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1.
Figure 6

Figure 6. From: Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Transcriptional repression by ySir2 in vitro reflects silencing activity in vivo. Reactions were carried out as in Fig. 2D (“Sir2 before assembly”) with dSir2 (100 nM) or the indicated ySir2 proteins (150 nM).

Xuejun Huang Parsons, et al. Proc Natl Acad Sci U S A. 2003 February 18;100(4):1609-1614.
2.
Figure 3

Figure 3. From: Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Characterization of transcriptional repression by dSir2 in vitro. (A) Effect of dSir2 concentration on transcriptional repression. Reactions were performed as in Fig. 2D with the indicated final concentrations of purified dSir2. (B) The extent of histone deacetylation by dSir2 roughly correlates with the amount of transcriptional repression. HDAC assays were performed, as in Fig. 1, with the indicated final concentrations of dSir2 or dHDAC1. (C) dSir2 does not repress transcription with hyperpropionylated core histones. Transcription reactions were carried out as in Fig. 2D. The properties of hyperpropionylated core histones (prepared by incubation of core histones with propionic anhydride) were compared with those of hyperacetylated core histones (prepared with acetic anhydride).

Xuejun Huang Parsons, et al. Proc Natl Acad Sci U S A. 2003 February 18;100(4):1609-1614.
3.
Figure 5

Figure 5. From: Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Histone deacetylation by Drosophila HDAC1 does not repress transcription or generate a nuclease-resistant histone-DNA complex. In these experiments, Drosophila HDAC1 (dHDAC1) and dSir2 were each used at a final concentration of 100 nM. (A) Deacetylation of core histones by dHDAC1. Histone deacetylation reactions were carried out as in Fig. 1B. (B) dHDAC1 does not generate a nuclease-resistant structure. Reactions were carried out as in Fig. 2C. The histones that were treated with both dHDAC1 and dSir2 (middle lanes) were first incubated with dHDAC1 for 30 min before the addition of dSir2 and NAD at the onset of the chromatin assembly reaction. (C) dHDAC1 does not repress transcription with hyperacetylated histones. Reactions were performed as in Fig. 2D.

Xuejun Huang Parsons, et al. Proc Natl Acad Sci U S A. 2003 February 18;100(4):1609-1614.
4.
Figure 4

Figure 4. From: Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Deacetylation of histones by dSir2 and NAD yields a fast-sedimenting histone–DNA species. (A) Sucrose gradient sedimentation analysis. The indicated components were used in the same relative proportions as in the transcription reactions (Fig. 2). The migration of DNA was monitored by agarose gel electrophoresis and staining with ethidium bromide. (B) Sucrose gradient sedimentation analysis was carried out with template DNA assembled with hyperacetylated histones and dSir2, as in Fig. 2D. DNA was visualized by ethidium fluorescence, core histones were monitored by staining with Coomassie blue, and dSir2 was detected by Western blot analysis. (C) Sucrose gradient sedimentation analysis was performed as in B, except that NAD was also included in the assembly reaction.

Xuejun Huang Parsons, et al. Proc Natl Acad Sci U S A. 2003 February 18;100(4):1609-1614.
5.
Figure 1

Figure 1. From: Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

dSir2 catalyzes the deacetylation of a broad range of lysine residues in core histone tails. (A) Synthesis and purification of dSir2. His-6-tagged, full-length dSir2 was purified and then subjected to 8% polyacrylamide-SDS gel electrophoresis and staining with Coomassie brilliant blue R-250. (B) Deacetylation by dSir2 requires NAD and is inhibited by coumermycin A1. Purified Drosophila core histones were hyperacetylated with acetic anhydride and then used as substrates for deacetylation by dSir2. Where indicated, dSir2 (100 nM), NAD (100 μM), and coumermycin A1 (100 μM) were included in the reactions. The extent of deacetylation at the indicated lysine residues was monitored by Western blot analysis with antibodies that specifically recognize the acetylated form of the residues. The amount of total histone polypeptides in the reaction mixtures was monitored by 15% polyacrylamide-SDS gel electrophoresis and staining with Coomassie brilliant blue R-250. The asterisk denotes a contaminant in the preparation of coumermycin A1. (C) dSir2 deacetylates histones when added prior to chromatin assembly, but not subsequent to chromatin assembly. Chromatin assembly reactions were performed with hyperacetylated histones, ACF, NAP-1, plasmid DNA, and ATP. dSir2 (100 nM) and NAD (100 μM) were added, as indicated, either before or after the chromatin assembly reactions. The deacetylation of specific lysine residues was detected by Western blot analysis as in B.

Xuejun Huang Parsons, et al. Proc Natl Acad Sci U S A. 2003 February 18;100(4):1609-1614.
6.
Figure 2

Figure 2. From: Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex.

Transcriptional repression by dSir2 requires NAD and hyperacetylated histones. (A) dSir2 and NAD do not disrupt the periodicity of nucleosomes assembled with ACF and NAP-1. Chromatin was assembled with ACF, NAP-1, purified native Drosophila core histones, ATP, and pHIV template DNA. Where indicated, purified dSir2 (100 nM) and NAD (100 μM) were added before chromatin assembly. The resulting samples were subjected to micrococcal nuclease (MNase) digestion analysis. (B) dSir2 does not inhibit transcription of chromatin assembled with native (and mostly unacetylated) core histones. Aliquots of the same chromatin samples used in A were subjected to in vitro transcription analysis. Sp1 (10 nM) and NF-κB p65 (100 nM) were added to the chromatin templates, and transcription was carried out with a HeLa nuclear extract. Where indicated, purified dSir2 (100 nM) and NAD (100 μM) were added either before or after chromatin assembly. The resulting transcripts were detected by primer extension analysis. Transcriptional activity is reported as relative to that observed with in the absence of dSir2 and NAD, which is designated as “(100).” (C) dSir2 and NAD mediate the formation of a nuclease-resistant structure with hyperacetylated histones. Chromatin assembly and micrococcal nuclease digestion analysis was performed as in A, except that hyperacetylated core histones were used instead of native core histones. (D) dSir2 is a potent NAD-dependent repressor of transcription with hyperacetylated core histones. Aliquots of the same chromatin samples used in C were subjected to in vitro transcription analysis as in B.

Xuejun Huang Parsons, et al. Proc Natl Acad Sci U S A. 2003 February 18;100(4):1609-1614.

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