Tau Stabilizes Chromatin Compaction

Abstract

An extensive body of literature suggested a possible role of the microtubule-associated protein Tau in chromatin functions and/or organization in neuronal, non-neuronal, and cancer cells. How Tau functions in these processes remains elusive. Here we report that Tau expression in breast cancer cell lines causes resistance to the anti-cancer effects of histone deacetylase inhibitors, by preventing histone deacetylase inhibitor-inducible gene expression and remodeling of chromatin structure. We identify Tau as a protein recognizing and binding to core histone when H3 and H4 are devoid of any post-translational modifications or acetylated H4 that increases the Tau's affinity. Consistent with chromatin structure alterations in neurons found in frontotemporal lobar degeneration, Tau mutations did not prevent histone deacetylase-inhibitor-induced higher chromatin structure remodeling by suppressing Tau binding to histones. In addition, we demonstrate that the interaction between Tau and histones prevents further histone H3 post-translational modifications induced by histone deacetylase-inhibitor treatment by maintaining a more compact chromatin structure. Altogether, these results highlight a new cellular role for Tau as a chromatin reader, which opens new therapeutic avenues to exploit Tau biology in neuronal and cancer cells.

Keywords: Tau protein (Tau); cancer biology; chromatin regulation; chromatin remodeling; histone (de)acetylation; histone deacetylase inhibitor (HDAC inhibitor); histone modification and chromatin structure.

FIGURE 1

Tau inhibition increases MCF7 breast cancer cell line sensitivity to TSA. (A) Tau protein expression in MCF7, MCF7shctrl, and shTau were quantify by ELISA. (B) Effect of 50 and 100 nM TSA (48 h) on cell death in the MCF7shctrl and MCF7shTau subclones. Cell death was determined by staining and flow cytometric analysis as described in section “Materials and Methods.” (C) Effect of 50 and 100 nM TSA (48 h) on apoptosis in the MCF7shctrl and MCF7shTau subclones. Apoptosis was determined by flow cytometric analysis of the PI-positive and Annexin-V-positive cells as described in section “Materials and Methods.” (D) Cell cycle distribution was determined by FACS analysis of combined propidium iodide and EdU staining in MCF7shctrl and MCF7shTau in the absence or presence of 100 nM TSA, 48 h. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ^###P < 0.001. All results are representative of three independent experiments.

FIGURE 2

Tau expression prevented TSA-dependent chromatin remodeling. (A) Representative confocal sections of MCF7shctrl and shTau cells untreated or treated with 100 nM TSA for 24 h. DNA was revealed by DAPI and HP1α was immuno-localized. (B) Quantification of HP1α clusters per nuclei visualized as described previously and realized on three independent experiments. (C) GADD45a and (D) p21 (CDKN1a) expression in MCF7shctrl and shTau cells untreated or treated with 100 nM TSA for 24 h were analyzed by real-time PCR and normalized to RPLO. Results are expressed, relative to the basal activity set to 1, as the mean ± SD of three independent assays. (E) Tau tethering prevents adjacent reporter gene activity induced by TSA. GAL4UAS responsive luciferase reporter Hela stable cell line was transfected with GAL4DBD (GAL4) or GAL4DBD-Tau4R and then, 24 h later, treated with TSA (600 nM) for 24 h. The luciferase activity was determined as described in in section “Materials and Methods.” (F) Ectopic Tau4R expression prevents adjacent reporter gene activity induced by TSA. The GAL4UAS responsive luciferase reporter Hela stable cell line was transfected with a plasmid encoding Tau4R for 24 h. Then, cells were treated with an increasing concentration of TSA, as indicated for 24 h. The luciferase activity was determined as described in section “Materials and Methods.” Data are mean ± SD,^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. control.

FIGURE 3

Histone H3 acetylation and Tau occupancy at different promoters. (A) Chromatin immunoprecipitations-qPCR analysis of H3 acetylation and Tau occupancy on the GAPDH promoter in MCF7shctrl cells in the indicated conditions. MCF7shctrl cells were subjected to cross-linking by 1% formaldehyde. Chromatin fragments were then immunoprecipitated using antibodies (Ab) against acetylated H3 (ac-H3) or Tau and analyzed by quantitative PCR for the presence of the GAPDH promoter. Quantification of enrichment is represented as fold-enrichment relative to IgG. (B) Functional organization of the p21 promoter. (C) ChIP-qPCR analysis of H3 acetylation in MCF7shctrl or shTau cells or (D) Tau occupancy in MCF7shctrl cells on the p21 promoter. Quantification of enrichment is represented as fold-enrichment relative to IgG. Data are mean ± SD over IgG control, **P < 0.01, ***P < 0.001. (E) ChIP-qPCR analysis of H3 acetylation or Tau occupancy on the stably transfected GAL4UAS responsive luciferase reporter transfected, or not, with Tau4R, then 24 h later treated with 100 nM TSA for 24 h. Cells were then subjected to cross-linking by 1% formaldehyde. Chromatin fragments were then immunoprecipitated using antibodies (Ab) against acetylated H3 or Tau and analyzed by quantitative PCR for the presence of the GAL4 UAS promoter. Quantification of enrichment is represented as fold-enrichment relative to IgG. Data are mean ± SD,**P < 0.01, ***P < 0.001.

