Histone Variant H2A.Z Nucleosome Incorporation Confers Unique Structural Characteristics of Chromatin

Tyler Lewis1a, Dr. Vladyslava Sokolova1b, and Dongyan Tan2.

1a. Stony Brook University, Molecular and Cellular Pharmacology, Stony Brook, USA 1b. Stony Brook University, Molecular and Cellular Pharmacology, Stony Brook, USA 2. Stony Brook University, Molecular and Cellular Pharmacology, Stony Brook, USA

All core histones except for histone H4 have nonallelic isoforms referred to as histone variants[1], [2]. A major mechanism for regulation of chromatin accessibility involves the exchange of canonical core histones with their variants to confer unique chemical and structural features to nucleosomes. Histone variant exchange is involved in a variety of cellular processes, including replication, transcription and heterochromatin formation and maintenance[1], [2]. H2A.Z is an evolutionarily conserved histone variant that is critical for normal mammalian development[3]. Interestingly, H2A.Z shares 60% amino acid sequence similarity with canonical H2A histone, yet displays unique surface properties which allow for distinct functional properties[4]. Compared with H2A, histone H2A.Z-H2B dimer displays an extended acidic patch on the nucleosome surface. The extended acidic patch likely serves as an interface to interact with histone tails and other protein factors, which in turn, promotes H2A.Z-unique biological functions[5].

Of particular interest, histone variant H2A.Z containing nucleosomes display high occupancy near promoters regions[1], [2]. This suggests that H2A.Z occupancy might allow for efficient recruitment of Pol II [7]. In stark contrast, H2A.Z has also been shown to be incorporated into heterochromatin regions such as telomeres, which suggests a repressive role of this variant[8]. Surprisingly, X-ray crystallographic studies of H2A.Z-containing mononucleosome revealed only subtle structural variations in the docking domain when compared to the major nucleosome structure [5]. Thus, how the H2A.Z incorporation structurally confers distinct functional characteristics of chromatin remains unclear.

We hypothesize that H2A.Z deposition enhances chromatin structural dynamics at the nucleosome level while simultaneously promoting the formation of a compact, higher-order chromatin structure. We resolved high-resolution, single-particle cryo-EM structures of nucleosomes containing either canonical H2A or histone variant H2A.Z at 3.8 Å and 3.7 Å respectively. Interestingly, structural analysis of each mononuclesome suggests that the entry/exit DNA at super-helical location (SHL) -6 appears to display increased flexibility in H2A.Z containing mononucleosome compared to canonical mononucleosome (Fig. 1). This is not observed in the H2A.Z crystal structure (PDBID: 1F66), perhaps due to packing constraints in the crystal. We propose a model where H2A.Z incorporation near the nucleosome-free region (NFR) at the promoter increases DNA flexibility of the +1 nucleosome and thus facilitates RNA polymerase recruitment.

To interrogate how H2A.Z incorporation and enrichment influences hierarchal chromatin structure, we generated reconstituted chromatin fiber on a DNA template containing 12 repeats of 601 Widom nucleosome positioning sequence to mimic chromatin secondary structure. Here, we have obtained two preliminary single-particle cryo-EM structures of chromatin fiber containing either H2A.Z or H2A (Fig. 2). Interestingly, H2A.Z incorporation appears to promote increased fiber compaction and increased overall twisting of fiber architecture relative to fiber generated with H2A. We propose that H2A.Z induced, increased chromatin compaction, may rationalize the propensity of H2A.Z occupancy in heterochromatic regions outside of promoter and enhancer regions of DNA.

References:

[1] B. Li et al., “Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling.,” Proc. Natl. Acad. Sci. U. S. A., vol. 102, 2005. [2] P. B. Talbert and S. Henikoff, “Histone variants on the move: substrates for chromatin dynamics,” Nat. Rev. Mol. Cell Biol., vol. 18, 2017. [3] R. Faast et al., “Histone variant H2A.Z is required for early mammalian development,” Curr. Biol., vol. 11, 2001. [4] H. Zhang, et al, “Genome-Wide Dynamics of Htz1, a Histone H2A Variant that Poises Repressed/Basal Promoters for Activation through Histone Loss,” Cell, vol. 123, 2005. [5] K. Luger, et al., “Crystal structure of the nucleosome core particle at 2.8 Å resolution,” Nature, vol. 389, 1997. [6] B. Guillemette et al., “Variant Histone H2A.Z Is Globally Localized to the Promoters of Inactive Yeast Genes and Regulates Nucleosome Positioning,” PLoS Biol., vol. 3, 2005. [7] M. Adam, et al, “H2A.Z is required for global chromatin integrity and for recruitment of RNA polymerase II under specific conditions.,” Mol. Cell. Biol., vol. 21, 2001. [8] M. Nekrasov et al., “Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics,” Nat. Struct. Mol. Biol., vol. 19,. 2012.

Figure 1. A) H2A.Z mononucleosome 3D classes used to generate the final map. B) Final H2A.Z mononucleosome map resolved to 3.7 Å fitted with H2A.Z crystal structure (1F66) (histone H2A.Z is colored green). Final H2A mononucleosome map resolved to 3.8 Å. B) Final H2A mononucleosome map resolved to 3.8 Å

Figure 2. A) Cryo-EM map of H2A.Z dodecanucleosome resolved to ~ 12 Å. B) Cryo-EM map of canonical dodecanucleosome resolved to ~ 16 Å.