Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus.

0.98 MB·English

by  Darst, Russell P.

Published online 29 January 2013 Nucleic Acids Research, 2013, Vol. 41, No. 5 2993–3009
doi:10.1093/nar/gkt033
Epigenetic diversity of Kaposi’s sarcoma–associated
herpesvirus
Russell P. Darst1,2, Irina Haecker2,3, Carolina E. Pardo1,2, Rolf Renne2,3 and
Michael P. Kladde1,2,*
1Department of Biochemistry and Molecular Biology, 2033 Mowry Road, Box 103633, University of Florida
College of Medicine, Gainesville, FL, 32610, USA, 2University of Florida Shands Cancer Center Program in
Cancer Genetics, Epigenetics and Tumor Virology, University of Florida College of Medicine, Gainesville, FL,
32610, USA and 3Department of Molecular Genetics and Microbiology, 2033 Mowry Road, Box 103633,
University of Florida College of Medicine, Gainesville, FL, 32610, USA
Received May 16, 2012; Revised November 27, 2012; Accepted January 8, 2013
ABSTRACT INTRODUCTION
Spontaneouslytic reactivation ofKaposi’s sarcoma– Kaposi’s sarcoma–associated herpesvirus (KSHV) infec-
associatedherpesvirus(KSHV)occursatalowratein tion causes sarcoma and lymphoproliferative disorders
in immunocompromised individuals [reviewed in (1)]. In
latently infected cells in disease and culture. This
thelatentphase,KSHVgenomesresidewithinthenucleus
suggests imperfect epigenetic maintenance of viral
as multicopy circular extrachromosomal elements or
transcription programs, perhaps due to variability in
episomes. Both micrococcal nuclease cleavage patterns
chromatinstructureatspecificlociacrossthepopu-
andimmunoprecipitationofhistoneproteinsdemonstrate
lation of KSHV episomal genomes. To characterize that episomal DNA is incorporated into nucleosomes
this locus-specific chromatin structural diversity, (2–4). Lytic reactivation, thought to be triggered by
we used MAPit single-molecule footprinting, which multiple cellular stress pathways in vivo, is associated
simultaneously maps endogenous CG methylation with changes in chromatin structure (3,4). Furthermore,
and accessibility to M.CviPI at GC sites. Diverse histonedeacetylaseandDNAmethyltransferase(DNMT)
chromatin structures were detected at the LANA, inhibitors can reactivate the lytic cycle in latently infected
cell culture, as can phorbol esters, e.g. 12-O-tetra-
RTA and vIL6 promoters. At each locus, chromatin
decanoylphorbol-13-acetate (TPA), which are protein
ranged from fully closed to fully open across the
kinase C agonists [reviewed in (5)]. Thus, episomal chro-
population. This diversity has not previously been
matin contributes to maintenance of the latent state and
reportedinavirus.PhorbolesterandRTAtransgene
the repression of lytic transcription.
induction were used to identify chromatin conform- Chromatin intrinsically regulates gene expression, as
ations associatedwithreactivationoflytictranscrip- many transcription factors exhibit reduced access to
tion, which only a fraction of episomes had. binding sites incorporated into nucleosomes [reviewed in
Moreover, certain chromatin conformations corre- (6)]. Furthermore, chromatin components often have
lated with CG methylation patterns at the RTA and post-synthesis chemical modifications (DNA methylation
vIL6 promoters. This indicated that some of the and acetylation, methylation, etc. of histones) that can
activate or repress transcription locally, as well as recruit
diverse chromatin conformations at these loci were
their own modifying enzymes. Chromatin modifications
epigeneticallydistinct.Finally,bycomparingchroma-
can thus constitute a self-perpetuating state. Such states
tin structures from a cell line infected with constitu-
are termed epigenetic if maintained throughout the cell
tively latent virus, we identified products of lytic
cycle, as in the case of DNA methylation [reviewed in
replication. Our findings show that epigenetic drift (7)]. Epigenetics underpin the differentiation of isogenic
can restrict viral propagation by chromatin compac- cells during development, as well as inactivation of one
tion at latent and lytic promoters. of two X chromosomes in female mammals. Imperfect
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: kladde@ufl.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
(cid:2)TheAuthor(s)2013.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
by-nc/3.0/),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
2994 NucleicAcidsResearch,2013,Vol.41,No.5
maintenanceofchromatinstates,i.e.epigeneticdrift,may with 10% (v/v) fetal calf serum (FCS), 2mM glutamine,
contribute to aging and cancer [reviewed in (8)]. 1 mM sodium pyruvate, 100 U/ml penicillin and
Epigenetic drift could be important for viral biology 100mg/ml streptomycin at 37(cid:2)C under 5% CO . TREx
2
as well. DNA methylation gradually builds over much of BCBL1-RTA cells for doxycycline-induced expression
the KSHV genome after de novo infection (3), indicating of RTA (31) were cultured in RPMI 1640 medium
that epigenetic changes occur. However, the phenotypic supplemented with 10% (v/v) FCS or tetracycline-free
consequences of this drift are unknown. Generally, DNA FCS, 2mM glutamine, 1mM sodium pyruvate,
methylationislinkedtotranscriptionalsilencing[reviewed 100U/ml penicillin and 100mg/ml streptomycin at 37(cid:2)C
in(9)],suggestingthatitcouldinactivatethevirus.Onthe under 5% CO . Telomerase-immortalized vein endothe-
2
otherhand,itmightprotectagainstthehostinnateimmune lial long-term–infected (TIVE-LTC) cells (32) were
responsetounmethylatedDNA(10). cultured in Dulbecco’s modified Eagle’s medium
Recently, ithasbecomepossible tocharacterize hetero- (DMEM) supplemented with 10% (v/v) FCS, 4.5g/l
geneity of chromatin structure at select loci within popu- glucose, 2mM glutamine, 1mM sodium pyruvate, 100
lations of mammalian nuclei by treatment with U/ml penicillin and 100 mg/ml streptomycin at 37(cid:2)C
recombinant M.CviPI DNMT [reviewed in (11–13)]. under 5% CO .
