Organisation of Chromosomes

252 Pages·2013·6.461 MB·1-254\252

by  Rossen Donev (Eds.)

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PREFACE
Chromosomesundergostructuralreorganizationsthroughoutthecellcycle
toenabletheirphase-specificfunctions.Thesereorganizationsareverycom-
plexandnumerous.Thus,inthisvolumeoftheAdvancesinProteinChemistry
andStructuralBiologydedicatedtotheorganizationofchromosomes,wedis-
cussonlyafewaspectsofchromosomeandcytoskeletonstructuralalterations
enablingchromosomerearrangements.
Chapters1and2discusstheroleoftandemlyrepeatedDNAsequences
inchromosomeorganizationandfunction.Thetandemlyorganizedhighly
repetitivesatelliteDNAisthemainDNAcomponentofcentromeric/per-
icentromeric constitutive heterochromatin. For a very long time, these
sequences have been considered as a “junk DNA.” However, it is now
established that each chromosome possesses a unique code madeup of dif-
ferent large tandem repeats. Tandem repeat multiformity tunes the devel-
oped nuclei 3D pattern by sequential steps of associations. Some satellite
DNAs are also found transcribed during different phases of development
andcellcycle,suggestingtheirimportanceforchromosomereorganization.
InChapters3and4,detailedreviewsontheroleofhistonevariantsand
zinc-fingerproteinsinelucidationofthestructuraldynamicsofnucleosomes
andchromosomesarepresented.Thepresenceofvarianthistoneproteinsin
the nucleosome core raises the functional diversity of the nucleosomes in
gene regulation and has the profound epigenetic consequences of great
importance for understanding the fundamental issues such as the assembly
of variant nucleosomes and chromatin remodeling. Zinc-finger DNA-
bindingproteinshaveshowntobeimportantorganizersofthe3Dstructure
of chromosomes and as such are called the master weaver of the genome.
Chapter 5 focuses on the latest developments in unconventional actin
configurations.Theexistenceofactininthenucleuswasfirstreportedmore
than40yearsago.However,theideaofanuclearactinmetwithskepticism
for decades. Nuclear preparations were thought to be contaminated with
actinfromthecytoplasm.However,aftertheidentificationofactininsev-
eral nuclear complexes, which implicates it in diverse nuclear activities
including transcription, chromosome remodeling, and nucleocytoplasmic
trafficking,skepticismhasfinallygivenwaytothechallengingsearchforspe-
cificstatesand/oruniqueconformationsactinmayadopttofulfillitsnuclear
functions.
vii
viii Preface
Finally, Chapter6 presentsourcurrent understandingof the chromatin
condensationduringmitosiswithparticularattentiontothemajormolecular
playersthattriggerandmaintainthiscondensation.Furthermore,themech-
anismsthattakecaretoensurethatmitoticchromosomecondensationand
theblockoftranscriptionduringmitosisdonotwipeoutthecellidentityare
also discussed.
Itismysincerehopethatthisoverviewofthebasicmechanismsinvolved
in the regulation of the dynamic chromosome organization would inspire
futuretranslationalstudiesfocusingonthefineregulationofthedysregulated
cellcycleindiseasessuchascancer,Alzheimer’s,etc.
