1. Volume 16, Issue 1, Pages 93-105 (October 2004)
Human SirT1 Interacts with Histone H1 and Promotes Formation of Facultative Heterochromatin 
Alejandro Vaquero, Michael Scher, Donghoon Lee, Hediye Erdjument-Bromage, Paul Tempst, Danny Reinberg 
Molecular Cell 
Volume 16, Issue 1, Pages (October 2004)
DOI: /j.molcel

2. Figure 1 Characterization of SirT1 Antibodies
(A) Localization of epitope for SirT1 antibody in the C-terminal region of SirT1.
(B) 50 ng of purifed baculovirus-expressed SirT1 (bSirT1), 5 μg of HeLaS3 nuclear extract (NE), and cytoplasmic (Cyt) fractions resolved by SDS-PAGE and monitored by Western blot using SirT1 antibody. A Coomassie blue stained gel containing 500 ng of bSirT1 is shown as control (left).
(C) 50 μg of E. coli extracts each expressing a different human SirT (SirT1–SirT5) protein resolved by SDS-PAGE and monitored by Coomassie blue staining (right) and Western blot using SirT1 antibodies (left).
(D) Immunostaining of HeLa, 293T, and HepG2 cells using SirT1 antibodies. Cells were stained with DAPI to visualize DNA and SirT1 antibody (Rhodamine), shown separately or together (merge).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
NAD+-Dependent Core Histone Deacetylation by SirT1 In Vitro and In Vivo
(A) Upper panel, scheme for the nicotinamide exchange reaction (NER). Lower panel, 0.5 nmol of SirT1 assayed in NER using no substrate (−), 4 μg of acetylated and nonacetylated BSA, and 4 μg of hypo- and hyperacetylated native core histones purified from HeLa cells (see Experimental Procedures).
(B) TAU (triton, acetic acid, urea) gel analysis of hyperacetylated core histones treated with the indicated amounts of SirT1 in the presence and absence of NAD+. Levels of acetylation of the corresponding histones are indicated on the right side of the panel.
(C) Hyperacetylated core histones were used as substrate in the SirT1-mediated deacetylation reaction in the presence and absence of NAD+. Products of the SirT1 reaction were analyzed by Western blot using antibodies against specific acetylated residues in H3 and H4 (see Experimental Procedures).
(D) The graph shows the extent of deacetylation of 8 μg of core histones with 0.7 nmol SirT1 in the presence of NAD+ as a function of time. Time points were taken every 10 min during 60 min and analyzed as in the right panel experiment. Quantitations were performed as indicated (Experimental Procedures).
(E) RNAi experiments using Smartpool of dsRNA for SirT1 (Dharmacon). U2OS cells were transfected with (SirT1) or without (C) the SirT1 Smartpool and analyzed by Western blot for the presence of SirT1, actin, H4-AcK16, H4-AcK8, H4-mono-MeK20, H3-AcK9, H3-Tri-MeK9, H3-Di-MeK79, and H3. H4 was analyzed by Coomassie blue staining.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Purification of SirT1 and SirT1-Interacting Proteins
(A) Nuclear extracts from HeLaS3 cells fractionated on a gel filtration column (Superose 6) and analyzed by NER and Western blot using SirT1 antibody.
(B) Endogenous SirT1 from HeLa nuclear fractions was purified by several chromatographic steps indicated in the purification scheme (left). Right panel, last step of the purification (MonoQ) resolved on a silver-stained gel and Western (below). The bands present in fractions 24–25 were analyzed by mass spectrometry techniques (see Experimental Procedures and text). In addition to SirT1, the presence of glucosidase IIα, HSPgp96, ef-2kinase, Tat-SF1, and BiP were detected, although none of these proteins cofractionated with SirT1.
(C) Left panel, purification scheme of SirT1-interacting proteins from 293 FLAG-tagged SirT1 stable cell line. Right panel, silver-stained gel of the last step of the purification (DEAE cellulose). The activity of the fractions was monitored by NER and the bands indicated in the gel were also identified by mass spectrometry.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 SirT1 Interacts with H1b through Its N-Terminal Region
(A) 293F cells were transfected with HA-tagged H1b, and extracts were immunoprecipitated with anti-HA resin. Input (I), flowthrough (FT), and elution (E) were monitored by Western blot for the presence of H1 and SirT1.
(B) Recombinant HA-H1b and baculovirus-purified FLAG-SirT1 were incubated and immunoprecipitated as indicated using anti-FLAG or anti-HA resins and monitored by Western blot for the presence of SirT1 and H1.
(C) Scheme of the constructs used in (D) and (E). The isoelectric point of the N- and C-terminal regions is indicated.
(D) Immunoprecipitation of recombinant HA-H1b incubated with either SirT1 or SirT1Δ(1-268) using anti-SirT1 antibody crosslinked to protein G. Interaction experiments were performed and monitored as in (D).
(E) Interaction between HA-H1b and SirT1(1-268) monitored by immunoprecipitation using FLAG or HA resin as indicated.
