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Supplementary data

H3.3 actively marks enhancers and primes gene transcription via opening

higher-ordered chromatin

Ping Chen1,5, Jicheng Zhao1,5, Yan Wang1,2,5, Min Wang1,2, Haizhen Long1,2, Dan Liang1,2, Li Huang1, Zengqi Wen1,2, Wei Li3, Xia Li4, Hongli Feng1, Haiyong Zhao1, Ping Zhu1, Ming Li3, Qian-fei Wang4, Guohong Li1,*

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Supplemental Figures and Legends

Figure S1: Single molecule analysis using magnetic tweezers. Related to Figure 1.

A. Schematic views of the setup of the magnetic tweezers (not to scale) B. The real-time step-by-step displacement of the

nucleosomes from the single canonical nucleosomal array was observed by increasing the DNA tether length using

magnetic tweezers.

Figure S2: The effects of H2A.Z and H3.3 on the folding of chromatin arrays induced by H1. Related to Figure

2.

A. The S20,w values of canonical and variant-containing nucleosomal arrays are shown as a function of the Ratio

H1e/Octamer. B. SDS-PAGE analysis showed that the variants do not have any significant effects on the affinity of

H1e to the chromatin arrays.

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Figure S3: Specific functions of the 4 different amino acids in H3.3 vs. H3.1 on chromatin folding properties.

Related to Figure 3.

The sedimentation coefficient distribution plots for canonical H2A (A-D, I-J) and H2A.Z (E-H, K-L) containing

nucleosomal arrays with the single point mutation in H3 on 4 different amino acid residues from H3.1 to H3.3,

including H3(A31S, S87A, V89I and M90G) and two double mutations H3(A31SS87A and V89IM90G) at 0, 0.5 and

1.5 mM MgCl2.

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Figure S4: In vitro transcription and nucleosome remodeling assays. Related to Figure 3.

A. MNase digestion analysis of the canonical and variant-containing chromatins assembled by dNAP1/ACF on the

DNA template pG5MLP for in-vitro transcription investigation. B. The effects of H2A.Z, H3.3 and the double variant

on the transcriptional activity on the chromatin templates with H1e compared to canonical chromatin. The

transcription assays have been performed independently for three times. C. The effects of H2A.Z, H3.3 and the

double variant on nucleosome remodeling efficiency by ACF. Remodeling assays were performed as described in

Supplementary Methods. The molar ratio of ACF to nucleosomes and positions of nucleosomes were indicated in

figure.

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Figure S5: Dynamic regulation of H2A.Z and H3.3 on the chromatin structures during HoxA1 gene activation

in mES cells (R1). Related to Figure 4.

A. The relative levels of HoxA1 mRNA (a) and nascent RNA (b) at different time points during the tRA induction

process as measured using RT-real-time-PCR. The levels were normalized as n-fold changes relative to those in

non-induced cells. B. The enrichment of H3.3 and H2A.Z on the enhancer and promoter regions of HoxA1 in mouse

ES cells (R1). Positions of the primer sets used in ChIP are indicated in (C). C. Schematic diagram of the positions of

the primer pairs used in the ChIP analysis on the HoxA1 gene. The primer pair (pD1) amplified the promoter

downstream region of HoxA1, and pHR1 amplified the enhancer region of HoxA1. D-F. The dynamics of H2A.Z,

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H2A and H2B (D), H3.3, H3 and H4 (E), RARα and Pol II (F) on the promoter (a) and enhancer (b) regions of the

HoxA1 gene during tRA induction. Primer pairs used in real-time PCR are shown in the schematic diagram (C). G.

EpiQ analysis of the accessibility of chromatin on the promoter and enhancer regions of the HoxA1 gene during tRA

induction. The results were normalized to the reference Rho gene. All of the data shown were expressed as the mean

± SD (standard deviation) of three independent biological replicates.

Figure S6. H3.3–dependent recruitment of histone acetyltransferase and chromatin remodelers on the

promoter regions of Cyp26A1 and HoxA1 genes. Related to Figure 5.