2
This enzyme, which we cloned from Chlorella virus, ForTPA-inducedreactivation,BCBL1cellsweregrown
methylates cytosine in GC dinucleotides to G-m5C (14). to a density of 2–3(cid:3)105 cells/ml. TPA was added to a
The methyl marks can be read by bisulfite genomic final concentration of 20ng/ml and cells were incubated
sequencing [BGS; (15)] and unequivocally distinguished atstandardconditions(37(cid:2)C,5%CO )for6h.Cellswere
2
from endogenous mammalian methylation at H-m5CG pelleted at 247g for 5min and resuspended in the same
(by convention, H equals A, C, or T). As nucleosomes volume of fresh RPMI medium and grown for another
and DNA-bound factors block access of exogenous 6h before nuclei isolation for MAPit. For reactivation
DNMTprobestoDNA(16–19),theresultantGCmethy- by doxycycline-inducible RTA, TREx BCBL1-RTA cells
lation pattern is used to infer chromatin structure. The were cultured for at least 2 days in tetracycline-free
combination of high-resolution probing with M.CviPI medium before performing the induction. Cells at a
and BGS is termed MAPit, for methylation accessibility density of 4–5(cid:3)105 per milliliter were then treated with
probingforindividualtemplates,developedbyourlabora- 1mg/ml doxycycline and 250mM foscarnet for 6h.
tory (20). By sequencing clonally expanded individual Foscarnet was used to suppress viral lytic replication;
molecules, MAPit allows detection of multiple chromatin quantification of viral load by real-time polymerase
states at selected loci within a cell population. This is in chain reaction (PCR) indicated that viral replication was
contrast to techniques such as chromatin immunopre- largely blocked (not shown). Cells were pelleted at 247g
cipitation, which report the population average for 5min and resuspended in the same volume of fresh
[reviewed in (11)]. Although new, MAPit is rapidly being RPMI medium with 250mM foscarnet and grown for
adapted to the study of diverse genomic loci (20–26). another 6h before nuclei isolation for MAPit.
RelatedstudiessupporttheabilityofMAPittoaccurately Uninduced BCBL1 or TREx BCBL1-RTA cells (treated
map gaps between nucleosomes across the genome (27) with250mMfoscarnet),respectively,servedasthe0htime
and track heterogeneity of chromatin structure (28,29). point.
Hypothesizing that KSHV populations would exhibit
epigenetic drift, we examined promoters and 50 ends of
genes of interest in latently infected cells by MAPit. The MAPit single-molecule footprinting
degree of diversity seen was surprising. Both fully closed
MAPit was performed as described previously (12,13).
andfullyopenconformationswerepresentatthreediffer-
Briefly, BCBL1 or TREx BCBL1-RTA cells were har-
ent KSHV promoters. Treatment with TPA and induced
vested at a density of 3–6(cid:3)105 cells/ml, washed twice
expression of the replication and transcription activator
with ice-cold phosphate-buffered saline (PBS), counted
(RTA) were used to test which of the diverse chromatin
and resuspended at 106 cells/ml in PBS. Four to five
structures found were linked to reactivation during lytic
million cells were stored as pellet at (cid:4)80(cid:2)C for later
transcription. At the time point tested, only a subset of
RNA extraction (see below). For each MAPit assay, 106
episomes appeared to have remodeled chromatin in
cellswerepelletedandresuspendedin200mlcoldBufferA
response to lytic reactivation by both means. We also
[(10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
observed a subset of episomes associated with lytic repli-
acid (HEPES)-KOH, pH 7.5, 2 mM MgCl , 10mM
cation, and identified a link between chromatin compac- 2
KCl, 2mM dithiothreitol (DTT), 0.2mM phenylmetha-
tion,CGmethylationandinhibitionofreactivation.These
nesulfonyl fluoride (PMSF)]. Cells were incubated on ice
findings suggest that latent virus is subject to progressive
epigenetic inactivation. for 10min to release nuclei by hypotonic lysis, then
vortexed 10s at medium velocity and centrifuged for 10s
at16000g.Tocheckforcompletecellularlysis,analiquot
MATERIALS AND METHODS ofnucleiin0.2%(w/v)trypanbluewereexaminedbylight
microscopy. Nuclei were only used for MAPit if the lysis
Cell culture
was complete, and nuclei were evenly round and granular
Body-cavity-based lymphoma cell line 1 (BCBL1) cells in appearance. Nuclear integrity was also confirmed in
(30) were cultured in RPMI 1640 medium supplemented parallel by Hoechst staining, which dyes DNA, and
NucleicAcidsResearch,2013,Vol.41,No.5 2995
fluorescence microscopy. Nuclei were washed twice with Bisulfite genomic sequencing
500ml cell resuspension buffer [20mM HEPES-KOH,
To deaminate, 2–4mg DNA was denatured for 5min at
pH7.5,70mMNaCl,0.25mMethylenediaminetetraacetic
95(cid:2)Cinfreshlymade0.3NNaOH,thenincubatedfor6h
acid (EDTA), 0.5mM ethylene glycol tetraacetic acid
at50(cid:2)Cinasaturatedmetabisulfitesolutionaspreviously
(EGTA), 0.5% (v/v) glycerol, 10mM DTT, 0.25mM
described(33).Bisulfite-convertedDNAwasdesulfonated
PMSF] and resuspended in 90ml methylation buffer (cell
and concentrated with a commercial kit (Zymo Research
resuspension buffer supplemented with 160mM S-
catalog no. D5026). Target loci were amplified from 20–
adenosyl-L-methionine).
100ngbisulfite-treatedDNAwith3UHotStarTaqDNA
TIVE-LTC cells were harvested at 100% confluency,
polymerase (Qiagen) and 250nM primers in 10mM
washedtwicewithicecoldPBS,countedandresuspended
tris(hydroxymethyl)aminomethane (Tris)–HCl, pH 8.6,
at106cells/mlinPBS.Cellswerepelleted(5(cid:3)106cellsfor
50mM KCl, 3 mM MgCl and 200mM of each 2’-
2
each MAPit assay), washed once in 250ml cell resuspen-
deoxynucleoside 5’-triphosphates (dNTP). Primers se-
sion buffer and lysed in 100 ml cell lysis buffer [cell resus-
quences are given in Supplementary Table S1.
pension buffer plus 0.25% (v/v) Nonidet P-40] for 10min
Amplification was performed in 2–3 separate reactions
onice.Nucleiwerepelletedat1000gfor5minat4(cid:2)C,then
to minimize stochastic variation in PCR amplification,
washed twice with 500ml cell resuspension buffer and re- then pooled for cloning. Cycling conditions were 5min
suspended in 90ml methylation buffer. at 95(cid:2)C, followed by 40 cycles of 1min at 95(cid:2)C, 1min at
To methylate accessible GC sites, 30U recombinant 56(cid:2)C–60(cid:2)C, (optimized for each primer pair), 3min at
M.CviPI-maltose binding protein (MBP) fusion [New 72(cid:2)C, and a final extension of 5min at 72(cid:2)C.
