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. 2018 Oct;19(10):e44445.
doi: 10.15252/embr.201744445. Epub 2018 Sep 3.

MacroH2A Histone Variants Limit Chromatin Plasticity Through Two Distinct Mechanisms

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Free PMC article

MacroH2A Histone Variants Limit Chromatin Plasticity Through Two Distinct Mechanisms

Marek Kozlowski et al. EMBO Rep. .
Free PMC article

Abstract

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP-ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1-dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform-specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.

Keywords: DNA damage; PARP1; heterochromatin; histone variants; macroH2A.

Figures

Figure 1
Figure 1. MacroH2A isoforms show specific structural differences in their macrodomains that translate into functional plasticity

Surface representations of the macrodomain structures for the human macroH2A isoforms macroH2A1.1 (PDB ID 1ZR3) and macroH2A2 (PDB ID 6FY5), respectively, with electrostatic potential included. A yellow‐framed box indicates the location of the major surface pocket accommodating ADP‐ribose in macroH2A1.1.

Close‐up view of the nucleotide binding region of ADP‐ribose bound by human macroH2A1.1 (C) overlaid with the corresponding region of macroH2A2 in orange (D). An important structural difference in macroH2A2 that distorts de nucleotide binding loop is highlighted in magenta, and glycines in the nucleotide binding loop are depicted as spheres (G312 and G314). The ADP‐ribose ligand is included in (D) (shaded light gray) to facilitate orientation and comparison. Dashed lines indicate polar interactions.

Figure EV1
Figure EV1. MacroH2A histones show tissue‐specific expression profiles

Western blot showing the expression of macroH2A1.1, macroH2A1.2, and macroH2A2 in different male mouse tissues. Samples of HepG2 cells expressing ectopic FLAG‐tagged forms of the three macroH2A isoforms are included as antibody specificity controls and to aid comparisons. H3 is used as a loading control. A representative blot of three independent analyses is shown.

In situ mRNA hybridizations of murine macroH2A2 on 20 μm cryosections of E18 mice reveal a high concentration of macroH2A2 mRNA in the entire central nervous system, kidney, and testis. Scale bar is 5 mm.

Immunohistochemical staining for histone macroH2A1.1 and macroH2A1.2 in paraffin‐embedded human testis sections and lymph nodes. Images reveal a distinct staining consistent with Sertoli cells for macroH2A1.1 (arrows), while macroH2A1.2 is also expressed in proliferating germ cells (arrowheads). In the lymph node, proliferating cells positive for Ki67 express low levels of macroH2A1.1 while they are positively stained for macroH2A1.2. Scale bars are 50 μm.

Source data are available online for this figure.
Figure 2
Figure 2. Binding of the NAD metabolite ADP‐ribose is specific to macroH2A1.1 and conserved

Isothermal calorimetry (ITC) assays using ADP‐ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.

In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25, and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant, and macroH2A2. PARP1 activity is assessed by its auto‐PARylation detected by an anti‐PAR immunoblot. The naphthol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.

Source data are available online for this figure.
Figure EV2
Figure EV2. The capacity of macroH2A1.1 to bind ADP‐ribose is conserved in fish

Isothermal calorimetry demonstrates the capacity of fish macroH2A1.1 macrodomains to bind ADP‐ribose with similar affinity as the human form. Representative blots of more than three independent experiments are shown.

Conservation scores resulting of aligning macroH2A1.1 and macroH2A2 protein sequences of human, zebrafish, and medaka all together or mH2A1.1 and mH2A2 separately. The conservation score reflects the conservation of physico‐chemical properties in each position of the alignment on a scale that ranges from 0 to 11 and is represented both in the height and color brightness of the bar. The top schematic displays the different domains of macroH2A. The alignment was generated with the online alignment tool PRALINE 59 using the protein sequences with the following accession codes: H. sapiens macroH2A1.1 (NP_613075.1), D. rerio macroH2A1 (NP_001035451.1), O. latipes macroH2A1.1 (XP_011481424.1), human macroH2A2 (NP_061119.1), D. rerio macroH2A2 (NP_001020673.1), and O. latipes macroH2A2 (XP_004076965.2). The alignment and conservation track were edited with Jalview 60.

