Identification of Factor-nucleosome Interactions in vivo
We employed an in vivo factor-nucleosome interaction assay, which is derived from the standard ChIP assay involving protein-DNA crosslinking. In this assay, the chromatin was solubilized into nucleosome core particles using high levels of MNase (Yuan et al., 2005
), rather than fragmented via sonication. We also employed multiple purification steps, associated with the use of TAP-tagged proteins. The resulting immunoprecipitated factor-bound mono-nucleosomal DNA was detected by LM-PCR as a nucleosomal-sized band (), and ultimately mapped across the genome using massively parallel DNA sequencing (AB SOLiD) and verified with high-density tiling microarrays (Affymetrix, 5 bp probe spacing). These genome-wide methods are expected to define a subset of all nucleosome positions in the genome that are in very close proximity (few angstroms) to the tested factor.
Identification of Factor-nucleosome Interactions in vivo
We detected nucleosomal crosslinks for representatives in each type of interaction (, and quantified in data column 1): (i) Htz1, Srm1, Vps72 and Bdf1; (ii) Rap1 and Reb1; and (iii) Rpo21 (RNA polymerase (Pol) II). No crosslinks were detected using an untagged (BY4741) control. No crosslinks were detected with the general transcription factor Sua7 (TFIIB), indicating that not all nuclear proteins are in close crosslinkable proximity to nucleosomes. TFIIB binds in the middle of the NFR (~100 bp from −1 and ~40 bp from +1) and thus is not expected to interact with nucleosomes (Venters and Pugh, 2009
). These findings substantially increase the number of proteins demonstrated to crosslink with nucleosomes in vivo, rather than with DNA only, which the standard ChIP assay does not distinguish.
Summary of factor-nucleosome interactions
A number of addressable caveats are associated with the factor-nucleosome LM-PCR assay. First, it does not distinguish between a protein bound directly to a nucleosome vs. a protein bound to the adjacent linker/NFR regions, but close enough to be crosslinked. Below, we provide a means to distinguish these possibilities for sequence-specific DNA binding factors. Second, without demonstration that binding is actually measurable in a standard ChIP assay, a negative result is not interpretable. Moreover, any crosslinking that is detected represents a net effect of intrinsic crosslinking (i.e., ChIP efficiency) and actual nucleosomal binding.
To distinguish between ChIP efficiency and actual nucleosome binding, we measured intrinsic crosslinking by standard genome-wide ChIP-chip experiments where the chromatin is fragmented by sonication rather than by MNase over-digestion. In this assay, all binding events (nucleosomal and non-nucleosomal) are measured. To assess intrinsic ChIP efficiency, we calculated the ratio of hybridization values at the top 1% of bound sites (after probe normalization) to the bottom 10%, which we take to represent background levels of binding. ChIP efficiency is reported in data column 2 in . Factors like Rap1, Reb1, and Sua7/TFIIB have very high intrinsic ChIP efficiencies (40–70 fold over the control BY4741).
We next calculated the Nucleosome Interaction Ratio (data column 3 in ), which equals the observed LM-PCR nucleosomal interaction signal normalized to ChIP efficiency (essentially data column 1 divided by data column 2). As expected, the highest Nucleosome Interaction Ratio was seen with Htz1/H2A.Z, which is a nucleosome subunit. The lowest ratio was Sua7/TFIIB, indicating that despite its strong ChIP signal, it does not crosslink to nucleosomes. Thus, despite the nucleus being crowded with nucleosomes, not all competent gene regulatory factors will crosslink with nucleosomes. We conducted further analysis to assess the physiological and mechanistic significance of such interactions.
Bdf1 interacts with NuA4-acetylated nucleosomes in vivo
Bdf1 (type I interaction) is a component of SWR-C/SWR1 (Kobor et al., 2004
; Krogan et al., 2003
), which is responsible for incorporating H2A.Z into nucleosomes at promoters. Bdf1 binds to acetylated lysines on isolated histone H4 tails (Jacobson et al., 2000
; Matangkasombut and Buratowski, 2003
), and this acetylation is catalyzed by the Esa1 subunit of the NuA4 complex (Allard et al., 1999
). As further validation of Bdf1-nucleosome interactions in vivo, we found that Bdf1TAP
-nucleosomal interactions were lost in a catalytically dead esa1-414
mutant (, lane 8 vs 10). As expected, H2A.Z incorporation was also lost (lane 7). Bdf1 also interacts with TFIID (Matangkasombut et al., 2000
; Sanders et al., 2002
), which is responsible for assembling the pre-initiation complex. However, loss of the main TFIID subunit in a taf1-2
strain failed to eliminate Bdf1-nucleosomal interactions (lane 9). Together, the results indicate that Bdf1-nucleosomal interactions are mediated through NuA4-directed histone acetylation rather than TFIID. Thus, the factor-nucleosome interaction assay is further validated by the demonstration that the expected NuA4-dependent Bdf1-histone interactions that have been largely defined in vitro, produce the expected dependencies in vivo.
