Evolution of high mobility group nucleosome-binding proteins and its implications for vertebrate chromatin specialization

Abstract

High mobility group (HMG)-N proteins are a family of small nonhistone proteins that bind to nucleosomes (N). Despite the amount of information available on their structure and function, there is an almost complete lack of information on the molecular evolutionary mechanisms leading to their exclusive differentiation. In the present work, we provide evidence suggesting that HMGN lineages constitute independent monophyletic groups derived from a common ancestor prior to the diversification of vertebrates. Based on observations of the functional diversification across vertebrate HMGN proteins and on the extensive silent nucleotide divergence, our results suggest that the long-term evolution of HMGNs occurs under strong purifying selection, resulting from the lineage-specific functional constraints of their different protein domains. Selection analyses on independent lineages suggest that their functional specialization was mediated by bursts of adaptive selection at specific evolutionary times, in a small subset of codons with functional relevance-most notably in HMGN1, and in the rapidly evolving HMGN5. This work provides useful information to our understanding of the specialization imparted on chromatin metabolism by HMGNs, especially on the evolutionary mechanisms underlying their functional differentiation in vertebrates.

Keywords: HMGN; chromatin; episodic adaptive selection; high mobility group proteins; long-term evolution; nucleosome-binding domain; purifying selection.

Fig. 1.

Schematic representation of the interactions of HMGNs with chromatin. (A) Interaction of HMGN2 with the nucleosome (Kato et al. 2011; Kugler et al. 2012). The core histones are depicted in different light colors: H3: blue, H4: green, H2A: yellow, H2B pink. The green oval indicates the approximate location of the acidic patch (Luger et al. 1997). The colors for the HMGN2 molecule correspond to the different structural regions along its amino acid sequence (as indicated in fig. 2). Interaction of the NBD of HMGNs with the nucleosome positions their C-terminal domain near the nucleosome dyad. This results in an impairment of the proper binding of the winged histone domain (WHD) (Kasinsky et al. 2001) of linker histones to this region (Zhou et al. 2013). (B) Interaction of HMGN5 with chromatin results in a relaxed open conformation of the chromatin fiber, which prevents histone H1 binding. Such an unfolding stems from the binding competition between HMGN5 and histone H1 for the dyad region of the nucleosome, and/or from the juxtaposition of their respective negatively and positively charged C-terminal domains (Rochman et al. 2009, 2010). The red circle in the histone H1 molecule represents its highly characteristic WHD. The double arrow underscores the highly dynamic nature of the interactions of histone H1 and HMGNs with the chromatin template (Kugler et al. 2012).

Fig. 2.

HMGN1 and HMGN2. (A) Protein sequence alignment for a representative organism of each of the five vertebrate classes: Zebrafish, Danio rerio (fish); African clawed frog, Xenopus laevis (amphibian); Carolina anole, Anolis carolinensis (reptile); chicken, Gallus gallus (bird); and mouse, Mus musculus (mammal). The combined Logos representations, using alignments from supplementary figure S1, Supplementary Material online, are also shown. (B) Western-blot analysis of HMGN2 from liver tissue-PCA extracts from each one of the vertebrate representatives in (A). A coomassie blue-stained replica SDS–PAGE corresponding to the histone H1 fraction coextracted in this way is also shown. (C) Coomassie blue stained SDS–PAGE and Western-blot analysis of HMGN2 PCA extracted from different mouse tissues (liver, brain, testis, kidney, lung, and gut). In (B) and in (C), histones H1 were used for protein loading normalization purposes.

Fig. 3.

HMGN3, HMGN4, and HMGN5. Protein sequence alignment for different representative organisms: chicken, Gallus gallus; cow, Bos taurus; mouse, Mus musculus; Rhesus macaque, Macaca mulatta; chimpanzee, Pan troglodytes; orangutan, Pongo abelii; human, Homo sapiens. The combined Logos representations, using alignments from supplementary figure S1, Supplementary Material online, are also shown.

Fig. 4.

Phylogenetic maximum likelihood (ML) relationships among vertebrate HMGN protein lineages. The numbers for interior branches represent nonparametric bootstrap (BS) probabilities based on 1,000 replications, followed by Bayesian posterior probabilities (only shown when BS ≥ 50% or posterior probability ≥ 0.5). Two black circles at internal nodes indicate subtrees at which the molecular clock hypothesis was rejected (P < 0.001) after testing for the presence of local molecular clocks.

Fig. 5.

Estimated rates of evolution for HMGN proteins. Evolutionary rates for the fast-evolving chromosomal proteins histone H2A.Bbd, as well as histone H1 and histones H2A/H2B (dashed lines) are included as reference. HMGN4 is not shown, due to its very slow rate of evolution.

Fig. 6.

Selection episodes involved in the evolution of mammalian HMGN lineages. (A) ML gene tree depicting episodes of diversifying selection during HMGN differentiation in mammals. Numbers for interior branches are indicated as in figure 4. Deviations from the molecular clock at internal subtrees are indicated by one (P < 0.01) or two (P < 0.001) black circles at the corresponding internal braches. The strength of selection at significant branches is represented in red (ω>5), gray (ω = 1), and blue (ω = 0), with the proportion of sites within each class represented by the color width. Thicker branches have been classified as undergoing episodic diversifying selection at corrected P ≤ 0.001 (thickest branches), P ≤ 0.01 (medium thickness), and P ≤ 0.05 (thin branches). (B) Phylogenetic location of mutations involved in diversifying selection episodes during the evolution of HMGN genes. Branches in red account for higher numbers of nonsynonymous mutations, whereas branches in blue indicate higher numbers of synonymous mutations, and branches in green represent cases with equal numbers of nonsynonymous and synonymous mutations. Codon 49 is located within the highly conserved NDB region.