Lewis TS et al. (2021), Structural basis of chromatin regulation by his...

XB-ART-58762

Nucleic Acids Res 2021 Nov 08;4919:11379-11391. doi: 10.1093/nar/gkab907.

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Structural basis of chromatin regulation by histone variant H2A.Z.

Lewis TS , Sokolova V , Jung H , Ng H , Tan D .

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The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z's ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription.

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Species referenced: Xenopus laevis
Genes referenced: h2ac21

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	Figure 1. Cryo-EM structures reveal flexible DNA terminus in H2A.Z nucleosomes. (A) Cryo-EM density map of the canonical nucleosomes at 3.8 Å resolution: disc view (top) and side view (bottom). (B) Cryo-EM map of H2A.Z nucleosome at 3.7 Å resolution: disc view (top) and side view (bottom). (C) Atomic model of H2A.Z nucleosome: disc view (top) and side view (bottom).
	Figure 2. Structural variability of the H2A.Z nucleosome. (A) superposition of the H2A.Z nucleosome with the canonical nucleosome structure (PDB ID: 3LZ0), disc view (top) and side view (bottom). The H2A.Z nucleosome DNA in purple are shown in surface mode. The canonical nucleosome DNA in grey are in ribbon mode. DF1 and DF2 are labeled. The region with the flexible DNA terminus on DF1 is highlighted with orange box. (B) Close-up view of the DNA-histone interaction as highlighted in the orange box in (A) to show the missing density of 11 bp DNA and part of the docking domain in H2A.Z. (C) Close-up views of the DNA-histone interaction in the same region as in (B) but on the DF2, to show visible DNA end and similar conformation of the C-terminal loop between H2A.Z and H2A. Part of the H2A.Z docking domain (orange) missing in (B) is also visible. (D) Conserved residues at helix αC of H2A.Z that are part of the acidic patch. (E) Conserved residues in H2A.Z L1 loop different from the canonical H2A.
	Figure 3. DNA terminus on H2A.Z nucleosome is accessible. (A) Sequence alignment of H2A and variant H2A.Z across species. The docking domain, L1 loop, extended acidic patch, M6-M7 cassette and C terminus are indicated. (B) 601 Windom sequence with the two restriction-enzyme cutting sites indicated. (C) Representative acrylamide gel of Hinf I DNA accessibility assay with the H2A.Z nucleosomes and canonical nucleosomes. (D) Representative acrylamide gel of the Nsil DNA accessibility assay with the H2A.Z nucleosome and canonical nucleosome. (E) Quantification of the HinfI DNA accessibility assay with canonical nucleosome (red), wild-type H2A.Z nucleosome (blue), and H2A.Z nucleosomes with L1 loop mutations (purple), with acidic patch mutations (pink), and with M6/7 cassette mutations (light blue). The graph shows the fraction of digested nucleosomes as a function of time. The data points are the average of different biological replicas. Data are mean ± SEM, n = 5. (F) Quantification of the Nsil DNA accessibility assay with canonical nucleosome and H2A.Z nucleosomes. Data are mean ± SEM, n = 3. (G) Quantification of the HinfI DNA accessibility assay with canonical nucleosome, wild-type H2A.Z nucleosome, and H2A.Z nucleosomes containing C_terminus mutant. Data are mean ± SEM, n = 6. H2A.Z C terminal mutant data shows statistically significant difference from H2A data (P = 0.0115) and H2A.Z data (P = 0.0027). (H) Quantification of the HinfI DNA accessibility assay with canonical nucleosome, wild-type H2A.Z nucleosome, and H2A.Z nucleosome with C_terminus_extended mutantations. Data are mean ± SEM, n = 6. H2A.Z nucleosome containing C_terminus_extended mutant data shows statistically significant difference from H2A data and H2A.Z data (P value for both < 0.0001).
	Figure 4. Cryo-EM structures of chromatin fibers containing H2A.Z and H2A nucleosomes. (A) Representative micrographs of vitreous sample of H2A fiber with selected 2D classes. Particles that share the same view as the 2D classes are color coded the same way and highlighted with either a circle or a square. Scale bar = 25 nm. (B) Representative micrographs of vitreous sample of H2A.Z fiber with selected 2D classes. Scale bar = 25 nm. (C) Schematic of dodeca-nucleosome fiber. A di-nucleosome structural unit is highlighted in an orange square. (D) cryo-EM map of multi-body refined H2A fiber, side and top view; Nucleosome 3 to 10 are shown. Linker DNAs are highlighted with red lines and the fiber axis is shown as a dotted line. Scale bar = 10 nm. Resolution of individual nucleosome ranged from 10 to 14.8 Å. (E) cryo-EM map of multi-body refined H2A.Z fiber, side and top view. Scale bar = 10 nm. Resolution of individual nucleosome ranged from 7.5 to 12.5 Å.
	Figure 5. Chromatin fiber containing H2A.Z is more compact (A) pseudo-atomic models of z-fiber containing nucleosome N3–N10, top view, front view and the side view. In side view, only one fiber strand is shown. (B) pseudo-atomic models of c-fiber; containing nucleosome N3- N10, top view, front view and the side view. In side view, only one fiber strand is shown. (C) Schematic of the chromatin model to depict inter-nucleosome distance x, shift d and rotation β between two adjacent di-nucleosome units as mentioned in the text. (D) comparison of di-nucleosome from three chromatin fiber models. DNA exit angle γ is shown. LS denotes the straight linker DNA and LB denotes the bend linker DNA in the tetra-nucleosome crystal structure (PDB ID: 1ZBB).
	Figure 6. Model of H2A.Z-mediated transcription regulation. Incorporation of variant H2A.Z leads to a more labile nucleosome with flexible DNA termini. Flexible DNA terminus of the +1 H2A.Z nucleosome (+1N) preceding the transcription start site (TSS) lower the activation energy for RNA Polymerase II (Pol II) during transcription initiation. The less rigid DNA terminus on a poly-nucleosome containing H2A.Z also enables the formation of a more compact and repressive chromatin domain.

References [+] :

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed