H3.3/H2A.Z double variant-containing nucleosomes mark ‘nucleosome-free regions’

of active promoters and other regulatory regions in the human genome

Chunyuan Jin, Chongzhi Zang, Gang Wei, Kairong Cui, Weiqun Peng, Keji Zhao and

Gary Felsenfeld

Supplementary Note

‘Nucleosome-free regions’ are occupied by H3.3/H2A.Z nucleosomes

Using NCPs isolated under high salt conditions without formaldehyde crosslinking, we1

had shown in human that H2A.Z nucleosomes flank ‘nucleosome-free regions’ (NFRs).

It had also been shown in Drosophila2 that H3.3 is enriched at promoter regions of active

genes, but with a distinct minimum in the 0 to -100 bp region. However, these studies

used micrococcal nuclease digested nucleosomes that were at some point exposed to

buffers containing 350 mM or higher NaCl. Since H3.3/H2A.Z NCPs are extractable at

this high salt, it is not surprising that we and others detected a depletion of H2A.Z or

H3.3 NCPs at the sites. In this study, we successfully duplicated the earlier results,

creating H2A.Z NCP-free promoters, by using the higher salt concentrations (Fig. 1g,h).

Under this condition, the double variant NCPs are also lost from CTCF-binding sites,

DNse I hypersensitive sites, gene bodies and 3’ TTSs (Fig. 2c,d and Supplementary Figs.

9,10,13). Other previous studies have employed in vivo formaldehyde cross-linking to

produce a genome-scale profile of H2A.Z nucleosomes. The results are contradictory.

Mavrich et al. reported the depletion of H2A.Z NCPs at the -1 and even -2 positions in

Drosophila embryos but suggested that different species might have different chromatin

structures3. In contrast, it was reported recently4 that in formaldehyde cross-linked

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C.elegans embryonic cells, H2A.Z is preferentially concentrated over transcription start

sites of active genes, consistent with our results, though no attempt was made to detect

double variant nucleosomes and the size of chromatin fragment tested was larger. These

results might reflect differences among species, but we note that in the latter case,

samples were crosslinked for twice as long as in the case of Drosophila, which might

explain why more H2A.Z was detected. While this manuscript was in preparation,

Henikoff et al., reported that under 80 mM salt conditions they could detect high

enrichment of H3.3, but not H2A.Z at the -1 position in Drosophila, supporting the idea

that H2A.Z/H2B would dissociate first from H3.3/H2A.Z NCPs5, which would leave a

hexamer or an H3.3/H4 tetramer.

Relative abundance of H3.3/H2A.Z nucleosomes at TSS

Gene-specific and genome-wide surveys from yeast to human have reported that there is

a ‘nucleosome-free region’ immediately upstream of transcription start sites, and that the

degree of nucleosome depletion is correlated with the level of gene expression. Some of

these studies measured levels of total histone H3, H4, H2B or nucleosomal DNA at the

sites, which could not distinguish among the variants. Using this approach, we showed

earlier6 that in NCPs isolated from human T cells under low salt conditions, the -1

position of expressed genes was depleted of NCPs by about 40% compared to the -2

position. This would be consistent with 60% occupancy of the TSS (-1 nucleosome) by

NCPs, which could include variants of all kinds. In other experiments, Ozsolak et al.7

mapped nucleosome positions at human promoters using MNase digested NCPs isolated

under higher salt conditions, in which H3.3/H2A.Z NCPs should be lost. We note that in

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several human cell lines they observed that -1 nucleosome density was about 70% lower

than -2 nucleosome density in expressed genes. This suggests that no more than 30% of

the TSS are occupied by the more stable H3/H2A NCPs in expressed genes, which could

represent canonical NCPs that have not yet been replaced by variant NCPs after DNA

replication. Promoters of expressed genes are thus likely to be occupied to a considerable

extent by the labile H3.3/H2A.Z NCPs depending on the level of activity and perhaps the

species.