ROSSEN DONEV
Institute of Life Sciences, College of Medicine
Swansea University, United Kingdom
Clinic and Polyclinic for Psychiatry and Psychotherapy
Rostock University, Germany
CHAPTER ONE
Large Tandem Repeats Make
up the Chromosome Bar Code:
A Hypothesis
Olga Podgornaya*,†,1, Ekaterina Gavrilova†, Vera Stephanova*,
* *
Sergey Demin , Aleksey Komissarov
*
InstituteofCytologyRAS,St.Petersburg,Russia
†FacultyofBiologyandSoilSciences,St.PetersburgStateUniversity,St.Petersburg,Russia
1Correspondingauthor:e-mailaddress:[email protected]
Contents
1. Introduction 2
2. SatelliteDNAintheMouseGenome 3
2.1 SatelliteDNAandtandemrepeats 3
2.2 Mousegenomeasamodelforthetandemrepeatsearch 4
2.3 Lackoftandemrepeatsinthereferencegenome 5
2.4 LargetandemrepeatsinWGSassemblies 6
2.5 Largetandemrepeatgraphicrepresentation 9
2.6 TEs-relatedtandemrepeats 10
2.7 FamilyTRPC-21A-MM 12
3. TandemRepeatPositionDefinedbyFISH 13
3.1 Chromosomesofbonemarrowcells:firstsetofprobes 13
3.2 Chromosomesofembryonicfibroblasts:secondsetofprobes 15
3.3 Hybridizationsummary 18
4. “BarCode”Hypothesis 19
4.1 Internallargetandemrepeatsinhumanandmousereferencegenomes 19
4.2 Proteinsspecificinbindingtotandemrepeats 21
4.3 RNA-mediatedmechanismoftandemrepeatassociation 22
4.4 “Barcode”asmorphogeneticprogram 24
Acknowledgments 25
References 25
Abstract
Muchoftandemrepeats’functionalnatureinanygenomeremainsenigmaticbecause
there are only few tools available for dissecting and elucidating the functions of
repeated DNA. The large tandem repeat arrays (satellite DNA) found in two mouse
whole-genome shotgun assemblies were classified into 4 superfamilies, 8 families,
and62subfamilies.Withthesimplifiedvariantofchromosomepositioningofdifferent
AdvancesinProteinChemistryandStructuralBiology,Volume90 #2013ElsevierInc. 1
ISSN1876-1623 Allrightsreserved.
http://dx.doi.org/10.1016/B978-0-12-410523-2.00001-8
2 OlgaPodgornayaetal.
tandem repeats, we noticed the nonuniform distribution instead of the positions
reportedformousemajorandminorsatellites.Itisvisiblethateachchromosomepos-
sessesakindofuniquecodemadeupofdifferentlargetandemrepeats.Thereference
genomesallowmarkingonlyinternaltandemrepeats,andevenwithsuchalimiteddata,
thecolored“barcode”madeupoftandemrepeatsisvisible.Wesupposethattandem
repeatsbarethemechanismforchromosomestorecognizetheregionstobeassociated.
Theassociations,initiallyestablishedviaRNA,becomefixedbyhistonemodifications(the
histoneorchromatincode)andspecificproteins.Insuchaway,associations,beingatthe
beginningflexibleandregulated,thatis,adjustable,appearasirreversibleandinheritable
incellgenerations.Tandemrepeatmultiformitytunesthedevelopednuclei3Dpattern
bysequentialstepsofassociations.Tandemrepeats-basedchromosomebarcodecould
bethecarrierofthegenomestructuralinformation;thatis,theorderofprecisetandem
repeatassociationistheDNAmorphogeneticprogram.Tandemrepeatsarethecoresof
thedistinct3Dstructurespostulatedin“genegating”hypothesis.
1. INTRODUCTION
Tandemlyrepeatedsequences(TRs)arecommoninhighereukaryotes
andcancompriseupto10%ofthegenomes.MuchofTRs’functionalnature
inanygenomeremainsenigmaticbecausethereareonlyfewtoolsavailable
fordissectingandelucidatingthefunctionsofrepeatedDNA(Blattesetal.,
2006).Itissupposedthatacertainamountofconstitutiveheterochromatinis
essential in multicellular organisms at two levels of organization: chromo-
somalandnuclear.Atthechromosomallevel,TRscanbeofprofoundstruc-
tural as well as evolutionary importance, as genomic regions with a high
densityofTRs(e.g.,telomeric,centromeric,andheterochromaticregions)
oftenhavespecificpropertiessuchasalternativeDNAstructureandpackag-
ing (Kobliakova, Zatsepina, Stefanova, Polyakov, & Kireev, 2005;
Podgornaya, Voronin, Enukashvily, Matveev, & Lobov, 2003; Ushiki &
Hoshi,2008;Vogt,1990).
At the nuclear level of organization, constitutive heterochromatin may
helpmaintaintheproperspatialrelationshipsnecessaryfortheefficientoper-
ationofthecellthroughthestagesofmitosisandmeiosis.Theassociationof
satellite(orotherhighlyrepetitive)DNAwithconstitutiveheterochromatin
is understandable, as it stresses the importance of the structural rather than
transcriptional roles of these entities. In the interphase nucleus, satellite
DNAs (satDNAs) have one property in common despite their species
specificity, namely, heterochromatization (Yunis & Yasmineh, 1971).