Protein sequence alignment of the amino acids that form the binding pocket of the macrodomain of macroH2A1.1 and macroH2A2 from human (H. sapiens), zebrafish (D. rerio), and medaka (O. latipes). Critical amino acids for ADPr binding conserved in macroH2A1.1 are indicated, as well as the amino acids in macroH2A2 cited in the main text. The top amino acid coordinates refer to macroH2A1.1 sequences while the bottom ones refer to macroH2A2 sequences. The alignment was generated as in (B) and colored using the ClustalX color scheme available in Jalview. In summary, amino acids are colored according to their physico‐chemical properties only if that position in the alignment fulfills a conservation criteria for that amino acid type: blue—hydrophobic, red—positive charge, magenta—negative charge, green—polar, pink—cysteine, orange—glycine, yellow—proline, cyan—aromatic, white—no criteria met.

Figure 3
Figure 3. MacroH2A proteins repress chromatin expansion at DNA damage sites; macroH2A1.1 being the most potent in the reduction

The diameter (d) of the fluorescence signaling of H2B tagged with photo‐activatable GFP (PAGFP‐H2B) is used to quantify chromatin expansion across 120 s after induction of DNA damage using intercalating DNA dye and local irradiation with a laser.

Chromatin expansion in absence and presence of intercalating DNA dye (− and + DNA damage) and pre‐treatment of 1 h with 1 μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 s. post‐DNA damage from three biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median, and cross indicates the average values. The whiskers show the highest and lowest data point within the 1.5× interquantile range of the upper and lower quartile, respectively (*P < 0.05 using unpaired, two‐tailed Student's t‐test).

Quantified chromatin expansion in cells not transfected (control) and cells transfected with each mCherry‐tagged histone (as indicated). Data are represented as in (B) (n > 30 cells, 3 bio. repl.; *P < 0.05 compared to mCherry‐H2A‐expressing cells using unpaired, two‐tailed Student's t‐test).

Chromatin expansion in Hela cells containing a doxocycline (doxo.)‐inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 h after induction, and expression was controlled by anti‐macroH2A1.1‐immunoblotting. Data are represented as in (B) (n > 30 cells, 3 bio. repl.; *P < 0.05 using unpaired, two‐tailed Student's t‐test).

Source data are available online for this figure.
Figure EV3
Figure EV3. Exogenous macroH2A has low mobility in U2OS cells

The comparison of the endogenous levels of all three macroH2A proteins in U2OS cell lysates with cells expressing tagged proteins for reference shows that U2OS cells predominantly express macroH2A1.2, little macroH2A2 while macroH2A1.1 was not detectable. Arrowheads indicate the size of endogenous proteins, asterisk indicates an unspecific band.

The mCherry‐tagged constructs of H2A and macroH2A proteins used in Fig 4C were transiently expressed in U2OS cells, and migration sizes were examined by immunoblotting.

Using experimental conditions as in Fig 3A, the fluorescence of mCherry‐tagged H2A and macroH2A proteins is not recovered in the bleached area after 120 s, indicative of stable chromatin incorporation of the exogenously expressed constructs.

Source data are available online for this figure.
Figure 4
Figure 4. MacroH2A histones repress chromatin expansion via the basic linker and the macrodomain capable of ADP‐ribose binding

Scheme of macroH2A and H2A variants constructs used in the study: full‐length wild type (WT), constructs with deleted macrodomain (ΔM), constructs with deleted linker and macrodomain (ΔLM), or hybrids consisting of the H2A histone fold fused to the linker of macroH2A1.1 (H2A + L) or the H2A fold fused to the linker and macrodomain of macroH2A1.1 (H2A + LM).

Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 s. post‐DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median, and cross indicates the average values. The whiskers show the highest and lowest data point within the 1.5× interquantile range of the upper and lower quartile, respectively (*P < 0.05 using unpaired two‐tailed Student's t‐test comparing to H2A; n.s., not significant).

Source data are available online for this figure.
Figure 5
Figure 5. The linker domain of macroH2A2 is able to maintain heterochromatic structures

Western blot showing the expression of macroH2A proteins in control HepG2 cells, knockdown for macroH2A1 and macroH2A2 (DKD) and DKD with ectopic expression of the indicated YFP‐tagged constructs. H3 is used as a loading control.

H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed‐in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z‐projections of confocal stacks.

Quantification of the nuclear area and number of H3K9me3 foci per nucleus, respectively (n > 50 cells, 2 biol. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median, and rhombi indicate the average values. The whiskers show the highest and lowest data point within the 1.5× interquantile range of the upper and lower quartile, respectively (*P < 0.05 using unpaired two‐tailed Student's t‐test; n.s., not significant).

Source data are available online for this figure.

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