England Biolabs, hereafter M.CviPI; (14)] were added Amplified DNA was separated from unincorporated
per 106 nuclei (based on cells counted after all washes primers by electrophoresis in 1% (w/v) agarose buffered
completed), which were then incubated at 37(cid:2)C for withTris-acetate-EDTA,excisedandpurifiedfromthegel
15min. Reactions were stopped by addition of an equal and ligated into a cloning vector using topoisomerase
volume of 1% (w/v) sodium dodecyl sulfate (SDS), (TOPO TA; Invitrogen). Calcium-competent DH10b or
100mM NaCl, 10 mM EDTA and incubated overnight at TOP10 cells were transformed with 10–50ng plasmid
50(cid:2)C with 100mg/ml proteinase K. DNA was then phenol and plated on lysogeny broth (LB) medium with 100mg/
extracted(partitionedwithanequalvolumephenol:chloro- ml ampicillin and 40mg/ml 5-bromo-4-chloro-3-indolyl-b-
form:isoamyl alcohol::25:24:1) and ethanol precipitated. A D-galactopyranoside (X-gal) for blue/white screening.
0UM.CviPIcontrolreactionwasalsoperformedtogauge Colony PCR with M13 forward and reverse primers was
endogenous GC methylation (not shown; none was also used to test cloning efficiency. Colonies containing
observed at any locus). the insert were suspended in 100ml LB plus 100mg/ml
To ensure comparable results between experiments, ampicillin and grown at 37(cid:2)C overnight in 96-well plates
M.CviPI activity was calibrated between aliquots of without shaking. Glycerol was then added to 10% (v/v)
enzyme using methylation of a HaeIII site in the and the plates were incubated at (cid:4)80(cid:2)C for at least 2h,
GRP78 promoter in BCBL1 cells as a standard. MAPit whereuponplasmidwassubjectedtoTempliPhiTMrolling
experimentswerealsoconductedat10ratherthan70mM circle amplification and automated BigDye sequencing
NaCl, with 3-fold higher and lower effective M.CviPI (Applied Biosystems) at the University of Florida
concentration, and over a 3-fold range in M.CviPI incu- Interdisciplinary Center for Biotechnology Research
bation time (Supplementary Figures S1 and S2). A (http://www.biotech.ufl.edu).
non-linear relationship was seen between M.CviPI con-
centration and degree of methylation. This matches a Sequence analysis
simple model in which DNA molecules adopt multiple
The genomic sequences corresponding to BGS amplicons
chromatin conformations that vary in accessibility to
are listed in Supplementary Table S2. Sequence files in
M.CviPI. ABI format were aligned in SequencherTM (Genecodes)
and are available on request. MethylViewer [http://dna.
leeds.ac.uk/methylviewer; (22)] was used to ascertain
Restriction (R) endonuclease R.HaeIII probing of nuclei
percent cytosine conversion and tabulate methylation
Direct treatment of nuclei with R.HaeIII confirmed status of each site in each sequence read (i.e. molecule).
relative differences in accessibility between the LANA Moleculeswith<95%conversionofcytosinesoutsidepo-
promoter and genomic loci (Supplementary Figure S3). tential methylation sites were discarded. Molecules from
Briefly, 4(cid:3)105 nuclei were prepared as above and sus- thesameinitialDNAsamplethathadidenticalpatternsof
pended in 100ml 20 mM HEPES-KOH, pH 7.5, 70mM both methylation and unconverted cytosines were
NaCl, 10mM MgCl , 0.5% (v/v) glycerol, 10mM DTT, presumed to be duplicate clones, and all copies but one
2
0.25mM PMSF, plus 0-100 U R.HaeIII (New England were then discarded. Molecules with either zero methyla-
Biolabs) or 50% (v/v) glycerol in 10ml. After 30min at tionor100%conversionofcytosineswerenotscreenedin
37(cid:2)C, reactions were stopped by addition of an equal thisway, asthere were insufficient groundstoinfer dupli-
volume 1% (w/v) SDS, 100mM NaCl, 10mM EDTA cation. Duplicates made up 0–10% of molecules in each
and incubation for 20min at 80(cid:2)C. Genomic DNA was dataset.
preparedasdescribedaboveandpercentdigestquantified The tables of methylation status were used to generate
by real-time PCR. digitalmethylationmapsforbothCGandGCmethylation
2996 NucleicAcidsResearch,2013,Vol.41,No.5
foreachmoleculeinExcel(Microsoft). Withinthe LANA, SYBR Green Master Mix and a StepOnePlus real-time
RTAand vIL6 promoters,CGmethylation was rare; thus, PCR instrument (both Applied Biosystems). Cycle condi-
GCGsiteswerecountedasGCsitestoincreaseresolution. tions were initially 95(cid:2)C for 10min, 40 cycles of 95(cid:2)C for
For the amplicon overlapping the LANA open reading 15s, 60(cid:2)C for 1min, followed by melting curve analysis.
frame (ORF), GCG sites were excluded from the analysis. For quantification of M.CviPI-mediated protection
Digital methylation maps were clustered hierarchically from R.HaeIII digest, 1mg undeaminated DNA was
using Cluster [http://rana.lbl.gov/EisenSoftware.htm; (34)], digestedwith10UR.HaeIII,aspertheenzymemanufac-
and printed as color maps with MapleTree (http:// turer’s directions, or mock-treated with an equal volume
mapletree.sourceforge.net). Dendrograms were split by of 50% (v/v) glycerol. For each sample, three wells with
eye into clusters of interest. Molecules in each cluster 10ng DNA apiece were quantified by real-time PCR, as
originating from each experiment were counted. above.