Heterochromatization appears to involve RNA interference-mediated
LargeTandemRepeatsandtheChromosomeBarCode 3
chromatin modifications (Alleman et al., 2006; Martienssen, 2003). The
RNAipathwayisrequiredfortheformationofheterochromatinandsilenc-
ingofrepetitivesequencesinDrosophilamelanogaster(Grewal&Elgin,2007).
In mammalian cells, an RNA component is required for the association of
HP1 with pericentric heterochromatin (Lu & Gilbert, 2007; Maison et al.,
2002; Muchardt et al., 2002). Moreover, the strand-specific burst in tran-
scription of pericentric satellites is required for chromocenter formation
andearlymousedevelopment.Specificexpressiondynamicsofmajorsatel-
lite (MaSat) repeats, together with their strand-specific control, represent
necessary mechanisms during a critical time window in preimplantation
development that are of key importance to consolidate the maternal and
to set up the paternal heterochromatic state at pericentric domains (Probst
et al., 2010). Such an important and crucial finding is based on the known
sequenceof themouse MaSat.MostoftheothermouseTRscouldnotbe
tested in similar experiments being undescribed and unclassified.
TheanalysisoftheTRcontentofgenomesisimportantforunderstanding
theevolutionandorganizationofgenomesaswellasgeneexpression.The
computation approaches to the TR analysis on the genome level gradually
appear with the genome sequencing advanced (Alkan et al., 2007; Ames,
Murphy, Helentjaris, Sun, & Chandler, 2008; Mayer, Leese, & Tollrian,
2010; Warburton et al., 2008). Current review is dedicated to the large
TRsorsatelliteDNA.
2. SATELLITE DNA IN THE MOUSE GENOME
2.1. Satellite DNA and tandem repeats
SatDNAswereidentifiedabout50yearsagoasanadditional,“satellite,”frac-
tionofgenomicDNAduringtheequilibriumcentrifugationinCsClgradient
(Kit, 1961, 1962). SatDNAs have been found in all the high eukaryotic
species investigated by advanced centrifugation methods (Beridze, 1986).
ReassociationkineticstestsshowthatsatDNAisthehighlyrepetitivesequence,
builtupto10%ofthetotalDNA(Britten&Kohne,1968;Corneo,Ginelli,&
Polli, 1967, 1970). Cytologically distinguished domains for satDNAs have
beenshownwithmouseMaSatwithinitiallydeterminedcentromericlocali-
zation (Pardue & Gall, 1970). Simple sequence satellites with monomers of
5–15bphavebeenfoundwiththesamemethodofequilibriumcentrifugation
of human DNA (Gosden, Mitchell, Buckland, Clayton, & Evans, 1975).
TheinventionoftherestrictionendonucleasesallowsfindingothersatDNA
families. Main human alpha-satDNA has been found with EcoRI and XbaI
4 OlgaPodgornayaetal.
restriction endonucleases (Jabs, Wolf, & Migeon, 1984; Manuelidis, 1976,
1978a,1978b). Long satDNA blocks of up to millions of base pairs built of
relativelyshort,fromseveraluptohundredsofbasepairs,monomerstandemly
arranged“headtotail.”Induetime,theterm“satDNA”hasbeenbroadenedto
comprisenearlyalltandemlyorganizedsequencesfoundincludingmini-and
microsatellites,whicharelocatedineuchromatin.Itcausessomeconfusion,for
mini-andmicrosatellitespossessfeaturesquitedifferentfromclassicalsatDNA
(Lo´pez-Flores&Garrido-Ramos,2012;Vogt,1990).
TandemlyrepeatedDNAmakesupasignificantportionofthemammal
genome.However,thereisnothingforsatDNAtoabuttoortobe“satellite”
ofanythingintheassembledgenomes;thearraysoftandemrepeatsjustcon-
tinuetheeuchromaticgene-containingregions.Nowadays,theterm“large
tandemrepeats”lookslikemoreadequateandweuseitinthistext.Theterm
“satDNA”couldnottotallybedrawnaway,forsomepreciselargetandem
repeatshaveitincludedintheirnameshistorically(e.g.,mouseMaSat,human
satellite3(HS3)).Ontheotherhand,thebulkofinformationobtainedabout
classicalsatDNAbyscientificcommunityisquiteapplicabletothelargetan-
demrepeats.