(F) Comparative binding of SirT1 to other H1 subtypes. Baculovirus-purified FLAG-SirT1 was incubated with either recombinant H1b-HA or H1o-HA, and interactions were monitored by immunoprecipitation using HA resin and resolved by Western blot as in (B)–(E).
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 H1 Is Acetylated In Vivo and Can Be Deacetylated by SirT1
(A) Upper panel, scheme of H1 deacetylation experiment. Lower-left panel, NER assay in the presence or absence of SirT1, as indicated using 20 and 40 μg of calf thymus H1 previously treated (H1-T1) or untreated (H1) with SirT1. Right panel, 5 μg of purified core histones (Core Hist.) and 80 μg of calf thymus H1 were analyzed by Western blot using a combination of H3 and H4 antibodies.
(B) H1 from 293 cells treated or untreated with nicotinamide was purified using perchloric acid extraction. The amount of H1 added to reactions was normalized by Western blot using anti-H1 antibodies (lower panel), and equal amounts were used in the NER assay. Quantitations were performed as indicated (Experimental Procedures). This assay was performed twice with almost identical results.
(C) H1 previously acetylated in the presence of [3H]-AcCoA and p300 was incubated with different amounts of SirT1 and treated as indicated in the Experimental Procedures. Analysis of the 3H-labeled H1 remaining after treatment was quantified and represented in the graph as the percentage compared to no enzyme counts. The bars on the assay were derived from three independent experiments.
(D) Similar experiment as in (C) with three different amounts of recombinant SirT1, SirT2, and SirT3 with equal exchange activity (NER) incubated and analyzed as in (C).
(E) Dot-blot assay of H1 α-AcK26 antibodies using 0.5 and 1 μg of peptides containing the N-terminal region of histone H4 (1–20) acetylated either at K12 or K16, histone H3 (20–31) acetylated at lysine K27, or the histone H1b N-terminal sequence 21–31 either unmodified, trimethylated at K26, or acetylated at K26.
(F) Right panel, deacetylation of H1 by SirT1. HA-H1b (150 ng) purified from nicotinamide+TSA-treated 293 cells (lanes 1–6) was analyzed as a substrate using different amounts of SirT1 (0, 5.3, 29, and 290 pmol; lanes 1–4, respectively) in the presence of NAD+ and monitored by Western blot using H1 α-AcK26 antibodies. Left panel, equal amounts (150 ng) of either H1b purified from TSA+nicotinamide-treated 293 cells (lane 5) or recombinant H1b (lane 6) were probed in Western blot using either H1 α-AcK26 antibodies or α-H1 antibodies.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 SirT1 Activity In Vivo
(A) Transient cotransfections of the different Gal4-fusion proteins, as indicated, together with a luciferase reporter containing Gal4 DNA binding sites. A β-gal plasmid was cotransfected as control for transfection efficiency. Luciferase levels were normalized to the corresponding β-gal levels and represented as the percentage relative to the empty vector control.
(B) Scheme of the SirT1 inducible system in 293-TREX cells. In normal conditions, Tet repressors bound to TetO2 elements keep Gal4-SirT1 expression repressed. Upon induction by tetracycline, the repressors leave the DNA and Gal4-SirT1 is expressed, resulting in binding to the Gal4 binding sites to the luciferase reporter that is stably integrated in euchromatic DNA. Location of the regions amplified in the ChIP experiments are indicated.
(C) Western blot of cell extracts prepared before and after induction for the presence of Gal4-SirT1 using anti-Gal4 and anti-SirT1 antibodies.
(D) Luciferase activity measured under the same conditions as in (C). Luciferase levels were normalized by protein levels and represented as percentage, with uninduced levels set at 100%.
(E) Reverse-transcription PCR (RT-PCR) from the cells used in (B)–(D) monitoring levels of luciferase, Neo, and GAPDH expression.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
SirT1 Recruitment Results in Deacetylation of H4-K16, Recruitment of H1, Hypomethylation of H3-K79, and Enrichment of Methylated H3-K9 and H4-K20
(A) ChIP assays were performed with the Gal4-SirT1 inducible cells as indicated in the Experimental Procedures, analyzing the region between the TK promoter and the beginning of the luciferase-coding region using no antibody (C), and antibodies against Gal4BD (G4), acetylated histone H4 (acH4), and acetylated histone H3 (acH3).
(B) ChIP studies as in (A) but analyzing simultaneously three different regions of the TK-luc reporter: promoter region as in (A) (1), middle of coding region (2), and end of the coding region (3). The presence of H4 acetyl-K16 (acK16), histone H1 (H1), H3 dimethyl K79 (mK79), H3 trimethyl K4 (mK4), H3 trimethyl K9 (mK9), and H4 monomethyl K20 (mK20) was analyzed by PCR using 1× and 3× amount of eluted DNA for every sample and set of primers.
(C) A summary of the results is shown.
Molecular Cell  , DOI: ( /j.molcel )