Single variant NCPs

The distribution of single variant NCPs shows that there are distinct and probably

functionally relevant patterns for these nucleosomes as well. It is particularly interesting

that, in the neighborhood of the TSS, there are very few NCPs carrying the combination

H3.3/H2A (i.e. lacking H2A.Z), whether or not the gene is transcriptionally active. On

the other hand, a small fraction of H2A.Z-containing NCPs consist of H2A.Z complexed

with H3.1 or 3.2 at TSS, while the majority of TSS is occupied by H3.3/H2A.Z NCPs.

Given that most H2A.Z NCPs are lost near TSS at the higher salt condition (150 mM,

Fig. 1g), and that H3.1/H2A.Z NCPs appear to be at least as stable as those containing

H3.1/H2A under these conditions, as shown in our previous study8, we suggest that the

majority of H2A.Z only NCPs at the TSS of active genes may carry the H3.2/H2A.Z

combination, although the corresponding determination of stability has not been made for

NCPs containing this histone variant pair.

Distribution of H3.3 NCPs over gene bodies

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Remarkably, the abundance of the single variant H3.3 NCPs increases across the

transcribed regions of the most active genes (Fig. 4d). The pattern resembles that of

H3K36 trimethylation, which also accumulates toward the 3’ end of active genes. There

is some evidence to support the idea that this modification is carried principally by H3.3

9. Since H3.3 deposition on chromatin is mediated by the HIRA complex 10-15,

presumably after displacement of other histone H3/H4 tetramers, this enrichment may

reflect an increasing amount of displacement activity toward the 3’ end of genes.

Deposition of H2A.Z is thought to involve a separate delivery system, the SWR1

complex 14-19, in which H2A.Z/H2B dimers are exchanged for H2A/H2B dimers on

existing nucleosomes. The way in which these exchanges might be targeted to genomic

sites to deliver the observed patterns is not yet known. It seems unlikely that the ‘H3.3

only’ increasing gradient pattern is generated by displacement of H2A.Z/H2B dimers and

their replacement by H2A/H2B, since one would then expect to see a complementary

decreasing gradient of the doubly substituted NCPs across gene bodies, something which

is not observed (Fig. 4e).

Effects of DNA sequence on the stability of H3.3/H2A.Z-containing nucleosomes

Although DNA sequence has some effect on nucleosome stability (most evidence is from

studies in yeast or Drosophila), our earlier results and those of others have made it clear

that in vertebrates the identity of the histone variants has a major effect on histone

octamer stability. Several lines of evidence indicate that DNA sequence itself is not

likely the determinant of H3.3/H2A.Z NCP stability. First, the correlation between the

presence of H3.3/H2A.Z NCPs at a gene promoter and the corresponding level of

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expression of that gene already suggests that in another cell type, with different patterns

of expression, the distribution of such NCPs will be different. This confirms that the

histone variant occupying the site does depend on whether or not the gene is active.

Second, the distribution of DNase I hypersensitive sites supports this conclusion. It is

known that some ENCODE DNase I hypersensitive sites20 and most predicted enhancers

(marked by DNase I hypersensitivity etc.) are cell type specific21. To prove further that

unstable NCPs enriched at DNase I hypersensitive sites could be cell-type specific, we

examined the distribution of double variant NCPs in HeLa cells at CD4+ T cell specific

DNase I hypersensitive sites (hypersensitive in CD4+ T cells but not in HeLa). The

result clearly indicates the absence of H3.3/H2A.Z NCPs at such sites (Fig. 2f). We have

also observed that in chicken, H3.3 and H2A.Z are co-localized at a locus control region

(DNase I hypersensitive site) of β-globin genes in 6C2 cells but H3.3 is absent at the site

in DT40 cells (data not shown). These results also suggest that unstable nucleosomes are

selectively enriched at specific sites and are involve in determining the cell identity.

References for Supplementary Note

1. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823-37 (2007).

2. Mito, Y., Henikoff, J.G. & Henikoff, S. Genome-scale profiling of histone H3.3 replacement patterns. Nat Genet 37, 1090-7 (2005).