SequencesofsatDNAsaredifferentindifferentspecies,andtheleveloftheir
evolutional variability is high (Beridze, 1986; Podgornaia, Ostromyshenski˘ı,
Kuznetsova,Matveev,&Komissarov,2009).However,theyhavesomecom-
monstructuralfeatures(Fitzgerald,Dryden,Bronson,Williams,&Anderson,
1994;Lobov,Tsutsui,Mitchell,&Podgornaya,2001;Mart´ınez-Balba´setal.,
1990), and their positions at the chromosomes are fixed. A great part of
satDNAs of different families reside in the pericentromeric or subtelomeric
chromosome regions. All the eukaryote centromeric sequences investigated
uptonow,withtheexceptionofsimpleSaccharomycescerevisiaecentromeres,
contain satDNA as the main component (Choo, 1997; Pezer, Brajkovic´,
Feliciello,&Ugarkovc´,2012).
2.2. Mouse genome as a model for the tandem repeat search
Tandem repeat content on genome level is well investigated in the human
genome and shows a wide range of repeat sizes and organizations, ranging
frommicrosatellitesofafewbasepairstomegasatellitesofuptoseveralkilo-
bases.Microsatellitesandvariablenumberoftandemrepeats(calledVNTRs
or minisatellites) can be highly polymorphic and have an important use as
genetic markers (Ames et al., 2008; Warburton et al., 2008).
Thecentromericregionofhumanchromosomescontainsalpha-satDNA,
thelargestTRfamilyinthehumangenome.Thishasbeenextensivelystudied
LargeTandemRepeatsandtheChromosomeBarCode 5
andprovidesaparadigmforunderstandingthegenomicorganizationofTRs
(Schueler, Higgins, Rudd, Gustashaw, & Willard, 2001). Their arrays are
composed of either diverged monomers with no detectable higher-order
structureoraschromosome-specifichigher-orderrepeat(HOR)unitschar-
acterizedbydistinctrepeatinglineararrangementsofanintegralsetofbasic
monomers(Rudd&Willard,2004).TheHORstructureofhumancentro-
mericalpha-satellitehasbeenimplicatedasimportantincentromerefunction
(Schueleretal.,2001).
In humans, the pericentromeric regions consist of alpha-satDNAarrays
that are surrounded by arrays of “classical” satellites (e.g., human satDNA
1–4) (Choo, 1997; Lee, Wevrick, Fisher, Ferguson-Smith, & Lin, 1997;
Moyzis et al., 1987; Podgornaya, Bugaeva, Voronin, Gilson, & Mitchell,
2000; Prosser, Frommer, Paul, & Vincent, 1986). These pericentromeric
regionshaveaspecifichigh-orderchromatinstructureandmightberespon-
sible for chromatin spatial organization.
Inthehousemouse,Musmusculus,therearetwohighlyconservedTRs
known as centromeric minor and pericentromeric major satellites (MiSat
andMaSat,respectively,GSAT_MMandSATMINinRepbasenomencla-
ture). MiSat comprises an AT-rich, 120bp monomer that occupies
300–600kb of the terminal region of all mouse telocentric (single-armed)
chromosomesandisthesiteofkinetochoreformationandspindlemicrotu-
bule attachment (Kalitsis, Griffiths, & Choo, 2006; Kipling, Ackford,
Taylor, & Cooke, 1991; Wong & Rattner, 1988). MaSat (234bp) is more
abundant and resides adjacent to MiSat, and has a role in heterochromatin
formation andsister chromatidcohesion(Broccoli,Miller,&Miller,1990;
Broccoli, Trevor, Miller, & Miller, 1991; Guenatri, Bailly, Maison, &
Almouzni, 2004). Neither of these satDNA has been identified at the cen-
tromereofthemorphologicallydistinctacrocentricYchromosome(Ho¨rz&
Altenburger, 1981), which has a very small short arm that distinguishes it
fromthetelocentricautosomesandXchromosome.Recently,thechromo-
some Y centromere was shown to comprise a highly diverged MiSat-like
sequence (designated Ymin) with HOR organization previously not
describedformouseMiSatarrays(Pertile,Graham,Choo,&Kalitsis,2009).