The changes in the recruitments of histone acetyltransferase complex Tip60 and different chromatin remodelers, such

as SNF2H, SRCAP and BRG1 on the promoter regions of Cyp26A1 gene (A) and HoxA1 gene (B) in H3.3

knockdown in mES cells (R1) using ChIP assays. Fold=1 for IgG enrichment level.

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Figure S7. H2A.Z and H3.3 are important for HoxA1 gene induction by tRA in mES cells (R1). Related to

Figure 5.

A. The effect of H3.3 knockdown on the induction of the HoxA1 gene in mES cells (R1). B. A schematic diagram of

the primer pairs in the enhancer (RARE) and promoter regions used for the HoxA1 gene (a). The effect of H3.3

knockdown on the enrichments of H2A.Z (b,c) and RARα (d,e) on the enhancer and promoter regions of the HoxA1

gene in mES cells (R1) using ChIP assays. C. A schematic diagram of the primer pair in the promoter regions on the

HoxA1 gene (a). The effect of H3.3 knockdown on the recruitments of TBP (b) and Pol II (c) on the promoter region

of the HoxA1 gene during tRA induction in mES cells (R1) using ChIP assays. D. The effect of H2A.Z knockdown

on the induction of the HoxA1 gene in mES cells (R1). E. A schematic diagram of the primer pairs in the enhancer

(RARE) and promoter regions on the HoxA1 gene (a). The effect of H2A.Z knockdown on the enrichments of H3.3

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(b,c) and RARα (d,e) on the enhancer and promoter regions of the HoxA1 gene during tRA induction in mES cells

(R1) using ChIP assays. F. A schematic diagram of the primer pair in the promoter region on the HoxA1 gene (a).

The effect of H2A.Z knockdown on the recruitments of TBP (b) and Pol II (c) on the promoter region of the HoxA1

gene during tRA induction in mES cells (R1) using ChIP assays. G. EpiQ analysis of the accessibility of chromatin on

the promoter (b,d) and enhancer (c.e) of the HoxA1 gene before tRA induction in H3.3 (b,c) and H2A.Z (d,e)

knockdown cells. The results were normalized to the reference Rho gene. All of the data shown were expressed as the

mean ± SD (standard deviation) of three independent biological replicates. “*” and “**” indicated P<0.05 and P<0.01,

respectively.

Figure S8. The genome-wide correlation of the enrichment of H2A.Z and H3.3 with chromatin structures.

Related to Figure 6.

Among the MNase-sensitive peaks of the intergenic regulatory regions, the open chromatin structures correlated well

with the regions with high levels of H3.3 but low levels of H2A.Z (A). In the MNase-sensitive peaks on the promoter

region, the chromatin structures at the TSS were very open, with very low levels of H3.3 and H2A.Z, which may

have resulted from the depletion of nucleosomes (B).

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Supplemental Methods

Protein, DNA and antibodies

Recombinant histones and DNA templates were prepared as previously described (Li et al., 2010). To study in

vitro transcription, p300 (Pavri et al., 2005), ACF and Gal4-VP16 (Loyola et al., 2001) were purified as previously

described (Li et al., 2010). Drosophila NAP1 was expressed in SF9 cells and purified using Ni2+ NTA-resins

(Fyodorov and Kadonaga, 2003). The anti-H4 pan antibody (05-858) was obtained from Millipore. The anti-H2B

(ab1790), anti-H2A (ab18255), anti-H3 (ab1791) and anti-SNF2H (ab3749) antibodies were purchased from Abcam.

The anti-H2A.Z antibody (39113) was purchased from Active Motif, and monoclonal Anti-HA Agarose Conjugate

Clone HA-7 (A2095) was purchased from Sigma. The anti-RARα (sc-551), anti-SRCAP (sc-133312), anti-BRG1

(sc-10768x), anti-Tip60 (sc-5725x), anti-TFIID (TBP) (sc-273x) and anti-Pol II (sc-899) antibodies were purchased

from Santa Cruz. The monoclonal anti-H3.3 antibody (C352) was generated in-house.