Expectation values for the observed distributions were
computed as two-tailed Fisher’s exact tests. To generate
the accessibility profile (Figure 2D), the fraction of RESULTS
sequence reads bearing accessible spans of 1–100 or
Multiple chromatin conformations in the KSHV
101–400bp was computed at each base pair position.
latency-associated region
The difference in accessibility after TPA treatment was
then subtracted from that before treatment. Likewise, to LANA,vFLIP,vCyclin,Kaposinand12pre-microRNAs
generate occupancy profiles (Figures 3D, 5D and 8D), the are highly expressed off polycistronic transcripts arising
fraction of sequence reads bearing footprints of 4–39, from a single KSHV latency-associated region (KLAR)
40–100 or 101–200bp was computed at each base pair in tumor cells. During latency, host RNA polymerase II
position.Footprintsextendingtotheendsoftheamplicons initiates transcription of KLAR from the upstream latent
were not counted. To get the difference in occupancy, the transcription start site (TSS; Figure 1B). Additionally, a
fraction for one experimental condition was then sub- second downstream TSS is used during lytic reactivation
tracted from the fraction for another. (36). Three CCCTC-binding factor (CTCF) binding sites
are located between these two TSSs and the LANA open
RNA preparation for real-time reverse transcription readingframe(ORF73);theiroccupancybythechromatin
PCR (RT–PCR) insulator CTCF (37) contributes to LANA expression,
mediates chromosomal looping and plays a role in
BCBL1 and TREx BCBL1-RTA cells were treated as
episomal maintenance (38,39). All KSHV-infected cells
described above in two independent experiments
express LANA, which is required for replication and
(including different stocks of cells). From 4 to 5(cid:3)106
maintenance of viral episomes in latently infected cells
induced and uninduced cells, RNA was extracted using
[reviewed in (40)]. Yet, not all of the (cid:5)80 episomes
RNA Bee (TEL-TEST, Inc.) according to the manufac-
present in latently infected lymphoma cells need express
turer’s instructions. RNA was DNase I treated [10mg
LANA. We hypothesized that a subset of episomes might
RNA, 1ml RNasin(cid:3) (Promega), 1(cid:3)DNase I buffer, 2ml
provide LANA function for the rest, which could be
(4U)RNase-freeDNaseI(NewEnglandBiolabs)in50ml
epigenetically inactivated.
totalvolume]for25minat37(cid:2)Candrecoveredbyethanol
As a single-molecule technique, MAPit provided a way
precipitation after (cid:5)16h at (cid:4)20(cid:2)C. An aliquot of
totestthishypothesisdirectly.AkeyadvantageofMAPit,
DNase I–digested RNA was tested for RNA quality by
incontrasttopopulationaveragereadouts,istheabilityto
agarose gel electrophoresis and the rest stored at (cid:4)80(cid:2)C.
discover distinct subpopulations without enrichment.
RNA (1–3mg) was reverse transcribed using oligo dT
Nuclei from the KSHV-infected pleural effusion
primerandInvitrogenSuperscriptIIIreversetranscriptase
lymphoma (PEL) cell line BCBL1 were incubated with 0
(RT)ina20mlreactionmixtureasindicatedbythemanu-
or 30U M.CviPI, following the protocol outlined in
facturer (20ml reaction mixture per 1mg RNA; for more
Figure 1A. We cloned and sequenced 10 bisulfite-
RNA, the assay was scaled up accordingly). A reverse-
converted molecules encompassing the LANA promoter
transcription reaction without RT enzyme served as
from each sample. The patterns of CG and GC methyla-
control (no RT).
tion were drawn by MethylViewer (22), which represents
methylated cytosines as ball-and-stick symbols
Quantitative real-time PCR
(Figure 1B). Efficiency of conversion was estimated as
According to the MIQE guidelines for quantitative percent HCH (H is any base but G) conversion in each
real-time PCR [qPCR; (35)], we refer to reference genes sequence read (i.e. cytosine outside potential methylation
instead of housekeeping genes and Cq (quantification sites; blue vertical ticks mark unconverted HCH). As
cycle) instead of Ct (threshold cycle). All primers expected, without M.CviPI addition, methylation of
(Supplementary Table S1) were purchased from GCHsiteswasnotabovethebackgroundgivenbyincom-
Integrated DNA Technologies, Inc. and tested for their plete conversion. The M.CviPI-probed sample exhibited
efficiency (between 90 and 110%) and specificity prominent accessibility above background between and
(melting curve analysis). adjacent to three known CTCF sites. Some molecules
Per 12ml qPCR reaction, 0.5ml complementary DNA were also accessible near the latent TSS.
was used. Primers were used at a final concentration of Although MethylViewer is excellent for comparing dif-
200nM. Two-step qPCR was performed with the Fast ferentmethylationtypesonsinglemolecules,suchdetailed
NucleicAcidsResearch,2013,Vol.41,No.5 2997
Continued
A B
C
D
E
Figure 1. MAPit analysis of chromatin structural diversity. (A) MAPit workflow diagram, adapted from Darst et al. (13). Chromatin (e.g. nucleo-
somes)modulatesaccessibilityofDNAsitestosolubleproteins,suchas(i)transcriptionfactors(representedbyshadedrectangle)inlivecells,or(ii)
exogenous M.CviPI DNMT in nuclei. Bisulfite treatment (iii) records M.CviPI accessibility pattern in the DNA sequence by conversion of
unmethylated cytosine to uracil (cyan). Subsequent amplification and cloning (iv) allows recovery of this information from individual molecules.
Note that endogenous and probe methylation sites are distinguished by sequence specificity of the respective DNMTs (respectively, cyan and
magenta). The DNA sequences obtained are used to generate maps (v) of DNA methylation and accessibility. (B) MAPit at the KSHV gene
LANAdetectedendogenousmethylationandsporadicaccessibilityofthelatentpromoter.MAPitwasperformedinnucleiofBCBL1cells.Top:map
of genetic elements within the MAPit amplicon: LANA ORF (ORF73), three CTCF sites (ovals), lytic and latent TSSs (bent arrows) and TATA
boxes (triangles). Middle: MethylViewer (22) display of MAPit results from control nuclei probed as indicated with 0 U or 30 U M.CviPI. Blue
2998 NucleicAcidsResearch,2013,Vol.41,No.5
graphs areunwieldy forlarge-scale analysis. Wetherefore and cut its sites on most molecules. Thus, variation at
devised a three-color map scheme (Figure 1C), used LANA was not due to uneven probing of nuclei.
throughout the rest of this work. Patches of accessibility As indicated by the second locus shown, the GAPDH
to DNMT (M.CviPI and endogenous DNMTs) received promoter, heterogeneity of promoter chromatin structure
bright color (yellow and red, respectively), and patches of was not unique to LANA. Variation in chromatin state
protection were colored black. Gray color denoted between copies of the same gene can arise from several
borders between methylated and unmethylated patches. causes. Epigenetic variation occurs in X inactivation, im-
We used unsupervised hierarchical clustering to order printing and monoallelic expression. Dynamic variation
molecules, facilitating pattern recognition. Color maps occurs in periodic and sporadic cycles of chromatin re-
for both GC and CG methylation status were clustered modeling and transcription [for instance, (41)]. However,
together. The 16 GCG sites were disregarded in this none of these phenomena have been described for a viral
analysis, leaving 52 sites for GC and 36 for CG. genesuchasLANA.Inanycase,theimplicationwouldbe
The data showed ‘banded’ CG methylation across that the promoters in the closed conformation do not
almost all molecules in the LANA ORF. By contrast, support transcription. Thus, as hypothesized, not all
the 10 molecules varied in degree and pattern of episomes continuously express LANA. Because all
M.CviPIaccessibility.Thiscouldreflectvariationinchro- infected cells express LANA (32), this result implies het-
matin structure between episomes. An alternate explan- erogeneity of episomes within individual nuclei.
