Interaction between SirT1 and PRC components. (A) RNA interference (RNAi) experiments using SMARTpool of dsRNA for SirT1 (Dharmacon, Lafayette, CO) as described in ref. 16. HeLa cells were transfected with or without the SirT1 SMARTpool and analyzed by Western blot for the presence of SirT1, actin, H1, and H1-AcK26. (B) We transfected 293 cells with expression vectors as indicated. Extracts from transfected cells were immunoprecipitated with anti-FLAG antibodies. Aliquots of the immunoprecipitation input (in), flowthrough (ft), and eluate (ip) were analyzed by Western blot using the indicated antibodies. (C) Analysis of specificity of SirT1-Ezh2 interaction. Anti-FLAG immunoprecipitation was performed with extracts from 293 cells transfected with expression vectors encoding untagged Ezh2 and FLAG-tagged SirT proteins as follows: WT SirT1 (SirT1), active site mutant SirT1 (SirT1m), or WT SirT2 protein (SirT2). Inputs for immunoprecipitation (Input, Left) and anti-FLAG immunoprecipitates (α-FLAG ip, Right) were analyzed by Western blot using the indicated antibodies. (D) Recombinant Ezh2, Su(z)12, EED (30–535), or EED (95–535) were incubated separately with or without recombinant SirT1 protein and immunoprecipitated by using SirT1-specific antibody (2G10) coupled to beads. Inputs and elutions were analyzed by Western blot using the indicated antibodies. (E) Chromatin immunoprecipitation experiments by using an antibody to Gal4 were performed as described in ref. 16 in 293f cells expressing Gal4Sirt1 in the presence(+Tet) or absence (–Tet) of tetracycline. The precipitated chromatin was analyzed with PCR by using primers specific to the promoters targeted by PRC2/3 (MYT1, CNR1, KCNA1, and WNT1) and to the negative control promoters (DHFR and PLCB4) as indicated. The relative enrichment of the signal in the absence or presence of tetracycline (which causes the induction of Gal4-SirT1) is shown.

PRC complexes in 293f cells overexpressing Ezh2. Gel filtration of nuclear extracts from 293f (A) and 293-FLAG-Ezh2 (B) cell lines on Sephacryl-400 columns. Fraction numbers and molecular mass standards are indicated; “in” corresponds to column input. Western blots were probed with antibodies against proteins as indicated. The amount of protein analyzed in A was approximately twice that of B.

Characterization of histone H1 methyltransferase activity and PRC4 substrate specificity. (A) Histone H1 methylation assay of the first purification step, DE-52 column, showing separation of two histone H1-specific activities, H1KMT-1 and H1KMT-2. The assay was performed using H1-containing oligonucleosomes. Positions of H1 and core histones are indicated. Fraction numbers are indicated at the top. The fractions corresponding to H1KMT-1 activity were pooled as indicated. (B) Gel filtration analysis of the PRC4 complex. Fraction numbers and corresponding molecular mass standards are indicated at the top. The first five rows from the top are Western blots of column fractions using the indicated antibodies. The bottom three rows are HKMT assays of column fractions performed with substrates indicated on the left. Positions of core histones and histone H1 are indicated. A trace amount of nucleosomal H4 specific activity was detected in the fractions eluting at ≈1.5 MDa, likely due to a contaminating HMT activity. (C) H1 and native nucleosomes were assayed in the presence or absence of native PRC2. (D) PRC4 histone methyltransferase activity toward H1 in the presence and absence of native or recombinant oligonucleosomes. (E) Comparison of PRC2 and PRC4 methylation of H1 isoforms. Recombinant H1b, H1d, or H1^o were purified from Escherichia coli and used as substrate with native oligonucleosomes. (F) PRC4 methylated K26 residue on H1b. Recombinant H1b WT or mutant with a substitution of K26 to A were used as substrates for methylation by PRC4. On the right is a comparison of the amino acid sequences of H1b, H1d, or H1^o sequences with the K26 residue underlined.

Expression of PRC components is modulated as a function of differentiation and cellular transformation. (A) Western blot analysis of nuclear extracts from PGK12.1 ES cells and differentiated cells. Days of differentiation are indicated. The Western blot was probed with antibodies against SirT1, Ezh2, and Eed as well as control antibody directed against histone H3. (B) Western blot analysis of PRC components using HeLa cell nuclear extracts or whole-tissue extracts prepared from normal (N) and tumor (T) tissues obtained from a breast cancer patient and a colon cancer patient. Tissue samples obtained from the Cooperative Human Tissue Network were processed as described in ref. 21. The blots were probed with antibodies to Su(z)12, SirT1, Eed, and actin, which served as a loading control. A long exposure of the same blot containing the normal colon extract probed with Eed antibody is also shown.

PRC4 analyses in vivo. (a) Immunohistochemical detection of SirT1 and Ezh2 in PIN and cancer lesions. Sections from anterior prostates of WT and Nkx3.1^+/–; Pten^+/– mice were processed for hematoxylin/eosin (H&E) staining (A–C) or immunostained by using Ezh2 (D–F), SirT1 (G–I), or Ki67 (J–L) antisera. (A, D, G, and J) Sections from a WT Nkx3.1^+/+; Pten^+/+ mouse at 9 months of age. (B, E, H, and K) Sections from a Nkx3.1^+/–; Pten^+/– mouse at 12 months of age. (C, F, I, and L) Sections from a Nkx3.1^+/–; Pten^+/– mouse at 15 months of age. Note that Ezh2 immunostaining is infrequent in normal tissue but is more common in PIN and carcinoma, whereas SirT1 is broadly expressed in normal epithelium and stroma but is up-regulated in epithelial cells in PIN and carcinoma. (b and c) Expression profiling of PRC components and target genes from normal to cancer lesions of prostate from compound mutant mice performed by using RNA obtained by laser-capture microdissection. Tree (b) and quantitative (c) representations of microarray results.