Heterochromatin Formation in Mammalian Cells Anders Lade Nielsen, Mustapha Oulad-Abdelghani, José A Ortiz, Eumorphia Remboutsika, Pierre Chambon, Régine Losson  Molecular Cell  Volume 7, Issue 4, Pages 729-739 (April 2001) DOI: 10.1016/S1097-2765(01)00218-0

Figure 1 Nuclear Localization of Endogenous and FLAG-Tagged HP1 Porteins Interphase cells from the parental P19 EC cell line [P19 (wt)] or from the three distinct FLAG-tagged HP1-expressing P19 EC-derived cell lines [P19 (f:HP1α); P19 (f:HP1β), and P19(f:HP1γ)] were analyzed by indirect immunocytofluoresence. (A) through (H) show the Hoechst DNA staining. (I) through (P) correspond to immunodetection with specific mAbs, as indicated. A mouse IgG fraction was used as negative control in (I) Molecular Cell 2001 7, 729-739DOI: (10.1016/S1097-2765(01)00218-0)

Figure 2 Mouse HP1α, -β, and -γ Homo- and Heteromerize In Vivo as Well as In Vitro (A and B) FLAG-tagged HP1 proteins (f:HP1s) are associated with endogenous TIF1β (A) and HP1 (B) proteins. Nuclear extracts from the parental P19 EC (wt) cell line (control) or from each of the three P19 EC-derived cell lines that express the tagged proteins (f:HP1α, f:HP1β, and f:HP1γ) were analyzed by Western blotting either directly (Input) or following immunoprecipitation with the M2 anti-FLAG antibody (FLAG IP). Western blots were probed with mAbs against TIF1β (A) and HP1α, HP1β, or HP1γ (B). In all panels, the input corresponds to one twentieth of the amount of nuclear extract used for immunoprecipitation. (C–E) All three HP1s self-associate and bind directly to each other in vitro. Purified His-HP1α (C), His-HP1β (D), or His-HP1γ (E) was incubated in a batch assay with control GST (lane 2) or GST-HP1s (lanes 3 to 5). Bound His-HP1 was detected by Western blotting. Lane 1 shows one tenth of the amount of input His-HP1 Molecular Cell 2001 7, 729-739DOI: (10.1016/S1097-2765(01)00218-0)

Figure 3 HP1α, -β, and -γ Are Associated with Core Histones In Vivo and Interact with Tailed and Tailless Nucleosomes In Vitro (A) Detection of core histones in f:HP1 immunoprecipitates. Nuclear extracts prepared from either the parental P19 (wt) EC cell line (control, lane 1) or each of the three distinct tagged cell lines (f:HP1α, -β, and -γ, lanes 2–4) were immunoprecipitated with the M2 anti-FLAG antibody (FLAG IP). After washing, immunoprecipitated proteins were eluted with the FLAG peptide and analyzed by SDS–PAGE, followed by silver staining. A mixture of purified core histones H2A, H2B, H3, and H4 from calf thymus was run in parallel (lane 5). The position and migration of each of the core histones are indicated. (B) Sucrose gradient fractionation of nucleosomal core particles. Purified nuclei of P19 (wt) EC cells were partially digested with MNase, and the resulting oligonucleosomes were fractionated on a sucrose. The DNA content of the individual fractions was analyzed on a 1.5% agarose gel and visualized by ethidium bromide staining. Lane L, DNA from the sucrose gradient load; lane M, molecular weight markers (100 bp ladder). Lengths in base pairs are indicated on the right. (C and D) HP1 proteins interact with mono- and oligonucleosomes in vitro. Fractions highly enriched in mononucleosomes (F3, [C]) and oligonucleosomes (F6, [D]) were incubated in a batch assay with control GST (lane 2) or GST-HP1 (lanes 3–5). Bound nucleosomes were visualized by Western blotting with the anti-H2B mouse antibody LG11. Lane 1 shows one tenth of the amount of input nucleosomal particles. (E) HP1α can bind to mononucleosomes with trypsin-cleaved N termini. Mononucleosomes from fraction F3 prior to trypsinization (+ Tails) or after trypsinization (− Tails) were incubated in a batch assay with GST (lanes 2 and 6), GST-HP1α (lanes 3 and 7), or GST-HP1αV22M (lanes 4 and 8). Bound nucleosomes were detected by SDS–PAGE and Coomassie blue staining. Lanes 1 and 5 show one tenth of the amount of input nucleosomal particles Molecular Cell 2001 7, 729-739DOI: (10.1016/S1097-2765(01)00218-0)

