Together, our data suggest that Nap1 promotes nucleosome assembly not by delivering histones, but through disfavoring non-nucleosomal interactions between H2A–H2B dimers and DNA, both in vitro
and in vivo
. Several early studies have characterized the interactions of H2A–H2B dimer with DNA (Aragay et al., 1988
; Oohara and Wada, 1987
; Samso and Daban, 1993
). Our in vitro
experiments demonstrate that these histone-DNA interactions must be prevented for nucleosome assembly to occur. Moreover, predictions from our in vitro
experiments are in perfect accord with results obtained in vivo
, where we observe significantly enriched H2A and H2B levels (but not H3 levels) at endogenous genes in a nap1
Δ strain. This atypical H2A–H2B enriched chromatin appears to present a lower barrier for the transcription machinery, resulting in deregulated gene activation and repression. Accumulation of H2A–H2B is observed in the single knockout strain; thus, this particular function of Nap1 is non-redundant with the other histone binding proteins in yeast. It is also interesting to speculate that localized inhibition of Nap1 (perhaps through one of its many interaction partners) may be capable of locally generating this atypical chromatin architecture in a wild type cell. The demonstration of non-nucleosomal chromatin structures profoundly changes the view of histones and histone chaperones in regulating DNA accessibility. Many studies have used the occupancy of H3 in a chromatin immunoprecipitation assay to infer that no histones are bound to the DNA. The existence of the atypical chromatin complexes described here must be taken into account when interpreting these published results. Our thermodynamic data also explain why Nap1 is found predominantly in complex with H2A–H2B in vivo
(Ito et al., 1996
; Mosammaparast et al., 2001
), despite its high affinity for both H2A–H2B and H3–H4 in vitro
(Andrews et al., 2008
The thermodynamic assay developed and employed here is applicable to other histone chaperones and provides a means to clarify the nomenclature in the chromatin field. There are a plethora of proteins currently classified as histone chaperones, many of which may simply bind histones and/or aid in direct assembly. In keeping with our results, as well as the original definition of a histone chaperone by Laskey et al.
(Laskey et al., 1978
), we feel this term should be reserved for those proteins that prevent incorrect histone-DNA interactions. This terminology is also consistent with the functions of general protein chaperones, which prevent both newly synthesized polypeptide chains and assembled subunits from aggregating into nonfunctional structures. As such, and somewhat ironically because of its name, our thermodynamic analysis and in vivo
experiments assign Nucleosome Assembly Protein 1 (Nap1) as the first true histone chaperone.
The comparison of the thermodynamics of nucleosome assembly on two different DNA sequences confirms the notion that DNA sequence has a measurable effect on nucleosome stability under physiological conditions. Previous studies have used competitive salt gradients to determine that the 601 DNA sequence is a more favorable sequence for nucleosome formation than 5S by a ΔΔG° of −2.9 kcal/mol (Lowary and Widom, 1998
). However, these observations are the result of a convolution of K3
, and K6
and their respective salt dependence, while our experiments are independent of ionic strength and address each constant independently. Thus, while the two experimental approaches are expected to give the same trend, the ΔΔG° values cannot be compared directly.
In light of recent efforts to understand the relationship between DNA sequence and in vivo
nucleosome positions, our assay will undoubtedly be a useful tool to test key hypotheses stemming from whole genome analyses (e.g. Kaplan et al., 2008
; Zhang et al., 2009
). Our thermodynamic analysis also clarifies the role of H3K56 acetylation in promoting chromatin dynamics during transcription, DNA repair and replication. With the known turnover of H2A–H2B in vivo
(reviewed in Kimura, 2005
; Thiriet and Hayes, 2006
), our results with acetylated H3K56 indicate that this modification increases the fluidity of chromatin by disfavoring the association of the tetramer with DNA. This alone could explain the observed increased interaction of (H3K56ac-H4)2
tetramer with CAF-1 (Li et al., 2008
). The future use of our multidisciplinary approach will fill important gaps in the field of chromatin biology, as it will allow us to systematically test additional key hypotheses regarding the effect of DNA sequence, histone posttranslational modifications and histone variants on nucleosome stability under physiologically relevant conditions.