3. Mavrich, T.N. et al. Nucleosome organization in the Drosophila genome. Nature 453, 358-62 (2008).

4. Whittle, C.M. et al. The genomic distribution and function of histone variant HTZ-1 during C. elegans embryogenesis. PLoS Genet 4, e1000187 (2008).

5. Henikoff, S., Henikoff, J.G., Sakai, A., Loeb, G.B. & Ahmad, K. Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res (2008).

Nature Genetics: doi:10.1038/ng.409

6. Schones, D.E. et al. Dynamic regulation of nucleosome positioning in the human genome. Cell 132, 887-98 (2008).

7. Ozsolak, F., Song, J.S., Liu, X.S. & Fisher, D.E. High-throughput mapping of the chromatin structure of human promoters. Nat Biotechnol 25, 244-8 (2007).

8. Jin, C. & Felsenfeld, G. Nucleosome stability mediated by histone variants H3.3 and H2A.Z. Genes Dev 21, 1519-29 (2007).

9. Hake, S.B. et al. Expression patterns and post-translational modifications associated with mammalian histone H3 variants. J Biol Chem 281, 559-68 (2006).

10. Tagami, H., Ray-Gallet, D., Almouzni, G. & Nakatani, Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116, 51-61 (2004).

11. Loppin, B. et al. The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus. Nature 437, 1386-90 (2005).

12. Konev, A.Y. et al. CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo. Science 317, 1087-90 (2007).

13. Loyola, A. & Almouzni, G. Marking histone H3 variants: how, when and why? Trends Biochem Sci 32, 425-33 (2007).

14. Jin, J. et al. In and out: histone variant exchange in chromatin. Trends Biochem Sci 30, 680-7 (2005).

15. Henikoff, S. & Ahmad, K. Assembly of variant histones into chromatin. Annu Rev Cell Dev Biol 21, 133-53 (2005).

16. Raisner, R.M. et al. Histone variant H2A.Z marks the 5 ' ends of both active and inactive genes in euchromatin. Cell 123, 233-248 (2005).

17. Mizuguchi, G. et al. ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303, 343-8 (2004).

18. Kobor, M.S. et al. A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin. PLoS Biol 2, E131 (2004).

19. Krogan, N.J. et al. Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4. Proc Natl Acad Sci U S A 101, 13513-8 (2004).

20. Crawford, G.E. et al. DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods 3, 503-9 (2006).

21. Heintzman, N.D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature (2009).

Nature Genetics: doi:10.1038/ng.409

HeLa cells(H3.3-FLAG)

Purify NCPsat lower salt

(10 mM)

Purify NCPsat higher salt

(150 mM)

Nuclei

MNaseDigest

Mononucleosomal DNA

IP with H2A.Z antibodies

IP with FLAG antibodies

Sequential IP with anti-FLAG

and H2A.Z antibodies

IP with H2A.Z antibodies

Genomic DNAInput DNA ChIP DNA

Solexa sequencing

Map to genome

(10 mM NaCl)

Genomic

Input

H2A.Z

H3.3

Double (H3.3/H2A.Z)

H2A.Z (high salt)

*H2A.Z only

*H3.3 only

Sample names used

(10 mM NaCl)

Genomic DNA Sonication 200-300 bp DNA fragments

Supplementary Figure 1 Scheme of immunoprecipitation and analysis of histone variants.The flow chart of the experiments; the asterisks mark the two data libraries (H2A.Z only and H3.3 only) obtained by computational analysis using total H2A.Z, total H3.3 and Double (H3.3/H2A.Z) ChIP-seqinformation. Briefly, the ‘H2A.Z only’ or ‘H3.3 only’ libraries contain the subset of tags from the total H2A.Z or H3.3 libraries that do not overlap with any tags from H3.3 or H2A.Z respectively or with Double library (see Methods).

Supplementary Figures

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Supplementary Figure 2 Profile of the starting NCP population used as input for all of the immunoprecipitation studies. The same method used in Figure 4 is applied to make the profile.

