Swi6 to silentcalled chromatin. SUV39H1 Loss of association of Swi6 with centromeres shouldin mammals result in SUV39H1 expression of a normally silent marker gene embedded in centro- methylase Swi6 (yeast) is meric chromatin. Figure 4e shows that this is indeed the case. On HP1 HP1 HP1HP1 indicator plates, wild-type strains silence the centromericHP1 in ade6+ Ac Ac Me Me Me Me marker, which results in red, repressed coloniesmammals13; however, in Ac Ac strains lacking Clr4 (D) or strains defective in Clr4 methylase activity (G341D), this ade6+ gene is clearly expressed,chromodomain resulting in Boundary the formation of white colonies. Collectively theseof HP1 results is a show element that methylase activity of Clr4 is required for theH3K9me recruitment binder of Swi6 to silenced centromeric heterochromatin andNote: for today transcrip- we Active chromatin Spreading of silenced and tional silencing. know that Clr4/ HP1-coated heterochromatin The experiments described here ®t well with existingSUV39H data is also show- a ing that SUVAR39H1 and HP1 colocalize atchromodomain heterochromatic Figure 5 A model of heterochromatic self-maintenance by the SUVAR39H1/HP1 5 sites and interact biochemically . We show that HP1protein. recognizes complex. PROJECT EARLY SIMPLE MODEL a Peptide pull-downBannister, AJ, Zegerman, P, Partridge, JF, Miska, EA, Thomas, JO, Allshire, RC, Kouzarides, T (2001) Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature, 410:120–124. 100% DATE b CLIENT input CD CSD2001 NAME Lys 4 Lysmeth 9 methLys 4 Lysmeth 9 meth Methylated clr4 wt clr4 G341D H3 12 Swi6: CD CD CSD CSD H3-K4methH3-K9methH3-K4methH3-K9meth c WT clr4 G341D clr4! swi6! Anti-Swi6 DAPI Merged D clr4 G341D e d WT clr4! ! ! WT clr4 G341clr4 swi6 Cen Control cen1::ade6+ ade6+ Anti-Swi6: T IP T IP T IP Figure 4 Swi6 localization is dependent on the methylase activity of the Clr4 SET domain.
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  • Characterization of the NIPBL Protein Associated with Cornelia De Lange Syndrome

    Characterization of the NIPBL Protein Associated with Cornelia De Lange Syndrome

    Characterization of the NIPBL protein associated with Cornelia de Lange Syndrome A Thesis Submitted to the Faculty of Drexel University by Daniel K. Keter in partial fulfillment of the requirements for the degree of Master of Science June 2008 ii Acknowledgements I would like to thank Dr. Mark S. Lechner for his advice, my committee members, Dr. Mary K. Howett for her time and invaluable assistance, Dr. Aleister Saunders and Dr. Felice Elefant. My regards also to Dr. Ian Krantz and Dr. Matthew Deardorff of the Childrens Hospital of Pennsylvania for their assistance, and my family members for being by my side. iii TABLE OF CONTENTS List of Tables v List of Illustrations vi Abstract vii Introduction 1 Cornelia de Lange syndrome 1 NIPBL and its relation to CDLS 3 NIPBL homologs and its chromosomal and DNA repair roles 7 Chromatin 17 Chromatin and its importance in gene regulation 18 Heterochromatin Protein 1 (HP1) 21 HP1 binds other proteins by its chromoshadow domain 22 HP1 in epigenetic silencing, gene regulation and chromatin remodeling 28 Malfunction/mutation of chromatin in other disease syndromes 29 Rationale, Hypothesis and Experimental Objective 30 Materials and Methods 32 Plasmid construction 32 Cell culture and transfection 32 Immunoflourescence Analysis 33 Doubling time 33 Western blot analysis 34 Antibodies 35 Recombinant proteins 35 Mouse protein heart extracts 35 Metaphase spreads 36 iv Results 37 Characterization and morphological characteristics of CdLS cells 37 Specificity and colocalization of anti-NIPBL and anti-HP1 antibodies