ation would be a poor nuclear preparation, such that the
M.CviPI probe did not enter all nuclei. However, this MAPit survey of LANA promoter architecture
seemsunlikely as nuclei appeared intact and homogenous In addition to the latent TSS, the LANA promoter uses a
by vital (trypan blue) and Hoechst staining (not shown). lytic TSS on reactivation (36). Furthermore, transcription
Moreover, all 10 clones of the LANA amplicon exhibited starting at the latent TSS overlaps the lytic K14 promoter
GC methylation above background unconverted HCH on the opposite strand. We asked whether MAPit could
(Figure 1C, compare lower panels in 0 versus 30 U visualize changes in chromatin structure within this
M.CviPI). In addition, we performed BGS at select host complex regulatory region during lytic reactivation
loci from the same sample. Two representative loci are induced by phorbol ester treatment. To test this, we
shown (Figure 1D). At the more open locus, the cloned and sequenced 102 amplified deaminated LANA
promoter of GRP78, all 17 molecules sequenced had GC promoter molecules from M.CviPI-treated BCBL1 nuclei
methylation.Thislocuswaspreviouslyfoundtobehighly (0h or uninduced), and 110 molecules from
accessible to a DNMT probe in a colorectal line (28,29). M.CviPI-treated nuclei harvested after 12h of TPA treat-
Because MAPit requires amplification and cloning of ment of cells (12h or induced). Induced transcription of
bisulfite-converted DNA, it can be affected by sequence- two early lytic genes, RTA (ORF50) and vIL6 (ORF K2),
specific biases in either step. We therefore measured was demonstrated by real-time RT-PCR (Supplementary
M.CviPI methylation by R.HaeIII digestion of undea- Figure S4). An early time point was chosen to obtain in-
minated DNA from the same samples used for MAPit complete lytic reactivation. Otherwise, histone-depleted
(Figure 1E and Supplementary Figure S2). As R.HaeIII products of viral DNA replication would out-compete
doesnotdigestitssiteGGCCwhenthecentralcytosineis cloning of BGS products derived from chromatinized
methylated,theconcordancebetweenR.HaeIIIprotection viral episomes (4,42). Hence, these induction conditions
and BGS showed the latter to be unbiased. In turn, this did not have an obvious effect on M.CviPI accessibility
meant that M.CviPI efficiently permeated nuclei and averaged across the population (Figure 2A and B).
accessed the GRP78 promoter. We also probed nuclear We therefore asked whether differences might be
DNA directly with R.HaeIII, without prior M.CviPI detected in a single-molecule analysis. To determine
treatment (Supplementary Figure S3). The restriction which methylation patterns were enriched in one dataset
enzyme, which binds DNA cooperatively as a dimer and relative to the other, we pooled all reads from the 0- and
thus is similar in size to the monomeric M.CviPI fusion 12-h TPA treatment time points and clustered them hier-
protein,respectively,74.2and83.7kDa,wasabletoaccess archically as GC methylation plots (Figure 2C). We
Figure 1. Continued
verticalticksindicatepositionsofunconvertedcytosines,excludingGCandCGsites(i.e.HCH);percentconversionofallHCHcytosinesforeach
sequence clone is given on the right. Bottom (key): Circles and triangles, respectively, indicate CG and GC sites. Methylated sites are shaded.
Methylated GCG sites are shaded gray to indicate their ambiguity; however, comparison of sequences from nuclei with and without M.CviPI
treatment(middle,lowerandupperpanels)indicatedthatGCGsiteswithintheLANAORFwereendogenouslymethylated.(C)Condensedviewof
the data in B, to scale with locus map at top. Each row of pixels represents one cloned sequence, i.e. molecule. Each plot tracks only GC or CG
methylation;GCGsitesareignored.Asindicatedbykeyatbottom,spansofcolormark(cid:6)2contiguousmethylatedsites;blackmarks(cid:6)2contiguous
unmethylated sites; gray marks spans between methylated and unmethylated sites; white indicates missing or unaligned sequence. Positions of sites
areindicatedbycirclesandtriangles(HCGandGCH,respectively).(D)HostpromotersvariedindegreeofaccessibilitytoM.CviPI.MapsofGC
methylationattworepresentativepromotersareshown,toscalewithLANAamplicon.(E)MAPitatthethreelocishownwassemi-quantitative.At
the HaeIII sites indicated in red at GAPDH, GRP78 and LANA, 10 of 20, 15 of 17 and 1 of 10 molecules had a methylated GC site, respectively
(blue bars). R.HaeIII digestion followed by quantitative real-time PCR was used to measure bulk methylation of the same three sites in genomic
DNApurifiedfromM.CviPI-treatednuclei(redbars).Undertheseconditions,(cid:5)95%ofDNAfromuntreatednuclei(i.e.unmethylated)wasdigested
(notshown).TheconcordancebetweenBGSandR.HaeIIIdigestindicatedthatrepresentationofmethylatedandunmethylatedmoleculesinMAPit
was unbiased.
NucleicAcidsResearch,2013,Vol.41,No.5 2999
plotted accessibility to M.CviPI in each molecule as blue chromatin structure visible by MAPit. Induced reactiva-
(0h) or yellow (12h) to mark its source and split the den- tion would be predicted to shift these molecules to the
drogram into clusters of interest. To better visualize actively transcribing state, which might involve nucleo-
differences in chromatin structure between clusters, each some repositioning. We therefore cloned and sequenced
was rescaled vertically. The fraction of molecules present 114 and 112 molecules from M.CviPI-treated nuclei har-
in each cluster from each time point is given on the right. vested after 0- or 12-h TPA treatment, respectively
The KLAR amplicon in this experiment was positioned (Figure 3).