Bdf1 interacts with the +1 and +2 nucleosomes
The genomic locations of Bdf1-crosslinked nucleosomes were determined by sequencing 1,202,352 of these nucleosomes (examples of mapped positions are shown in ), and were verified by hybridization to high-density tiling arrays. Approximately 3% (1,853) of all 54,753 nucleosomes in the yeast genome were significantly crosslinked to Bdf1 (P
<0.05, Supplementary Fig. 1A
; and listed in Supplementary Table 1
), many of which may represent low levels of binding. We selected the genes having the strongest 150 Bdf1-bound nucleosomes as a robust subset for further analysis (listed in Supplementary Table 2
; cutoffs of 50, 450, and 1,853 produced essentially the same results, as shown in Supplementary Fig. 1
SWR1/Bdf1 are Enriched at the +1 and +2 Nucleosomes
Surprisingly, at individual genes, Bdf1 bound predominantly to either the +1 or the +2 nucleosome (upper vs lower panels in and Supplementary Figs. 1B, C
). This was not a consequence of mis-identifying the +2 nucleosome, because the hallmark of the +1 nucleosome, H2A.Z, was enriched at the +1 nucleosome in both cases (cyan filled plot in ). Moreover, the NFR that is adjacent to the +1 nucleosome is evident in both cases. We also found many cases where Bdf1 bound to the −1 and −2 positions, but these turned out to also be the +1 and +2 nucleosomes of divergently transcribed genes (Supplementary Fig. 2B
). Approximately 63% of the top 150 bound nucleosomes were found at the +1/+2 positions compared to 15% expected by chance (P
); 51% of all 1,853 significantly bound nucleosomes were at this position (P
= 0). Therefore, Bdf1 is selective for the +1 and +2 nucleosomes. Those not at +1/+2 positions may represent a combination of false positives, occupancy at non-protein encoding genes, and/or additional functionalities associated with Bdf1.
The selectivity of Bdf1 for the +1/+2 nucleosomes was not due to any intrinsically strong positioning of these nucleosomes, making them more detectable, because Bdf1-bound nucleosomes were about average for positioning strength when compared to all nucleosomes (Supplementary Fig. 1D
). Furthermore, the distribution of Bdf1-bound nucleosomes from −1 kb to +1 kb of the TSS did not follow the canonical distribution of all nucleosomes at the same set of genes (), which would be expected if the interactions were simply selecting the best-phased nucleosomes.
Since Bdf1 is part of the SWR1 complex, we examined the genome-wide distribution of SWR1-nucleosomal interactions (via its Vps72 subunit). Genes having Bdf1- and Vps72-bound nucleosomes were statistically co-incident (P ~10−99, ). Moreover, when Bdf1 was enriched at the +1 nucleosome so was SWR1 (Vps72), and when enriched at +2 so was SWR1 (Vps72) (, blue vs gold traces). This further supports the notion that the SWR1(Vps72)/Bdf1 complex together segregates between either the +1 or the +2 nucleosome, depending on the gene. Both clusters of genes tended to be transcriptionally active (red bar graph in ), indicating that the +1/+2 Bdf1 interactions are associated with transcription. However, neither group was differentially enriched with any Gene Ontology function, which is consistent with such interactions being associated with the transcription process rather than any gene-specific control mechanism. We also examined over two thousand genomic datasets in the public domain for differential properties between the two clusters. We found that cluster 1 tended to have higher levels of intergenic H4 acetylation (largely probing the status of the −1 and +1 nucleosomes) compared to cluster 2 (not shown), which is consistent with cluster 2 being relatively depleted of crosslinkable +1 nucleosomes, and cluster 1 having relatively high levels of acetylated +1 nucleosomes for Bdf1 binding.
Bdf1 Forms a Di-nucleosome Complex Specifically with the +1 and +2 Nucleosomes
We next sought to understand the relationship between Bdf1 binding to the +1 vs +2 nucleosome by biochemically isolating native Bdf1-nucleosomal complexes (i.e., no formaldehyde and use of a less chaotropic buffer). Surprisingly, these complexes were resistant to MNase (unlike other immunoprecipitated nucleosomal complexes), yielding predominantly di-nucleosomes rather than mononucleosomes (). This observation suggests that a native Bdf1-containing complex simultaneously binds to two nucleosomes and protects the intervening linker DNA from MNase digestion.