Figure 4 Direct Interaction between all Three HP1s and Histone H3 and between HP1α and Histone H1 (A–F) Far Western blot analysis of the interactions between HP1s and histones. In (A), GST-HP1 proteins that had been phosphorylated with [32P]ATP in vitro were analyzed by SDS–PAGE and autoradiography. In (B) through (F), equal amounts of purified calf thymus histones (2 μg of each) and 10 μg of a chromatin fraction prepared from P19 (wt) EC nuclei as described in the Experimental Procedures were electrophoresed on SDS–PAGE gels. In (B), the resolved histones were visualized by silver staining. In (C) through (F), the gels were electroblotted onto nitrocellulose filters and probed with 32P-labeled GST (C) or with 32P-labeled GST-HP1 fusion proteins (D–F) as indicated. 32P probe localization was performed by autoradiography. The positions of the histones are indicated. (G and H) GST pulldown analysis. Purified calf thymus histones H3 (G) and H1 (H) were incubated in a batch assay with control GST (lane 2), GST-HP1α (lane 3), GST-HP1β (lane 4), GST-HP1γ (lane 5), or GST-dHP1 containing Drosophila HP1 (lane 6). Bound histones were detected by Western blotting using the anti-histone mAb MAB052. Lane 1 shows one tenth of the amount of input histones Molecular Cell 2001 7, 729-739DOI: (10.1016/S1097-2765(01)00218-0)

Figure 5 Direct Binding of the HP1 Proteins to the Histone-Fold Domain of H3 (A) Schematic representation of histone H3. The acetylable lysines in the tail domain of H3 are indicated. Numbers refer to amino acid positions in the protein. (B–D) Residues 48–135 of H3 mediate interaction with HP1s. Full-length recombinant mouse His epitope B10–tagged H3 (r-H3, [B]) and two deletion mutants [r-H3(1–66), (C), and r-H3(48–136), (D)], all three purified from E. coli, were incubated in a batch assay with control GST (lanes 2) or GST-HP1 (lanes 3–5). Bound H3 was detected by Western blotting using the B10 antibody. Input lanes contain the equivalent of one tenth of the input protein used in each pulldown assay Molecular Cell 2001 7, 729-739DOI: (10.1016/S1097-2765(01)00218-0)

Figure 6 Separate Domains of HP1α Mediate Self-Association and Histone Binding (A) Schematic representation of the various GST-HP1α deletion mutants used in the Far Western blot experiment, with amino acid numbers below both ends; mutants that scored positive or negative for binding to HP1α, H1, and H3 are indicated on the right by plus or minus signs, respectively. (B–G) Mapping of the domains in HP1α required for interaction with HP1α, H1, and H3 by Far Western blot analysis. Increasing amounts (1 and 5 μg) of purified His-tagged HP1α (lanes 1and 2), calf thymus H1 lanes 3 and 4), and H3 (lanes 5 and 6) were electrophoresed on SDS–PAGE gels and electroblotted onto nitrocellulose filters. The replica filters were probed with 32P-labeled GST (B) or with different 32P-labeled GST-HP1α deletion derivatives (C–G) as indicated. 32P probe localization was performed by autoradiography. The positions of HP1α, H1, and H3 are indicated Molecular Cell 2001 7, 729-739DOI: (10.1016/S1097-2765(01)00218-0)

Figure 7 HP1α and Pc1/M33 CDs Are Necessary and Sufficient for H3 Binding (A) Amino acid alignment of the CDs. The sequences were aligned using the Clustal W program. Secondary structure elements shown were derived from mouse HP1β CD (Ball et al., 1997). Numbers refer to amino acid positions in the corresponding proteins. Invariant residues are highlighted in black. Conserved residues are shaded. The box encloses the structured part of the CDs. Mutations described in this paper are indicated below the alignment. (B and C) Both HP1α and Pc1/M33 CDs bind to histone H3. Purified calf thymus H3 was incubated in a batch assay with control GST (lane 2) or with different GST fusion proteins immobilized on glutathione S-sepharose beads. Bound H3 was detected by Western blotting using the mAb MAB052. Input lane 1 shows the equivalent of one tenth of the input protein used in each pulldown assay. (D) HP1α and Pc1/M33 bind to H3 in a CD integrity-dependent manner. Binding assays were done as in (B) with wild type and CD mutants for HP1α and Pc1/M33. (E) The CD of HP1α is required for interaction with the histones in mammalian cells. Nuclear extracts from P19 EC cells transfected with 10 μg of expression vector for unfused FLAG (control) or FLAG-HP1α mutant proteins were used for immunoprecipitation with the M2 anti-FLAG antibody. Nuclear extracts (Input) and immunocomplexes (IP) were analyzed by Western blotting using the anti-H2B antibody LG11. Inputs correspond to one two hundredth of the amount of nuclear extracts used for immunoprecipitation Molecular Cell 2001 7, 729-739DOI: (10.1016/S1097-2765(01)00218-0)