farther upstream from the LANA gene than that Accessibility to CG and GC DNMTs was computed at
analyzed in Figure 1, allowing better characterization of each position. Within ORF48 (50 of RTA), a peak of CG
accessibilityattheLANAlatentTSS.Strikingly,thethree methylationwasobservedupstream(313–284bpupstream
CTCF sites were seen to be occupied by footprints in of the RTA TSS) of a positioned nucleosome, hereafter
almost all molecules. Even in cluster i, which had N(cid:4)1 (Figure 3A and B). The same position had been
reduced M.CviPI methylation overall, the three CTCF previously shown to be endogenously methylated in
sites were dimly outlined. Importantly, this demonstrates BCBL1,althoughinalargerproportionofthepopulation
that M.CviPI permeated these nuclei, but was preferen- (53). A second notable feature of this plot was a broad
tially excluded from the latent TSS (especially compared peakinGCaccessibilitycentered(cid:5)100-bpupstreamofthe
with clusters iii and iv). RTATSS.Anopenchromatinstructurewasnotexpected
For each cluster, we found the probability P of having to be so common in the latent state. Moreover, there was
so skewed a distribution of molecules from the two no obvious difference between GC accessibility profiles
samples by chance. Although clustering revealed several before and after TPA treatment (Figure 3A). Thus, the
distinct chromatin conformations, TPA treatment bulk of the population was unchanged. As expected, the
increased representation of molecules only in cluster iii GAPDH(SupplementaryFigureS2C)andGRP78control
(by 13%) with borderline significance (P=0.05). To loci also showed no change in chromatin architecture in
further examine TPA-induced changes in LANA responsetoTPA(compareSupplementaryFigureS5with
promoter chromatin structure, we calculated accessibility Figure 1D, upper).
of lengths 1–100 and 101–400 bp occurring at each As before, molecules from the two time points were
position, and subtracted accessibility before TPA treat- clustered together to identify chromatin conformations
ment (0h) from that after (12h) (Figure 2D). TPA treat- enriched in either (Figure 3C). Cluster i, comprising
ment decreased accessibility adjacent to the CTCF sites (cid:5)50% of all molecules, was, as at KLAR, largely (but
(1–100bp, red curve) and just downstream of the LANA not completely) inaccessible to M.CviPI and presumably
latent TSS (101–400bp, blue curve) in (cid:5)10–15% of the transcriptionally inactive. Clusters ii–v all had an N(cid:4)1
molecules, corresponding well with the number of mol- footprint. Clusters ii and iii appeared similar to the eye:
ecules TPA increased in cluster iii. We conclude that both featured three evenly spaced positioned
TPA increased protection at the K14 TATA element, nucleosome-sized footprints. However, cluster iii was
mainly in cluster iii. more closely related to cluster v than ii in the original
Clustering also highlighted the large degree of diversity dendrogram (Supplementary Figure S6). The central
of chromatin structure at this locus. Both fully protected N+1 nucleosome in cluster ii covered several previously
and almost fully accessible molecules were detected. identified transcription factor binding sites upstream of
Cluster ix was of particular interest as it appeared to be the RTA TSS. The N+1 and N+2 nucleosomes seemed
nearly nucleosome free. Complete chromatin unfolding to shift slightly downstream in cluster iii versus ii. Cluster
and nucleosome displacement across a region of this size iv had a variably positioned N(cid:4)1 footprint, followed
downstream by a large open gap, then 2–3 variably pos-
has not been observed at any human chromosomal locus
itioned nucleosome-sized footprints. Cluster v molecules
studied by MAPit. We hypothesize that molecules of
shared a large M.CviPI-accessible linker upstream of
cluster ix originated from spontaneous lytic reactivation
N(cid:4)1, with chromatin that appeared to be denser down-
of KSHV, which occurs within 1–3% of PEL cells
streamovertheRTAtranscribedregion.Clustervilacked
(30,43,44). As is the case for other herpesviruses, the
N(cid:4)1. Lastly, cluster vii was, just as cluster v found at
KSHV genome is depleted for histones during lytic repli-
KLAR, almost entirely accessible and again comprised
cation before being packaged into virions (4,42). These
(cid:5)3% of all molecules.
results indicated that MAPit could discover chromatin
Clustersii,vandvihadPvaluesbelowtheusualthresh-
conformations comprising <(cid:5)5% of a complex
old for significance (<0.05). Because clusters ii and vi
population.
decreased in response to TPA, they may represent tran-
scriptionallypoisedstates,primedtorespondtoactivating
A subset of episomes undergoes changes in chromatin
stimuli. Cluster iv would then correspond to a transcrip-
structure during reactivation
tionallyactivestateorathermodynamicallystablestagein
The master regulator of lytic transcription is the replica- the cycle of RNA polymerase recruitment and release.
tion and transcription activator RTA (45). RTA directly Although it had too few molecules for statistical signifi-
activates several downstream viral genes, such as vIL6, as cance, cluster iii was also enriched for molecules from
wellastranscriptionofitsowngene(46).Wehypothesized TPA-treated cells and might also represent a stage in the
that a subset of latent episomes would be poised to tran- transcription cycle.Comparing theincrease incluster v to
scribe RTA (ORF50) and that these would have a distinct the decrease in clusters ii and vi, we conclude that at least
3000 NucleicAcidsResearch,2013,Vol.41,No.5
A 0 h HCG 12 h HCG 0 h GCH 12 h GCH A 0 h HCG 12 h HCG 0 h GCH 12 h GCH
60 50
C C
305m 5m25
% %
0
400 200 0 –200 0
Distance to LANA latent TSS (bp) –400 –200 0 200 400
Distance to RTA TSS (bp)
B LANA lytic TSS LANA latent TSS
CTCF B Asp718 HinCII
O A X RTA (ORF50)
ORF K14 ORF48 ORF49
Y S T
C
TPA
C 0 h 12 h P N–1 N+1 N+2
TPA
C 0 h 12 h P
i 44% 42% 0.78
i 48% 51% 0.79
ii 15% 11% 0.42
ii 9% 2% 0.03
iii 12% 25% 0.05 iii 4% 7% 0.25
iv 7% 10% 0.47 iv 18% 11% 0.18
v 11% 22% 0.03
v 5% 3% 0.48
vi 8% 1% 0.02
vi 4% 1% 0.19
vii 3% 6% 0.21
vii 5% 3% 0.48
–200 0 200
Distance to RTA TSS (bp)
viii 6% 4% 0.76 D 4-39 bp 121-200 bp
1150at 12 hat 0 h
ix 3% 2% 0.62 5ncy ncy
0aa
pp
400 200 0 –200 uu
Distance to LANA latent TSS (bp) – 5occocc
D 1-100 bp 101-400 bp 1012 h0 h –400 –200 0 200 400–10% –
at at Distance to RTA TSS (bp)
–100essibility essibility FaretidgRu;rT1e2A-3.h.(TIAnP)dAuPcrteordfiealeltymtoiecfnart,evaegcrrtaeigveenat)ieonannddoagleteexnrooegudesncoChurGosmG(0aC-thinmTsePttahAtyeltafrtreieoaqtnume(en0nc-thy,
–20accacc TPA treatment, black; 12-h TPA treatment, blue) across the locus.