Bdf1 Forms a Di-nucleosome Complex with the +1 and +2 Nucleosomes
To verify that the di-nucleosomal complex represents interactions at the +1 and +2 positions, as opposed to minor or nonspecific complexes at other locations, the di-nucleosomal DNA was mapped at high resolution to the yeast genome. The di-nucleosomal DNA mapped to a region spanning the +1 and +2 nucleosomes (, note that occupancy between −1 and −2 is due to +1/+2 occupancy of divergent genes), which demonstrates that the Bdf1-bound di-nucleosomal complex is indeed specific to the +1/+2 nucleosomes. Taken together, our findings suggest that the SWR1/Bdf1 complex binds to a NuA4-acetylated di-nucleosomal complex that resides at the +1 and +2 positions of active genes (). The SWR1 complex then inserts H2A.Z preferentially at the −1 and +1 nucleosomes.
The strong bias of Bdf1 binding towards the +1 vs. the +2 nucleosome position (or visa versa) at individual genes might be a consequence of greater intrinsic nucleosome occupancy levels at the biased position, as shown in (gray filled plot). To identify a possible source of this bias we hypothesized that as Pol II transcription moves through this region, the +1 acetylated histones are ejected but perhaps retained locally by the SWR1/Bdf1 complex bound at +2 (). These histones are returned to +1 and a reciprocal process happens at +2 as Pol II moves through the +2 region.
Because Bdf1-bound nucleosomes might present a stronger barrier to Pol II movement, such a model predicts that Pol II occupancy (measured by standard sonication-based ChIP) would be enriched just before the nucleosome that SWR1/Bdf1 is bound to. In addition, the same SWR1/Bdf1-bound nucleosome might be preferentially crosslinked to Pol II due to their close proximity. Indeed, we find evidence to support these predictions at both the +1 () and +2 () nucleosome positions, where a local enrichment of Pol II (red trace) is found at a fixed distance just upstream of a SWR1/Bdf1-bound (blue-filled plot) and Pol II-crosslinked nucleosome (dark red trace). Additional Pol II is found in the body of the genes, as expected of their transcriptionally active state. Interestingly, in examining over two thousand public genomic datasets for distinguishing features between cluster 1 and 2, one of the strongest distinctions was the enrichment of the Bye1 negative regulator of transcription at some cluster 1 genes (not shown), which might indicate that the hold-up of Pol II before the +1 nucleosome might be regulated at least in part through pol II at these genes.
The notion that Bdf1 might help retain nucleosomes at some promoters is in apparent conflict with the findings that nucleosomes are highly dynamic at promoter regions (Dion et al., 2007
; Rufiange et al., 2007
). We addressed this by comparing the dynamic state of Bdf1-bound nucleosomes to all other nucleosomes at the +1/+2 position. Strikingly, Bdf1-bound nucleosomes were as cold or even colder (i.e. slower exchange dynamics) than the coldest 5% of +1/+2 nucleosomes (). This finding lends further credence, from two independent data sets, to the idea that Bdf1 promotes retention of nucleosomes at promoters during the passage of Pol II.
Rap1 Selectively Binds to the −1 Nucleosome that is Shared Between Two Divergent Genes
As a representative of type ii nucleosome-interacting proteins, the sequence-specific DNA binding transcription factor Rap1 is both an activator and repressor of some of the most highly and lowly expressed genes in the cell (Kurtz and Shore, 1991
; Shore, 1994
). Rap1’s positive role in transcription might be to direct nucleosome disruption and/or recruit TFIID to promoters (Garbett et al., 2007
; Yu and Morse, 1999
), whereas its negative role, paradoxically, may be to promote nucleosome formation (Gartenberg, 2000
; Shore, 1994
). These apparent opposing functions remain enigmatic, but could be linked to the location of Rap1 and nucleosomes in promoter regions.