Cell culture

Mouse ES cells (line R1 and Milli TraceTM Nanog GFP Reporter Mouse Embryonic Stem Cells, SCR089) were

grown on gelatin-coated tissue culture plates in the presence of 1000 U/ml of leukemia inhibitory factor (LIF;

ESGRO, Millipore) in ES cell medium consisting of knockout Dulbecco’s minimal essential medium (DMEM;

GIBCO/BRL) or Dulbecco’s minimal essential medium (DMEM; Specialty mesia) supplemented with 15% FBS

(Hyclone), 100 mM MEM nonessential amino acids, 0.55 mM 2-mercaptoethanol, 2 mM L-glutamine, nucleosides

and antibiotics (all from Millipore).

Nucleosome and chromatin reconstitution

The respective histone octamers were reconstituted as previously described (Dyer et al., 2004). Equimolar

amounts of individual histones in unfolding buffer (7 M guanidinium HCl, 20 mM Tris-HCl, pH7.5, 10 mM DTT)

were dialyzed into refolding buffer (2 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM 2-mercaptoethanol),

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and purified through a Superdex S200 column. For in vitro structural investigation, chromatin arrays were assembled

using the salt-dialysis method as previously described (Dyer et al., 2004). The reconstitution reaction mixture with

octamers and 601 based DNA templates in TEN buffers (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 M NaCl) were

dialyzed over 16 hrs at 4°C in TEN buffer, which was continuously diluted by slowly pumping in TE buffer (10 mM

Tris-HCl, pH 8.0, 1 mM EDTA) to a lower concentration of NaCl from 2 M to 0.6 M. For histone H1 incorporation,

H1 with different molar ratios relative to the mononucleosomes was added at this step and further dialyzed in TE

buffer with 0.6 M NaCl for 3 hrs, followed by a final dialysis step in TE buffer for 4 hrs. The stoichiometry of histone

octamer binding to the DNA template was determined by EM images and AUC investigation. For in vitro

transcription study, the chromatin was assembled on a DNA template pG5MLP using histone chaperone NAP1 and

chromatin remodeling complex ACF as previously described (Li et al., 2010). A standard chromatin assembly

reaction containing 2.0 μg of DNA template, 2.0 μg of histone octamers, 0.4 μg of ACF and 6.0 μg of NAP1 was

performed at 30°C overnight in 150 μl of 10 mM HEPES (pH 7.5). For histone H1 incorporation, an equal molar

amount of histone H1 (relative to mononucleosomes) was added after the chromatin assembly reaction. The

assembled chromatin was purified by gel filtration (Agarose 2 column) (Loyola et al., 2001).

Electron Microscopy

Reconstituted chromatin samples were prepared using DNA concentrations of 20 µg/mL in measurement buffer

(10 mM HEPES, pH 8.0, 0.1 mM EDTA). The samples were fixed with 0.4% glutaraldehyde (Fluka) in the same

buffer on ice for 30 min. For the metal shadowing study, 2 mM spermidine was added into the sample solution to

enhance the absorption of the chromatins to the grids. The samples were applied to the glow-discharged

carbon-coated EM grids and incubated for 2 min and then blotted. The grids were washed stepwise in 20 ml baths of

0%, 25%, 50%, 75%, and 100% ethanol solution for 4 min, each at room temperature, air dried and then shadowed

with tungsten at an angle of 10° with rotation. For the negative staining study, the chromatin samples in fixative

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solution were incubated on glow-discharged carbon-coated EM grids for 1 min. The excess sample solution was

removed using filter papers. The grid was incubated in 2% Uranylacetate for staining for 30 sec twice, blotted with

filter papers and allowed to air-dry for several minutes. The samples were examined using a FEI Tecnai G2 Spirit 120

kV transmission electron microscope.

