Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin.
Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin.
PubMed ID: 24100296
PMC ID: PMC3798258
Article link: J Cell Biol.
Grant support: R01 GM052426 NIGMS NIH HHS
Genes referenced: cdk2 cfp mtor nucb1 pin1 ppp2ca
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|Figure 1. Pin1 interacts with histone H1. Coimmunoprecipitation experiments were performed to test whether Pin1 and H1 interacted with each other in vivo. “Total” refers to the total nuclear extract before the addition of the antibody, and “FT” refers to flow-through (∼3–6% of the total volume). The entire contents of the eluate were run on the gel. Black lines indicate that intervening lanes were spliced out, and arrows indicate bands that correspond to the protein being IB. Asterisks indicate heavy/light chain IgG antibodies that form part of the eluate. (A) Under the conditions used, both histone H1 and Pin1 did not bind beads nonspecifically. Histone H1 antibodies were used to immunoprecipitate (IP) H1 from mouse embryonic cells (B) and from Ciras-3 cells (C). Immunoblots (IB) reveal pull-down of Pin1 along with histone H1 demonstrating their association in vitro. Reciprocal experiments were performed using Pin1 antibody to pull down Pin1 from extracts prepared from 10T1/2 mouse embryonic cells (D), Ciras-3 cells (E), and Pin1wt cells (F) and Pin1−/− cells (G). RNA polymerase II, which is an established substrate for Pin1, was used as a positive control. Both H1 and RNA polymerase II form a part of the eluate in 10T1/2 and Ciras-3 cells, but not in Pin1−/− cells, demonstrating specific interactions mediated by Pin1. (H, top) Interaction between Pin1 and GFP-H1.1 in extracts prepared from T98G cells stably expressing GFP-H1.1. GFP-H1.1 was immunoprecipitated using GFP antibody coupled to magnetic particles (GFP-Trap). This interaction is dependent on the phosphorylation status of proteins (H, bottom) as treatment of the extracts with calf intestinal phosphatase (CIP), a general nonspecific protein phosphatase, abrogated the interaction between H1 and Pin1.|
|Figure 2. Pin1 promotes H1 dephosphorylation. (A) Histones were extracted from Pin1−/−, Pin1wt, and Ciras-3 cells, and were then probed with either pS173(H1.2/H1.5), pS187(H1.4), a phospho-specific stain that labels all phosphorylated proteins, or with a stain that labels total protein. Levels of pS173, pS187, and net H1 phosphorylation levels were found to be higher in Pin1−/− cells as compared with Pin1wt cells, similar to those observed in Ciras-3 cells (positive control). (B) Nuclear extracts from Pin1−/− cells and Pin1wt cells revealed that the levels of Cdk2 and PP2Ac were similar in both cells. (C) The dephosphorylation activity of PP2Ac activity was analyzed using purified H1 as a substrate. PP2Ac was immunoprecipitated from either Pin1wt cells or Pin1−/− cells and assessed for its ability to dephosphorylate pS187. The kinetics of this dephosphorylation reaction are plotted in E with each dot/square representing the average H1 phosphorylation level obtained from at least three independent experiments. The average intensity from the zero-minute time point is set as the maximum, against which all other time points are compared. (D) PP2Ac was immunoprecipitated from Pin1−/− cells and was mixed with a constant amount of H1, while levels of purified Pin1 were varied from 0.004 to 8 µg. The former corresponds to a molar stoichiometry of H1/Pin1 = 1:0.0005, whereas the latter corresponds to H1/Pin1 = 1:0.9. The kinetics of dephosphorylation is plotted in F and G, with the average intensity at the zero-minute time point set to 1. These curves were then submitted to a one-phase decay curve analysis and the rate obtained was plotted as a function of the amount of Pin1 added to the reaction (H).|
|Figure 3. Pin1 and H1 phosphorylation change the structure of the CTD. (A) The position of the Cy3 and Cy5 label are indicated in relation to the whole H1 molecule, not to scale (N, N-terminal; C, C-terminal; SP, Ser-Pro). (B) H1 labeled with Cy3 and Cy5 were treated with Cdk2 immunoprecipitated from Pin1−/− cells in the presence or absence of ATP and probed with a phospho-specific stain. These blots reveal successful phosphorylation of H1 in the presence of ATP (now referred to as phosphoH1), whereas Cdk2 was unable to phosphorylate labeled H1 molecules in the absence of ATP (now referred to as nonphospho H1). (C) Labeled phospho H1 molecules were then diluted either in solution (sol) or with reconstituted nucleosomes (nuc). A 514-nm laser was then used to excite the molecules and fluorescence emission spectra was obtained from 525–724nm (5-nm slit width). Fluorescence intensity was normalized to the total fluorescence intensity obtained from each spectrum. The spectra show a slight increase in FRET signal (peak at 671 nm) in the mono-labeled H1s (either Cy3 or Cy5) mixed with each other in 1:1 stoichiometry together with nucleosomes, indicating inter-molecular FRET, whereas this signal increases dramatically when both Cy3 and Cy5 are on the same H1 molecule. (D) FRET signal was compared between phosphorylated H1 and nonphosphorylated H1 in solution versus these molecules added to reconstituted nucleosomes. Although FRET signal remains the same when H1 is in solution, FRET signal is dependent on the phosphorylation status of H1 in the presence of reconstituted nucleosomes. (E) FRET signal was compared between phosphorylated H1 and nonphosphorylated H1 with reconstituted nucleosomes in the presence or absence of Pin1. Although phosphorylation alone increases the FRET signal, addition of Pin1 reduces this signal toward that of the nonphosphorylated H1 molecules.|
|Figure 5. Mobility shift assay for detecting phosphorylated H1 and FRAP analysis of H1.1 mutants. (A) FLAG-tagged H1.1 wt and H1.1 mutants were transfected in Pin1−/− and Pin1wt cells. Histones were then extracted using 0.4 N H2SO4 and the extracts were then run in a 10% acrylamide gel ± Phos-tag. Phos-tag is a ligand that interacts with phosphate molecules imparting shifts in mobility. H1.1wt migrates as two distinct species in the presence of Phos-tag, whereas H1.1T152S migrates as three distinct species (shown by arrows). In the absence of Phos-tag, all mutants migrate as a single band. (B) GFP H1.1 (i) or GFP H1.1 mutants (ii–ix) were expressed either in Pin1wt (black filled circles) or Pin1−/− (open circles) cells. FRAP experiments were performed to measure the dynamics of the H1 molecules. Each curve represents an average of ∼20 cells (total), three independent experiments. The inset is a diagrammatic representation of the genetic alteration and relative position of serines (S), theonines (T), prolines (P), and alanines (A). (ii–iv) Role played by serine at either position 183 or 152 in contributing toward Pin1 mediated changes in H1 dynamics. (v–vii) Role played by altering the Thr residue on H1.1 in Pin1-mediated changes in H1 dynamics. (viii) Recovery of H1.1 when the ser and thr positions are switched. (ix) Lack of any change in H1 dynamics when both the ser and thr residues are changed to ala.|