The genomic locations of Rap1-crosslinked nucleosomes were determined by sequencing 383,892 of these nucleosomes (), and were verified by hybridization to high-density tiling arrays. Approximately 0.4% (229) of all 54,753 nucleosomes in the yeast genome were significantly crosslinked to Rap1 (P
<0.05, Supplementary Fig. 2A
and listed in Supplementary Table 1
). Thirty percent of the previously determined Rap1-bound loci (Lieb et al., 2001
) overlapped with these nucleosomes (the remainder being nucleosome-free sites). The genes associated with the top 150 Rap1-crosslinked nucleosomes were selected for further study (listed in Supplementary Table 2
Rap1 Associates with a Specific Rotational Setting on the −1 Nucleosome
Approximately 43% of the Rap1-bound nucleosomes were at the −1 position (P
) ( and Supplementary Fig. 2B,C
). For the same reasons presented above for Bdf1, detection of the Rap1-crosslinked nucleosomes was not a consequence of biased selection of nucleosomes that are intrinsically the most detectable ( and Supplementary Fig. 2B–D
Rap1-nucleosome crosslinking was not a consequence of Rap1 binding to adjacent linker DNA and fortuitously crosslinking to a neighboring nucleosome because when crosslinking was omitted, Rap1-nucleosomal binding was still detected on fully digested nucleosome core particles (presumably eliminating linker sites) (Supplementary Fig. 2E
). Moreover, the MNase resistant DNA present in Rap1-bound nucleosomes was not longer than that found in other nucleosomes (see ), indicating that Rap1 was not protecting additional flanking sequence as a potential consequence of adjacent binding. More importantly, 80% of Rap1-bound nucleosomal DNA possessed a Rap1 binding site within it borders, and very few had sites in adjacent linker regions (, black filled plot). Rap1-bound sites (Buck and Lieb, 2006
) that were not detected as Rap1-bound nucleosomes in our study, were found adjacent to nearby nucleosomes (red trace). This further confirms that Rap1 in linker/NFR regions does not fortuitously crosslink to adjacent nucleosomes. Interestingly, telomeric Rap1 sites tended to be internal to nucleosomes (green trace), suggesting that nucleosomal Rap1 interactions may be different in telomeric regions compared to promoter regions.
Reb1 Associates Specifically with the −1 Nucleosome
Strikingly, 23% of Rap1-bound “−1” nucleosomes were shared between two divergently-transcribed genes (i.e., the same nucleosome serving the −1 role for both genes), compared to <5% expected by chance (P
) ( and Supplementary Fig. 2B
, and illustrated as the “√” configuration in ). In contrast, for 27% of all divergently transcribed genes, the +1 nucleosome of one gene is the −1 nucleosome of the other gene (illustrated as the “X” configuration in ). None of these genes harbored a Rap1-bound nucleosome (P
). Thus, Rap1 may place an evolutionary constraint on the spacing between two divergent Rap1-regulated promoters, such that promoter Rap1-nucleosomal interactions are restricted to configurations where the bound −1 nucleosome does not also serve as a +1 nucleosome.
Rap1 Binds to the First and Second Rotationally Exposed Major Groove inside either Nucleosome Border
We further examined the distribution of the 13-bp bipartite directionally-oriented Rap1 binding site (ACACCCRYACAYM) on the mapped Rap1-nucleosome positions at −1. The midpoint of the Rap1 sites peaked 14 bp from either nucleosome border (, black filled plot), and was independent of site orientation (not shown). This places the bipartite Rap1 DNA binding domain and the bipartite DNA recognition site on the first and second turn from the nucleosome border of the rotationally exposed major groove (), which biochemical studies have shown to be the preferred location for Rap1 binding (Rossetti et al., 2001
). Together these findings provide near base-pair resolution for the placement of Rap1-nucleosomal interactions in the yeast genome.
Reb1 Selectively Binds to the NFR-proximal Border of the −1 Nucleosomal DNA
As a second representative of type ii nucleosome-interacting proteins, the sequence-specific DNA binding transcription factor Reb1 is thought to bind promoter regions and promote NFR formation (Angermayr and Bandlow, 1997
; Hartley and Madhani, 2009
; Raisner et al., 2005
), although NFR formation may be Reb1-independent at some sites (Erkine et al., 1996
; Moreira et al., 2002
; Reagan and Majors, 1998
). Conceivably, Reb1 might promote NFR formation in part by creating a boundary to which a nucleosome may not encroach. In such situations Reb1 might reside at or near the NFR-proximal nucleosome border. Alternatively, instead of a boundary, Reb1 might position a nucleosome by engaging in specific contacts with histones at some position along the nucleosomal DNA.