Single molecule magnetic tweezer analysis

Single molecular manipulation of a nucleosomal array was performed on magnetic tweezers (Pico Twist

Company, France) as described previously (Gosse and Croquette, 2002; Meglio et al., 2009). Briefly, a single

nucleosomal array was bound at one end to a glass coverslip via the bonds between digoxigenin and anti-digoxigenin,

and the other end to a magnetic Dynabead (Invitrogen Norway) via biochemical reactions between biotin and

streptavidin (Smith et al., 1992) as shown in Fig. S1a. A 15-µl flow cell was constructed with a sandwiched

130-µm-thick double-sided tape (50×5 mm2, 3 M Corporation) using a thoroughly cleaned glass coverslip and Mylar

film. To eliminate the unbound anti-digoxigenin, anti-digoxigenin (10 mg/ml Roche) was injected into the flow cell

together with passivation buffer (10 mg/ml BSA, 1 mM EDTA, 10 mM phosphate buffer, pH 7.4, 10 mg/ml Pluronic

F127 surfactant (Sigma-Aldrich), 3 mM NaN3) overnight at 37°C. Subsequently, 2 µl chromatin/bead mixture was

injected into the flow cell for 10 minutes. The flow cell was finally rinsed with the buffer (10 mM Hepes, pH 7.5, 10

mM KCl, 0.5 mM EDTA, 10% Glycerol, 10 mM beta-glycerophosphate, 1 mM DTT, 0.2 mM PMSF) to eliminate

the nonspecific interaction with magnetic beads. Next, two small NdFeB controlled magnets were used to pull on the

Dynabead to stretch the chromatin molecule using a large force (F= 25pN). The real-time bead position was observed

using a microscope objective (Olympus 100× 1.2, oil immersion) and the sample image was projected onto a JAI

Giga-Ethernet CCD camera. The algorithm for tracking the bead position offered a remarkable accuracy (Gosse and

Croquette, 2002), where the measurement uncertainty was typically approximately 1 nm in the x, y and z directions.

The bead’s calibration images were recorded to store the information of the bead at various distances from the focal

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point of the objective. At least 5 beads were tracked at a same time in each experiment.

Nucleosome Remodeling Assay

Different variant-containing mononucleosomes were assembled by salt dialysis as described above, using a

Cy3-labeled 229 bp DNA fragment containing the 601 nucleosome positioning sequence. Nucleosome sliding assays

were performed in sliding buffer (10 mM Tris-HCL PH7.6，50 mM KCL，1.5 mM MgCl2，0.5 mM EDTA，10%

glycerol, 0.2 ug/ml BSA) at 30 for 1℃ hr in the presence of 2 mM ATP. In each reaction, 400 nmol monoculeosome

was incubated with indicated amounts of ACF, and the reaction mixtures were resolved on 5 % native polyacrylamide

gels in 0.5 × TBE.

Isolation of nascent RNA and mRNA and real-time PCR analysis

Nascent RNA was extracted as previously described (Khodor et al., 2011). R1 cells were lysed in ice-cold buffer

A (15 mM HEPES-KOH at pH 7.6, 10 mM KCl, 5 mM MgOAc, 3 mM CaCl2, 300 mM sucrose, 0.1 % Triton X-100,

1 mM DTT, protease inhibitors). The resulting lysate was divided into 0.5 mL aliquots and layered over 1 mL of

buffer B (15 mM HEPES-KOH at pH 7.6, 10 mM KCl, 5 mM MgOAc, 3 mM CaCl2, 1 M sucrose, 1 mM DTT, 1×

Complete protease inhibitors), and then centrifuged at 6200*g for 15 min at 4°C. The pellet was resuspended in 5

volumes of nuclear lysis buffer (10 mM HEPES-KOH at pH 7.6, 100 mM KCl, 0.1 mM EDTA, 10% glycerol, 0.15

mM spermine, 0.5 mM spermidine, 0.1 M NaF, 0.1 M Na3VO4, 0.1 mM ZnCl2, 1 mM DTT, protease inhibitors, 1

U/μL RNasin Plus [Promega]). We added 2× NUN buffer (25 mM HEPES-KOH at pH 7.6, 300 mM NaCl, 1 M Urea,

1% NP-40, 1 mM × Complete protease inhibitors) drop-wise at a 1:1 ratio to the nuclear suspension while vortexing,

and the suspension was placed on ice for 20 min prior to centrifugation at 17000*g for 30 min at 4°C. TRIzol reagent

(Invitrogen) was added to dissolve the DNA–Histone–Pol II-RNA pellets and RNA was extracted. The resulting RNA

was subjected to pA depletion with Oligo (dT).