2.3. Lack of tandem repeats in the reference genome
Thelargeregionsofclassicalheterochromatinarepoorlycoveredbyassem-
bled sequences (Warburton et al., 2008), and for the mouse genome even
lessis known.Mouseacrocentric chromosomeshave prolongedTRarrays
at the ends, and it is the reason why these regions are difficult to assemble.
6 OlgaPodgornayaetal.
Table1.1 LargeTRsintheregionadjustedtocentromericgap
Chromosome TRsubfamily Arraylength(kb) Coordinates(bp)
3 TRPC-21A-MM 33.6 3,000,001–3,033,629
4 TRPC-21A-MM 7.0 3,006,469–3,013,522
4 TR-22A-MM 4.9 3,104,899–3,109,811
6 TR-22A-MM 9.9 3,082,006–3,091,879
9 MaSat 38.4 3,000,003–3,038,419
11 MaSat 3.9 3,000,004–3,003,872
16 TRPC-21A-MM 9.0 3,232,335–3,241,336
17 TRPC-21A-MM 32.5 3,006,399–3,038,945
17 TR-27A-MM 4.6 3,070,530–3,075,093
18 TR-22A-MM 8.0 3,112,790–3,120,776
OnlyTRswiththearraymorethan3kbinthedistanceupto2Mbfromthecentromericgapareshown.
Coordinates,thearraypositiononchromosomes.
The mouse chromosomes end abruptly in 3Mb gaps reserved for centro-
mericregions.Wefoundoutthatonlyeightchromosomeendscontaindis-
tinctTRarraysandonlytwoofthemcontainMaSat(Table1.1).Theresult
obviously illustrated the bad assembly of the heterochromatic regions: the
rest of the chromosomes contain genes at the ends. The assemblies did
notcovereventhebeginningoftheregionsoftheconstitutiveheterochro-
matin (Komissarov, Gavrilova, Demin, Ishov, & Podgornaya, 2011).
Thesequencesfromthoseregionscanbefoundinwhole-genomeshot-
gun (WGS) contigs. The advantage of WGS is that they includethe entire
shotgunsequencingreads,assembledintocontigs.Bothassembliesrepresent
euchromatic and heterochromatic regions, even when not assembled into
continuouscontigsand/ornotanchoredonchromosomesyet.Theregions
enrichedinTRsaremostlynotanchored,althoughTRsand,inparticular,
large TRs are present in WGS due to their abundance in the genome.
2.4. Large tandem repeats in WGS assemblies
InWGSassemblies,theamountofallTRsislessthantheexperimentallydeter-
mined amount of the MaSat alone ((cid:1)8%) (Abdurashitov, Chernukhin,
Gonchar,&Degtyarev,2009),indicatingthateveninWGSassembliesTRs
remain underrepresented (Table 1.2). All large TRs found in the mouse
LargeTandemRepeatsandtheChromosomeBarCode 7
Table1.2 TandemrepeatsinmouseWGSassemblies
Assembly Size(bp) Contigs TRs(all) %ofassembly TRs(>3kb)
MGSC WGS 2,477,633,597 224,713 849,466 2.9 157
Celera WGS 3,003,109,157 837,963 1,084,552 5.0 784
Total WGS 5,480,742,754 1,062,676 1,944,018 3.8 941
TRs(all),totalamountfoundinassembly;MSGC,themousesequencinggenomeconsortium.
Figure 1.1 Schematic representation of the large tandem repeat classification
workflow.Overviewofthelargetandemrepeatanalysis.Foreachprogram,onlyparam-
etersthatwerechangedareshown.FamilynamesaregivenaccordingtoTable1.2.The
completedescriptionoftheworkflowisgiveninKomissarovetal.(2011).
WGSwereclassifiedinto4superfamilies,8families,and62subfamilies,includ-
ing60notdescribedyet,usingclassificationbasedonarraysimilarity,monomer
length,thedegreeofunitsimilarity,positiononthereferencegenomechro-
mosome assemblies, and GC content. Each new subfamily was named
accordingtothesuggestedcytogenetic-basednomenclature(theclassification
methodshownonFig.1.1).