The genomic locations of Reb1-crosslinked nucleosomes were determined by sequencing 7,004,145 of these nucleosomes (). Approximately 0.5% (281) of all detectable 54,753 nucleosomes in the yeast genome were significantly crosslinked to Reb1 (P
<0.05, Supplementary Fig. 3A
and listed in Supplementary Table 1
). The genes associated with the top 150 Reb1-crosslinked nucleosomes were selected for further study (listed in Supplementary Table 2
Remarkably, 82% of the Reb1-bound nucleosomes were at the −1 position (P
) and 94% of the associated genes were divergently transcribed (upper panels in and Supplementary Fig. 3B,C
). Thus, like Rap1, Reb1 strongly favors the −1 nucleosome of divergently transcribed genes. However, unlike Rap1, Reb1-bound nucleosomal DNAs were ~12 bp shorter than the expected length (), suggesting that Reb1 binding might promote MNase invasion by enhancing the breathing of DNA at the nucleosome border in accordance with the site exposure model (Polach and Widom, 1995
When the distribution of Reb1 binding sites were examined around Reb1-bound nucleosomes at the −1 position, the Reb1 sites were found to be enriched at the border (), and were independent of recognition motif orientation (not shown). Strikingly, they were particularly enriched at the NFR-proximal border. The increased nuclease accessibility of the borders of Reb1-bound nucleosomes, which could be particular to the Reb1-bound border, precluded an accurate determination of their position, and so we were less certain as to the rotational setting of the Reb1 binding site. Nonetheless, the NFR-proximal location of Reb1 binding is in accord with the notion of Reb1 setting a boundary for nucleosome positioning adjacent to an NFR (Hartley and Madhani, 2009
; Raisner et al., 2005
). Since we do not see enrichment of Reb1 at the +1 nucleosome, some other factor may be responsible for establishing the downstream border of the NFR.
Srm1 Abundantly but Non-selectively Occupies Nucleosomes Genome-wide
Srm1 (RCC1 in human) is a guanine nucleotide exchange factor that is thought to regulate chromatin condensation and nucleocytoplasmic shuffling (Aebi et al., 1990
; Hadjebi et al., 2008
). Importantly, Srm1 is nuclear and binds nucleosomes (Nemergut et al., 2001
). In our in vivo factor-nucleosome interaction assay, Srm1 generated the strongest interaction ratio ().
Genome mapping of Srm1-nucleosome interactions revealed a distribution pattern around genes that was essentially indistinguishable from bulk nucleosomes ( and Supplementary Fig. 4
). Thus, while Srm1 binds abundantly to nucleosomes it does not appear to bind specifically. This is in accord with a general role of Srm1/RCC1 in maintaining chromatin structure, particularly in light of the fact that an srm1-1
mutant displays gross chromosomal structural abnormalities (Aebi et al., 1990
Srm1 Binds Nucleosomes Broadly and Pol II-bound Nucleosomes are Delocalized
Pol II-bound Nucleosomes are Delocalized
As a representative of type iii nucleosome-interacting proteins, Pol II is not expected to stably bind to an intact nucleosome. However, due to the fact that it must translocate along DNA, Pol II might collide with nucleosomes, and this could present a barrier to elongation (Bondarenko et al., 2006
). Indeed, in Drosophila
, Pol II initiates transcription and then pauses as it contacts the +1 nucleosome (Mavrich et al., 2008b
; Muse et al., 2007
; Zeitlinger et al., 2007
). Continued transcription elongation requires that Pol II either eject a nucleosome barrier or traverse some remodeled state of the nucleosome.
The genomic locations of Pol II-crosslinked nucleosomes were determined by sequencing 5,097,371 of these nucleosomes (), and were verified by hybridization to high-density tiling arrays. Size selection after MNase digestion () ensured that intact nucleosomes were being examined.
The genes associated with the top 150 peaks were analyzed further (Supplementary Fig. 5A
and listed in Supplementary Tables 1
). The positions of the pol II-crosslinked nucleosomes lacked phasing ( and Supplementary Fig. 5B
), and so making consensus calls of their positions was not informative. Individual nucleosomal tags were not enriched at canonical locations, as evidenced by a lack of well-defined peaks and valleys of tags around the TSS ( and Supplementary Fig. 5C
). Genes that contained relatively high levels of Pol II-crosslinked nucleosomes were generally highly transcribed (red bars in ) and depleted of nucleosomes. We interpret these findings to suggest that during transcription, Pol II collides with nucleosomes (detected as Pol II-crosslinked nucleosomes), and this results in their random repositioning and ultimately their eviction or partial dismantling to allow passage of Pol II (hence nucleosome depletion). The enrichment of Pol II-nucleosomal interactions towards the 5′ end of genes might reflect slower release or a quicker return of nucleosomes at the 5′ end of genes upon transcription.