400 200 0 –200 % – GCG sites were not counted. Note locus of highest CG methylation
Distance to LANA latent TSS (bp) coincided with a peak of GC methylation; elsewhere, CG methylation
Figure 2. TPA treatment altered KLAR chromatin structure in (cid:5)10% was minimal. (B) Diagram of RTA locus amplified. Triangles mark
of BCBL1 episomes. Note that this KLAR amplicon, although some of the known or suggested transcription factor–binding sites:
overlapping the amplicon of Figure 1, was centered upstream to C/EBP (C) (47), OCT-1 (O) (48), YY1 (Y) (49), AP-1 (A) (50), Sp1/3
better observe accessible DNA at the latent TSS. (A) Profile of en- (S) (2), XBP-1 (X) (51) and TATA (T). A second RTA TSS was
dogenous CG (0–h TPA treated, red; 12-h TPA treated, green) and recently reported roughly 500-bp upstream of this amplicon (52).
exogenous GC methylation (0-h TPA treated, black; 12h TPA Below, inferred nucleosome positions are indicated as two overlapping
treated, blue) across the locus. GCG sites were not counted. grayovals,each150-bplong.(C)ClusteredGCmethylationmapsfrom
(B) Schematic of the analyzed amplicon. The LANA ORF begins left 0-h (blue) and 12-h (yellow) treatment with TPA. Methylation of GC
oftheamplicon.TheLANAlatentandlyticTSSsareindicated.TATA sites(includingGCG,asm5CGwasrare)wasplottedasinFigure1,to
boxes are indicated by triangles (upside down, latent and lytic LANA; scale with panels (A, B and D). Position of GC sites is indicated by
righted,ORFK14).(C)AllGCmethylationplotsfor0-and12-hTPA hashes at top. Maps from both data sets were sorted together by un-
treatment(blueandyellow,respectively),includingGCG,asm5CGwas supervised, hierarchical clustering. Seven clusters of interest are pre-
rarein(A),wereorganizedbyunsupervisedhierarchicalclustering.GC sented. On right are given the proportion of sequence reads from
sites are indicated above; plot is to scale with other panels. each data set present in each cluster, and estimate of expectation
Dendrogramwassplitintoninefractionsofinterest.Onright,propor- value P assigned as in Figure 2. Columns do not sum to 100% due
tion of sequence reads from each data set present in each cluster torounding.(D)Differenceinoccupancyfrom0to12hofTPAtreat-
(columns do not sum to 100% owing to rounding). Expectation value ment. Footprints in two size ranges, 4–39 and 121–200bp, were
Pisthechancethatasunevenadistributionofmoleculesfromthetwo counted at every position in each dataset, then the 0-h profile was
datasets would occur if molecules had been assigned to the cluster at subtracted from the 12-h profile. An intermediate size range,
random. (D) Difference in accessibility from 0- to 12-h of TPA treat- 40–120bp, showed no difference between the data sets.
ment. Cumulative accessibility in two size ranges, 1–101 and 101–
400bp, was tallied at every position in each dataset, then the 0-h
profile was subtracted from the 12-h profile.
NucleicAcidsResearch,2013,Vol.41,No.5 3001
10% of episomes remodeled RTA chromatin in response episomes to be expressing a downstream target of RTA,
to TPA. By the same token, a large fraction of molecules an immediate-early gene such as vIL6. We cloned 96 and
appearednottohaverespondedtoTPAatthistimepoint, 71 molecules amplified from bisulfite-converted DNA
includingthoseofclusteri,whichwerehighlycompacted. isolated from TPA-treated and untreated cells, respect-
Severalsmallfootprintscovering1–2GCsitesoccurred ively. Unlike at RTA, at the vIL6 promoter (within
at 93, 86 and 74bp upstream of the TSS (Supplementary ORF2), there was a wide peak of accessibility to
Figure S6), which might represent some of the transcrip- M.CviPI (Figure 4A and B). As with RTA, no change in
tionfactorsreportedtobindthisarea.Todeterminewhich bulk accessibility was seen on TPA treatment from the
footprints were more abundant in each dataset, we methylationplotsalone.However,clusteringofmolecules
counted footprints of lengths 4–39 and 121–200bp once again showed otherwise. As at KLAR and RTA,
occurring at each position and subtracted footprint there was both an abundant, highly inaccessible, likely
counts before TPA treatment from those after silenced cluster (Figure 4C, cluster i) and a rare, highly
(Figure 3D). The result is difference in occupancy of accessible cluster (v), suggesting that the underlying chro-
factors within each footprint size range at each base pair matinbehaviorwas episomewide. Again, neitherof these
position. This analysis revealed greater definition of the two clusters changed significantly on TPA treatment.
N(cid:4)1 footprint (broad peak in black spanning from However, cluster iv, lacking two or more nucleosomes
(cid:4)300 to (cid:4)100bp relative to the RTA TSS), repositioning within the vIL6 promoter, did increase significantly from
of nucleosomes inside the transcribed region of RTA 8 to 24% of molecules after induction (P=0.01). This
(sharp peaks on right), and changes in some suggests chromatin remodeling in response to activation
sub-nucleosomal footprints flanking these areas (in red), by RTA. Moreover, these numbers were consistent with
but not changes in the putative transcription factor foot- the estimate that (cid:5)10% of episomes were transcribing
prints. Nucleosome repositioning in the gene was consist- RTA at this time point. The observation that 8% of mol-
ent with the hypothesized shift from cluster ii to iii. ecules showed the same vIL6 chromatin accessibility
Together, these results suggested that only a subset of during latency is in congruence with a recent report of
KSHV episomes had responded to TPA by reorganizing low levels of vIL6 expression during latency (54). As at
RTA promoter chromatin by 12h of TPA induction. RTA, results were consistent with a model of heteroge-
From this, one would predict a similar proportion of neous reactivation of the lytic pathway, in which an
0 h HCG 12 h HCG 0 h GCH 12 h GCH
A 50
C
5
m25
%
0
–200 –400 –600 –800
Distance to vIL6 TSS (bp)
B
vIL6 ORF2 ORF K3
TPA
0 h 12 h P
C 15%
i 63% 49% 0.11
ii 16% 8% 0.24
iii 9% 13% 0.62
iv 8% 24% 0.01
v 4% 6% 0.72
0 –200 –400 –600 –800
Distance to vIL6TSS (bp)
Figure 4. TPA treatmentaltered chromatin structureatthevIL6gene in10–15%ofBCBL1episomes. (A)ProfileofaverageendogenousCG(0-h
TPA treatment, red;12-h TPAtreatment, green) andexogenous GCmethylation (0-hTPA treatment, black;12-h TPA treatment, blue)across the
locus. (B) Diagram of vIL6 locus amplified. Little has been reported of the structure of the vIL6 promoter, except for the two shown TSSs (bent
arrows) (74). (C) All GC methylation plots for 0- and 12-h TPA treatment (blue and yellow, respectively, including GCG) were organized by
unsupervised hierarchical clustering. GC sites are indicated above; plot is to scale with other panels. Dendrogram was split into five fractions of
interest. On right, proportion of sequence reads from each data set present in each cluster, and expectation value P calculated as in Figure 2.
