Histone tails and the H3 alphaN helix regulate nucleosome mobility and stability

Abstract

Nucleosomes fulfill the apparently conflicting roles of compacting DNA within eukaryotic genomes while permitting access to regulatory factors. Central to this is their ability to stably associate with DNA while retaining the ability to undergo rearrangements that increase access to the underlying DNA. Here, we have studied different aspects of nucleosome dynamics including nucleosome sliding, histone dimer exchange, and DNA wrapping within nucleosomes. We find that alterations to histone proteins, especially the histone tails and vicinity of the histone H3 alphaN helix, can affect these processes differently, suggesting that they are mechanistically distinct. This raises the possibility that modifications to histone proteins may provide a means of fine-tuning specific aspects of the dynamic properties of nucleosomes to the context in which they are located.

FIG. 1.

Effect of histone tail deletions on thermal nucleosome sliding. (A) Two picomoles of truncated and full-length octamers was assembled onto differentially labeled fluorescent DNA, mixed, and incubated at 47°C for the specified amount of time. Nucleosomes at different translational positions were separated on a native polyacrylamide gel, and the individual types of nucleosomes were visualized using the appropriate excitation and emission settings as illustrated. The majority of nucleosomes assembled at a position +70 relative to the MMTV transcriptional start site; following thermal incubation these equilibrated to a more favorable location at +22 (16). However, a proportion of nucleosomes (indicated by an asterisk) was deposited at a less strongly favored location at +47 (53). (B to M) Individual tail deletions have distinct effects on the rate of nucleosome repositioning. The graphs show the fraction of nucleosomes repositioned as a function of time. The data points are the average of three independent repeats, with the exception of panel F, and the curve describes the line of best fit used to calculate the initial rates. (N) Initial rate of repositioning for globular nucleosomes relative to a full-length control. WT, wild type.

FIG. 2.

Thermal repositioning of H3 truncated nucleosomes. (A) Cartoon illustration of the nucleosome core particle and the relative positions of residues R26, historically considered the last tail residue, and K37, which is the last residue. (B to E) Two picomoles of nucleosomes with either full-length or the indicated truncated H3 was incubated for 1 h at the temperatures indicated. Deletion of the entire H3 tail results in the nucleosomes' becoming diffusely distributed about the start position rather than moving to a new location.

FIG. 3.

Deletion of the H3 tail affects DNA end-to-end FRET. (A) Nucleosomes were assembled onto DNA containing Cy3 on one end and Cy5 on the other. Once assembled, the two arms of DNA were brought into close enough proximity to generate a FRET signal. (B) gH3 nucleosomes are not able to constrain their arms of linker DNA, as indicated by the greatly reduced FRET signal. This does not occur if the H4 tail is deleted instead. The measurements were made in 50 mM Tris (pH 7.5)-1 mM MgCl₂ with 60, 160, 260, 360, 460, and 2,010 mM NaCl. WT, wild type.

FIG. 4.

Deletion of the H3 tail affects histone-DNA contacts in the outer gyre. (A) Cysteine-EDTA nucleosome mapping reagent was conjugated to nucleosomes via cysteine residues introduced either at the dyad (H4 S47C) or the outer DNA gyre (H2B T87C). (B) Nucleosomes were incubated for 1 h at the indicated temperatures, followed by initiation of the Fenton reaction which causes site-specific DNA cleavage. The absence of the H3 tail results in the preferential loss of histone-DNA contacts at H2B T87C (cleavage sites in red box). Compare lanes 1 and 2 with lanes 5 and 6. In contrast, the DNA contacts in the region of the nucleosome dyad (H4 S47C contacts in blue box) are relatively unaffected.

FIG. 5.

Measuring histone dimer stability with a dimer exchange assay. (A) Histone octamers containing Cy5-labeled H2B were assembled onto a 219-bp DNA fragment and incubated at 47°C with histone H3-H4 tetramers assembled on a 147-bp DNA fragment. Any histone dimers that dissociate from the donor nucleosome are likely to associate with the tetrasome which acts as a “sink.” (B) Deletion of the H3 tail results in increased transfer of histone dimers to the acceptor chromatin. WT, wild type.

FIG. 6.

Point mutations in the H3 αN helix affect nucleosome dynamics. (A) Wild-type and H3 I51A nucleosomes were incubated for the indicated amount of time at 47°C. H3 I51A nucleosomes slide approximately ninefold faster than wild type. (B) H3 Q55A nucleosomes show decreased dimer stability. After 60 min at 47°C, H3 Q55A nucleosomes have lost almost threefold more dimers than wild type. (C) Specific point mutations within the αN helix reduce DNA end-to-end FRET (R49A), indicating that DNA wrapping is severely altered in these nucleosomes. WT, wild-type.

FIG. 7.

Interactions of the H3 αN helix with DNA, H2A, and H4 help to explain the effects of mutations in this region on nucleosome dynamics. (A) Overview of where the H3 αN helix sits within the nucleosome and an enlargement illustrating some of the regions it interacts with. These include the DNA entry gyre, the central DNA gyre, the H2A C-terminal extension, or the H4 α1 helix. Residues oriented on the same surface of the helix make similar contacts so the residues within the helix can be assigned into these four interaction surfaces. In panel B the residues within each horizontal bar interact with a common adjacent region of the nucleosome. This helical net annotation provides a means of relating how the different interaction surfaces affect the different aspects of nucleosome dynamics we have studied. Residues H3 R40 to P43 do not form part of the αN helix but are shown for completeness. S57 is joined with a dotted line to K56 to indicate that it is in an unusual conformation. The four interaction surfaces are displayed linearly; however, due to its circular nature, residues pointing toward the DNA central gyre are also close to the H4 α1 helix. (C) Data from Table 1 are superimposed onto the helical net to uncover patterns in how residues affect the dynamic properties of nucleosomes. This analysis reveals that DNA end FRET is most influenced by mutation to residues pointing toward the entry DNA gyre, while dimer exchange is affected by residues interacting with the H2A C-terminal extension as well as the central DNA gyre. Nucleosome sliding is strikingly affected by residues contacting H2A and also by residues interacting with DNA.