The total RNA was extracted using TRIzol reagent (Invitrogen, USA), and the first strand of cDNA was reverse

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transcribed using 2 μg of RNA. cDNA products were used for quantitative real-time PCR using the SYBR Premix Ex

Taq (Takara, Japan). The sequences of mRNA detecting primers used in RT real-time PCR (ABI 7300, USA) were

the following:

Cyp26A1: sense 5’-GGTTTCGGGTTGCTCTGA-3’, antisense 5’-ACTATAAAGCGGTCGGGATT-3’;

HoxA1: sense 5’-ACGCCAGCCACCAAGAAG-3’, antisense 5’-TGTAGGGACGCGGCAATC-3’;

GAPDH: sense 5’-GCACAGTCAAGGCCGAGAAT-3’, antisense 5’-GCCTTCTCCATGGTGGTGAA-3’.

Chromatin immunoprecipitation (ChIP)

ChIPs and RT-PCR were performed as previously described (Margueron et al., 2008). R1 Cells were treated

with tRA and cross-linked with 1% formaldehyde. The nuclei were isolated by incubation in lysis buffer (50 mM

HEPES, 140 mM NaCl, 1 mM EDTA, 10 % glycerol, 0.5 % NP-40, 0.25 % Triton X-100, protease inhibitors), and

washed with wash buffer (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitors).

Chromatin was resuspended in the buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA), and sonicated to obtain

200–1000-bp-sized fragments. Twenty milligrams of sonicated chromatin was precleaned and incubated with

antibodies in incubation buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium

deoxycholate, 0.33% N-lauroylsarcosine, protease inhibitors) overnight at 4°C. Antibody–chromatin complexes were

captured using Protein A/G agarose beads and washed with modified RIPA buffer (50 mM HEPES pH 7.6, 500 mM

LiCl, 10 mM EDTA, 1 % NP-40, 0.7% sodium deoxycholate). Enriched DNA was analyzed using real-time PCR. For

ChIP with the HA agarose beads, chromatin were captured in incubation buffer (50 mM Tris-HCl pH 7.5, 1 mM

EDTA, 150 mM NaCl, 1% triton X-100), and the antibody–chromatin complexes were washed with TBST (0.05%

Tween-20). The primer pairs used for the real-time PCR experiments were the following:

Cyp26A1 RARE2 (pR2):

sense 5’-GCAGGCTGAACTTGGTGG-3’, antisense 5’- GCCCATTCCCAATCCTTTA-3’;

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Cyp26A1 RARE1 (pR1):

Sense 5’- CGGAACAAACGGTTAAAGATT-3’, antisense 5’- ATAAGGCCGCCCAGGTTA-3’;

Cyp26A1 +2000 (p+2k):

sense 5’- TACCCTTGAAGTCTTCCGTG-3’, antisense 5’-GTTGACGATTGTTTTAGTGCC-3’;

HoxA1 promoter (pD1):

sense 5’- GGTCCTATGGAGGAAGTGAGA-3’, antisense 5’-TGCCAAGGATGGGGTATT-3’;

HoxA1 RARE (pHR1):

sense 5’- GGTTGTTGAAAAGGCTGCTA-3’, antisense 5’- GGACTCATTCTAAAGTGACCCA-3’.

Genome-wide data analysis

Four public datasets for the ChIP-Seq were obtained from GEO with the accession numbers GSE36114

(GSM881348 for H2AZ), GSE16893 (GSM487544 for H3.3), GSE23830 (GSM587479 for H3) and GSE24211

(GSM595518 for Input). The mouse ES cell RNA-Seq expression data was obtained from GEO with accession

number GSE38596 (GSM881355).The promoter-like regions and enhancer regions were obtained from previous

studies (Chen et al., 2012). To identify the open region in genome chromatin, MACS was utilized to perform peak

calling (Zhang et al., 2008). The uniquely mapped reads from the mild digestion condition (open reads) was

normalized to the reads from the extensive digestion condition. The parameters were set as the following: effective

genome size = 2.7e+09; tag size = 100; nomodel; shift size = 75; P-value cutoff = 1.00e-05; ranges for calculating the

regional lambda were: peak region, 1000, 5000 and 10000. The genome-wide correlation of H2A.Z and H3.3

distribution with an open chromatin profile was analyzed in R 2.12.0 (http://www.r-project.org/) and displayed in the

software of Integrative Genomics Viewer (IGV) (Thorvaldsdottir et al., 2013).

