Volume 18, Issue 4, Pages 483-490 (May 2005) Regulation of Tissue-Specific and Extracellular Matrix-Related Genes by a Class I Histone Deacetylase  Johnathan R. Whetstine, Julian Ceron, Brendon Ladd, Pascale Dufourcq, Valerie Reinke, Yang Shi  Molecular Cell  Volume 18, Issue 4, Pages 483-490 (May 2005) DOI: 10.1016/j.molcel.2005.04.006 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Validation of HDA-1 Target Genes by RT-PCR (A) The hda-1 (RNAi) increased gene expression. Column 1 represents ECM-related genes. (B) Upon hda-1(RNAi), collagen gene expression increased compared to control treated worms (pL4440). The fold upregulation for these targets ranged from 1.4-fold to 10.0-fold and represent different mountains. The Wormbase names are indicated for each gene tested. Molecular Cell 2005 18, 483-490DOI: (10.1016/j.molcel.2005.04.006) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 HDA-1 Directly Associates with Promoters Identified from the Microarray Analysis (A) Only HDA-1 antibody (anti-HDA-1) resulted in detectable PCR amplifications from the ChIP reactions. (B) HDA-1 interactions are specific and are associated with less histone acetylation at target promoters. RNAi depleted HDA-1 from the promoter and increased H3K9 acetylation (anti-H3K9Ac; Upstate Biotechnologies). Chromatin was prepared from control gfp- and hda-1(RNAi)-treated embryos, and ChIP reactions were performed with no or IgG antibody controls. HDA-1 did not associate with a non-HDA-1 target gene, taf-1. All of the ChIP reactions were done at least twice. The input material was derived from chromatin used in the control immunoprecipitations (“IgG” or “No,” respectively). Molecular Cell 2005 18, 483-490DOI: (10.1016/j.molcel.2005.04.006) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 HDAC-1 Homologs and an HDA-1 Target Gene Regulate Mammalian Cellular Invasion (A) HDAC-1 homologs cause dramatic increases in cellular invasion. A2058 cells overexpressing either hHDAC-1 (260%, p < 0.0007) or CeHDA-1 (233%, p < 0.0001) invaded through Matrigel compared to control-treated cells, whereas the hHDAC-1 catalytic mutant (H141A) showed a significant reduction in cellular invasion (47% decrease). (B) Endogenous hHDAC-1 siRNA depletion resulted in a 30% (p < 0.005) reduction in cellular invasion. (C) An HDA-1 target gene regulates cellular invasion. K08B4.6 (a C. elegans cystatin) was overexpressed in A2058 cells and caused a 50% (p < 0.0005) reduction in invasion. (D) Overexpression or depletion of HDAC-1 homologs does not alter cell number or apoptosis. No statistically significant changes in total cell number were observed for either the overexpression or depletion at the time of the invasion assay or 24 hr later. (E) HDAC-1 RNAi did not cause increased levels of PARP cleavage in A2058 cells (compare lanes 2 and 3). UV-treated A2058 cells served as a positive control for PARP cleavage (lane 1). Each experiment was conducted at least three times in duplicate, and the error bars represent the standard deviation. Statistical significance was determined by Student’s t test. Molecular Cell 2005 18, 483-490DOI: (10.1016/j.molcel.2005.04.006) Copyright © 2005 Elsevier Inc. Terms and Conditions