Nor

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Input

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Downstream position relative to TSS (bp)

Nor

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ized

cou

nts

H2A.Z

+1 +2 +3

Supplementary Figure 3 High resolution map of H2A.Z nucleosome phasing downstream of TSS. The H2A.Z nucleosome pattern downstream of the TSS is less obvious in low salt (Fig. 1h) than in high salt (Fig. 1g), but the regularity is very similar in both. This figure reveals that in low salt, immediately downstream of the TSS, total H2A.Z NCPs are positioned essentially as others have observed in high salt. The H2A.Z remaining after exposure to high salt represents NCPs that do not also contain H3.3. This suggests that H2A.Z only NCPs (without H3.3) are better positioned than H3.3/H2A.Z NCPs downstream of TSS.

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Position relative to TSS (bp)

a H2A.Z

Nuc

leoo

som

ele

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Position relative to TSS

b 400 bp

Nuc

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Positioned nucleosomes

H2A.Z (high salt)

Nucleosomes not uniformlypositioned across region

160 bp

0.0

2.0

4.0

6.0

0 80 160 240 320 4000.0

1.0

2.0

3.0

4.0

0 80 1600.0

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0 80 160

0

Supplementary Figure 4 The irregular averaged pattern in low salt of H2A.Z-containing NCP positions at the TSS of active genes reflects the presence of labile NCPs. (a) Experimentally observed pattern on either side of the TSS for all H2A.Z-containing NCPs shown by nucleosome occupancy level. Nucleosome levels were obtained by applying a simple scoring function to the sequenced reads. A sliding window of 20 bp was applied across all chromosomes and at each window all reads mapping to the upper strand 80 bp upstream of the window and reads mapping to the lower strand 80 bp downstream of the window contributed equally to the score of the window. (b) Expected pattern for a single NCP (left), positioned NCPs (middle), and for the superimposition of one or two NCPsdistributed as shown (right, below graph). The pattern was generated by assuming an arbitrary shape for each NCP and summing all the signals, fitting the results to multi-peak Gaussian curves, and adjusting positions to give a reasonable fit to the data. This is not meant to be a unique solution, but shows that such patterns are easily generated when mobile NCPs are present. It must be kept in mind that each sequence tag contributing to the pattern in (a) is derived from a true NCP monomer, not from a smaller fragment. The result in (a) represents contributions from NCPs summed both over many copies of each gene’s TSS, and over all TSS of analyzed genes, just as diagrammed in (b). The data are consistent with a model in which one or two double variant NCPs can occupy ~400 bp of DNA at multiple positions. Thus, these special NCPs are not phased in any obvious way.

(bp)

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Supplementary Figure 5 High resolution map of H2A.Z nucleosome phasing downstream of CTCF-binding sites.

Downstream position relative to CTCF-binding sites (bp)

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H2A.Z

+1 +2 +3

Nature Genetics: doi:10.1038/ng.409

a H2A.Z

Nuc

leoo

som

ele

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Position relative to CTCF-binding sites (bp)

b 480 bp

0

1

2

3

4

0 160 320 480

Nucleosomes not uniformly positioned across region

Supplementary Figure 6 The irregular averaged pattern in low salt of H2A.Z-containing NCP positions at the CTCF-binding sites reflects the presence of labile NCPs. (a) Experimentally observed pattern on CTCF-binding sites for all H2A.Z-containing NCPs shown by nucleosome occupancy level.(b) Expected pattern for the superimposition of one or two NCPs distributed as shown (below graph). The pattern was generated by using the same method as for Supplementary Fig. 4b. The data are consistent with a model in which one double variant NCP can occupy multiple positions around CTCF-binding sites.