FIGURE 4

Tau4R is associated with condensed chromatin. (A) Schematic representation of salt fractionation of nucleosomes steps. Nuclei were isolated and digested with micrococcal nuclease and extracted successively with the indicated NaCl concentration. (B) DNA characterization from SH-SY5Y-(SBP)Tau4R cell chromatin fractions. DNA ladder obtained from micrococcal nuclease-treated nuclei purified as described in (A). Nucleosomes are indicated on the right. (C) Tau interacts with histones in the condensed chromatin and the transcriptionally active fractions. Equal aliquots of each salt fraction were resolved on 12% SDS-PAGE and visualized by immunoblot analysis with antibodies against Tau or H3 (input). The remainder was used for streptavidin pulldown of SBP (streptavidin binding peptide) tagged Tau4R. Bound fractions were resolved on 12% SDS-PAGE and visualized by immunoblot analysis with antibody against H3. (D) Analysis of the different histone H3 or (E) H4 post-translational modifications associated with Tau in the condensed chromatin fraction (600 mM). Eluted Tau4R complex obtained from the 600 mM fraction obtained as described in (A) were analyzed for H3 and H4 post-translational modifications using ELISA kits. Data are mean ± SD, **P < 0.01, ***P < 0.001. All results are representative of three independent experiments.

FIGURE 5

Tau4R binds directly to histones. (A) Tau4R interacts with core histones. GST-pulldown experiments were carried out using GST (1 μg) or GST-Tau4R (1 μg) and purified core histones (1 μg). Complexes were precipitated with Sepharose-glutathione beads, resolved by 12% SDS-PAGE and visualized by immunoblot analysis with antibodies against H3, H4, H2A, and H2B. (B) Tau4R interacts with histone H3 and H4. GST-pulldown experiments were carried out using 1 μg of GST or GST-Tau4R and 1 μg of recombinant H3, H4, H2A, or H2B. Complexes were precipitated with Sepharose-glutathione beads, resolved by 12% SDS-PAGE and visualized by immunoblot analysis with antibodies against H3, H4 H2A, or H2B. (C) Histone H4 tail peptides tested for Tau binding. Tau protein bound to biotinylated synthetic H4 peptides was detected by immunoblot. A representative western blot is shown and (D) is a quantification from three independent experiments. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 6

Frontotemporal lobar degeneration Tau mutations disrupt its interaction with histones. (A) P301L mutation abolished Tau/histone interaction. GST-pulldown experiments were carried out using GST (1 μg), GST-Tau4R (1 μg) or GST-P301L (1 μg), and purified core histones (1 μg). Complexes were precipitated with Sepharose-glutathione beads, resolved by 12% SDS-PAGE and visualized by immunoblot analysis with antibodies against H3, H4, H2A, and H2B. GST-Tau and GST-TauP301L loading was controlled by Ponceau red staining. (B) Single confocal sections of Hela cells transfected with GFP-HP1β, with or without Tau4R or TauP301L, and treated 24 h later with the TSA (300 nM) for 24 h. Tau C-terminus antibodies and GFP fluorescence were used to visualize total Tau protein and HP1β respectively. Representative images are shown. (C) Quantification of HP1β clusters per nuclei visualized as described previously and realized on three independent experiments. Data are mean ± SD, ^∗P < 0.05, ^∗∗∗P < 0.001.

FIGURE 7

Tau4R increases chromatin compaction. (A) Schematic representation of the experimental procedure. Nucleosome ladders obtained after micrococcal nuclease digestion of SH-SY5Y Tet-on Tau4R cells treated or not with tetracycline (Tet, 1 μg/ml, 24 h) then followed (B) or not (C) by TSA treatment (300 nM, 24 h). Digested chromatin was analyzed on a 1.5% agarose gel and revealed by ethidium bromide staining. Mono-, di-, tri, and tetra-nucleosomes are indicated on the right. One representative experiment out of three is shown. (D,E) Densitometric analysis of the 1N fragments obtained in control (B) and TSA-treated conditions (C) were calculated from three independent experiments. Data are mean ± SD,*P < 0.05, **P < 0.01. (F) Tau4R reduces chromatin template accessibility to micrococcal nuclease in vitro. Reconstituted nucleosomal arrays were incubated with 3 μg of GST or GST-Tau4R and subjected to micrococcal nuclease digestion for 2 or 4 min. Purified DNA fragments were then run on a 1.5% agarose gel in 0.5× TBE buffer and revealed by ethidium bromide staining. Mono-, di-, tri, and tetra-nucleosomes are indicated on the right. One representative experiment out of three is shown.

FIGURE 8

Proposed model for the role of Tau on chromatin. Tau binds to condensed chromatin regions through the interaction with unmodified histone H3, H4, or acetylated H4. The interaction between Tau and histones maintains the condensed chromatin state and prevents chromatin remodeling complexes accessing histones and gene expression. By an indirect mechanism, Tau also prevents heterochromatin decompaction and HP1s spreading induced by chromatin remodeling agents such as TSA, BIX 01294, and possibly others.