3002 NucleicAcidsResearch,2013,Vol.41,No.5
openchromatinstructuresuchasinFigure3C,clusterviis AP-1 to the promoter, but direct RTA induction acts
prerequisite.Weconcludethatchromatinstructurereflects through C/EBP (47,50).
ability of episomes to respond to inducers of lytic As in the BCBL1 cells assayed in Figure 3, a large
transcription. fraction of cloned sequences showed promoters with
As a protein kinase C agonist, TPA induces weak sig- compact chromatin, inaccessible to M.CviPI (Figure 5C,
naling that may resemble a pathway for spontaneous cluster i). The fraction of KSHV episomes with closed
KSHV reactivation in vivo. To further examine the chromatin did not significantly decrease in response to
extenttowhichthefractionofKSHVepisomes withrela- RTAinductionintransdespite30-foldincreased RTAex-
tivelyinaccessiblechromatin(i.e.inclusteri)wasresistant pression as compared with TPA induction. Thus, MAPit
to lytic reactivation, we performed an experiment with indicated heterogeneous reactivation of RTA in highly
TREx BCBL1-RTA cells. These cells, containing an induced TREx BCBL1-RTA cells, just as in TPA-treated
integrated doxycycline-inducible RTA transgene, over- BCBL1 cells.
express RTA after 12 h of treatment with doxycycline
and hence undergo efficient reactivation (31,55). We col-
lected cells after 12h of induction and performed MAPit,
using primers specific to the wild-type KSHV RTA
A
promoter. We cloned 51 and 45 molecules amplified
0 h HCG 12 h HCG 0 h GCH 12 h GCH
from bisulfite-converted DNA isolated from doxycycline- 100
treated and untreated cells, respectively (Figure 5). Fold C
5
m50
inductionoflyticgeneswas30-and6-foldhigherforRTA %
and vIL6 transcripts, respectively, in doxycycline-treated
0
TREx BCBL1-RTA cells than had been observed in –400 –200 0 200 400
parental BCBL1 cells treated with TPA (Supplementary Distance to RTA TSS (bp)
Figure S4). B Asp718 HinCII
Several differences in RTA promoter chromatin archi- O A X RTA (ORF50)
tecture were also observed between the cell lines. In the ORF48 ORF49
Y S T
C
TREx BCBL1-RTA cell line, the upstream peak of CG
N–1 N+1 N+2
methylation was absent (compareFigures 5Aand 3B,red Doxycycline
and green curves), and a footprint spanned the two GC C 0 h 12 h P
sites within the Sp1 binding site in 10–20% of molecules i 37% 25% 0.27
[Figure 5B and C, under triangle labeled ‘S’; (2)].
Although this footprint was slightly more common on ii 28% 10% 0.03
RTA induction, the increase did not pass the threshold
for significance (P=0.4). iii 12% 39% 4×10–3
As before, we noted a subset of episomes with highly
accessiblechromatin,clusterv.Theseweremoreabundant iv 7% 10% 0.73
than we had observed in the parent cell line BCBL1,
v 16% 16% 1.00
possibly due to higher basal expression of RTA in the
untreated TREx BCBL1-RTA cells. Consistent with this,
–200 0 200
5.6-fold more RTA transcript was observed in uninduced Distance to RTA TSS (bp)
(tetracycline-free FCS) TREx BCBL1-RTA versus
BityCTBthoLre1Mec.ceCllulvssitP(enIrsowtoefsrhmeoofwolenuc)nu.dle,siiw–iivth(Finitgeurrmee5dCia)t.eAacscseesesnibiiln- D 4-39 bp 121-200 bp –1 5050ncy at 12 hncy at 0 h
the presence of phorbol ester, induction of RTA in trans –10papa
caused a significant shift in abundance between two ––2105occuoccu
clusters (ii and iii). However, in this case, the fraction of –25% –
moleculesaffectedwaslarger(30%,i.e.increaseincluster –400 –200 0 200 400
Distance to RTA TSS (bp)
iii versus decrease in clusters i and ii), and the
Figure 5. Expressionofadoxycycline-inducibleRTAtransgenealtered
doxycycline-induced state (cluster iii) was more accessible
chromatin structure at the endogenous RTA promoter of 30–40% of
to M.CviPI than had been seen with TPA induction
episomes.(A)ProfileofendogenousCG(uninduced,red;12hofdoxy-
(Figure 3, cluster iv). Displacement of N(cid:4)1 occurred in cycline, green) and exogenous GC methylation (uninduced, black; 12h
20%ofmolecules(Figure5D),consistentwiththeshiftof of doxycycline, blue). GCG sites were not counted. (B) Schematic of
the RTA locus, as in Figure 3B. (C) GC methylation plots for 0- and
atleast20%ofmoleculestoclusteriii.Increasedchroma-
12-h RTA induction (blue and yellow, respectively). GC sites are
tin remodeling in TREx BCBL1-RTA is consistent with
indicated above; plot is to scale with other panels. Dendrogram was
the higher and more persistent levels of RTA expression split into five fractions of interest. On right, proportion of sequence
than are observed with TPA-induced reactivation readsfromeachdatasetpresentineachcluster,andexpectationvalue
(31,56,57). Alternatively, dissimilarities between the two P calculated, as in Figure 2. (D) Difference in footprint occupancy
from 0 to 12h of TPA treatment. Footprints in two size ranges, 4–39
experimental systems may reflect differences in transcrip-
and 121–200bp, were counted at every position in each dataset, then
tion factor and/or co-activator recruitment. TPA brings the 0-h profile was subtracted from the 12-h profile.