Nuc

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(bp)

Nature Genetics: doi:10.1038/ng.409

a

c

e

b

H3.3

Double (H3.3/H2A.Z)

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Position relative to TTS (bp)

H2A.Z

H3.3

Double (H3.3/H2A.Z)

Supplementary Figure 7 Genome-wide distribution of histone variants near the 3’ end of genes (TTS). (a-c) Profiles of histone variants (indicated above each panel) across the TTS of all genes. (d-f) H2A.Z, H3.3 and double variant nucleosome positioning near the TTS. The y axis shows the normalized number of sequenced tags from the upper strand (red) and the lower strand (green) of the DNA at each position, representing 5’ and 3’ boundaries of each NCP. (g,h) Profiles of Input and Genomic DNA around TTS.

g

d hH2A.Z Genomic DNA

Input

f

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H2A.Z only

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Supplementary Figure 8 H2A.Z only distribution normalized by ‘H2A.Z only’ library. The distribution pattern of 1000 highly active (red), intermediately active (green) or silenced (blue) genes across the transcribed regions as well as the 5’ and 3’ end. The profile was normalized by the total tag numbers in the ‘H2A.Z only’ library.

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Active gene Inactive gene

Double (H3.3/H2A.Z)

H2A.Z (high salt)

H2A.Z (low salt)

H3.3

CCT8 X TRIM42

20

114

1

9

1

14

1

Chr21: 29355000 I 29370000 I Chr3: 141885000 I 141900000 I

Supplementary Figure 9 A typical example of histone variant patterns at TSSs of active and inactive genes shown as custom tracks on the UCSC genome browser. A high concentration of H3.3/2A.Z NCPs is evident over the promoter of the active gene CCT8, and notably absent from the promoter of inactive TRIM42 (red rectangles). NCPs isolated in 150 mM NaCl lose H2A.Z at sites that normally carry the H3.3/2A.Z double variant (see also Fig. 1i).

Double (H3.3/H2A.Z)

H2A.Z (high salt)

H2A.Z (low salt)

H3.3

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Inactive gene

XCSRP3

16

119

1

24

1

15

1

Chr3: 150570000 I 150575000 I Chr11: 19165000 I 19175000 I

Active gene

TM4SF1

Supplementary Figure 10 A typical example of histone variant patterns at TTSs of active and inactive genes as shown as custom tracks on the UCSC genome browser. The TTS of the active, but not the inactive, gene is enriched in H3.3/2A.Z NCPs (red rectangles). Double variant NCPs are also more abundant over the TM4SF1 coding region.

Double (H3.3/H2A.Z)

H2A.Z (high salt)

H2A.Z (low salt)

H3.3

Double (H3.3/H2A.Z)

H2A.Z (high salt)

H2A.Z (low salt)

H3.3

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-FLAG-H3

-Exogenous H3-Endogenous H3

WT

eH3.

1

eH3.

3

α-FLAG

α-H3

Supplementary Figure 11 Relative abundance of FLAG-H3.3 to total H3.The acid extracts from HeLa S3 cells (WT), HeLa cells expressing FLAG-tagged H3.1 (eH3.1) or FLAG-tagged H3.3 (eH3.3) were separated on SDS gel and probed with antibodies against FLAG (upper panel) or total H3 (lower panel) respectively.

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Supplementary Figure 12 Validation of H2A.Z only library. This is an example of a gene (DDK1) that is enriched with H2A.Z only NCPs at the promoter region.

Double (H3.3/H2A.Z)

H2A.Z (high salt)

H2A.Z (low salt)

H3.3

28

112

1

7

1

7

1

Chr10: 53744500 I 53745500 I 53746500 I

DKK1

Nature Genetics: doi:10.1038/ng.409

Double (H3.3/H2A.Z)

H2A.Z (high salt)

H2A.Z (low salt)

H3.3

Supplementary Figure 13 A typical example of histone variant patterns at intergenic DNase I hypersensitive site in HeLa, shown as custom tracks on the UCSC genome browser.The loss of H2A.Z NCPs after exposure to high salt (top panel) is evident (red rectangle).

21

1

92

1

17

1

68

1

Chr11: 64075000 I 64080000 I 64085000 I

DNase HeLa RawP-Value

RefSeq Genes

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Library Unique Tag number

H2A.Z 21,429,267

H3.3 17,447,045

Double (H3.3/H2A.Z) 10,769,822

H2A.Z (high salt) 14,954,642

Input 7,211,194

Genomic 4,177,986

Supplementary Table 1 Unique Tag numbers in each library.

Nature Genetics: doi:10.1038/ng.409