The enhancer regions were classified into two groups, which named “open enhancer regions” and “rest enhancer

regions”, according to whether they have an overlap with the open region. The promoter regions were classified into

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two groups according to their expression levels, in which the “active promoter regions” were referred to the predicted

promoters which overlap with the TSS (+/- 2Kb) of the top 1/4 expression genes, and the “repressive promoter

regions” were referred to the predicted promoters which overlap with the TSS (+/- 2Kb) of the bottom 1/4 expression

genes. For each group of enhancers and promoters, we calculated the reads density of H2A.Z, H3.3, H3 and Input

sample in a range of -1Kb upstream and +1Kb downstream from the center of enhancer or promoter with 10-bp

window size. The reads density of H2A.Z, H3.3 and H3 were normalized with reads density of input sample, and

then the H3.3 enrichment level was further normalized with reads density of H3.

The MNase-sensitive peaks generated from our MNase-seq were also categorized into two classes: peaks within

TSS (with at least 1 bp overlapping with the region of 500 bp upstream to 500 bp downstream from TSS) and peaks

outside TSS (Note: the peaks at both the intron and exon regions have been filtered in this study). MNase-sensitive

peaks were then quantified in a 10-bp sliding window from -1 Kb upstream to +1 Kb downstream centered at the

summit coordinates generated by MACS. The H2A.Z and H3.3 reads in these two regions were calculated as

described above.

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Supplemental References

Chen, C.Y., Morris, Q., and Mitchell, J.A. 2012. Enhancer identification in mouse embryonic stem cells using integrative modeling of chromatin and genomic features. BMC Genomics 13, 152.

Dyer, P.N., Edayathumangalam, R.S., White, C.L., Bao, Y., Chakravarthy, S., Muthurajan, U.M., and Luger, K. 2004. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol. 375, 23-44.

Fyodorov, D.V., and Kadonaga, J.T. 2003. Chromatin assembly in vitro with purified recombinant ACF and NAP-1. Methods Enzymol. 371, 499-515.

Gosse, C., and Croquette, V. 2002. Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys. J. 82, 3314-3329.

Khodor, Y.L., Rodriguez, J., Abruzzi, K.C., Tang, C.H., Marr, M.T., 2nd, and Rosbash, M. 2011. Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila. Genes Dev. 25, 2502-2512.

Li, G., Margueron, R., Hu, G., Stokes, D., Wang, Y.H., and Reinberg, D. 2010. Highly compacted chromatin formed in vitro reflects the dynamics of transcription activation in vivo. Mol. Cell 38, 41-53.

Loyola, A., LeRoy, G., Wang, Y.H., and Reinberg, D. 2001. Reconstitution of recombinant chromatin establishes a requirement for histone-tail modifications during chromatin assembly and transcription. Genes Dev. 15, 2837-2851.

Margueron, R., Li, G., Sarma, K., Blais, A., Zavadil, J., Woodcock, C.L., Dynlacht, B.D., and Reinberg, D. 2008. Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Mol. Cell 32, 503-518.

Meglio, A., Praly, E., Ding, F., Allemand, J.F., Bensimon, D., and Croquette, V. 2009. Single DNA/protein studies with magnetic traps. Curr. Opin. Struct. Biol. 19, 615-622.

Pavri, R., Lewis, B., Kim, T.K., Dilworth, F.J., Erdjument-Bromage, H., Tempst, P., de Murcia, G., Evans, R., Chambon, P., and Reinberg, D. 2005. PARP-1 determines specificity in a retinoid signaling pathway via direct modulation of mediator. Mol. Cell 18, 83-96.

Smith, S.B., Finzi, L., and Bustamante, C. 1992. Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 258, 1122-1126.

Thorvaldsdottir, H., Robinson, J.T., and Mesirov, J.P. 2013. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14, 178-192.

Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nusbaum, C., Myers, R.M., Brown, M., Li, W., et al. 2008. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137.