Abstract 3013: Identification of Novel Genes Directly Upregulated by Class II Histone Deacetylases
Introduction: Class II histone deacetylases (HDACs) are considered transcriptional inhibitors that repress cardiac hypertrophy, yet their target genes are largely unknown. Transcriptional analysis of targets is complicated because HDACs can inhibit the expression of both activators and repressors. Thus, distinguishing between primary and secondary targets remain difficult. The aim of this study was to identify direct target genes for HDACs in cardiomyocytes.
Methods: A novel surrogate system for the chromatin immunoprecipitation (ChiP) technique, termed DamID, was used to map genome-wide occupancy of HDAC4 binding sites in rat cardiomyocytes using promoter microarrays. Expression analysis on microarrays was performed following adenoviral expression of HDAC4. This method generated a list of direct target genes whose promoter was occupied by HDAC4, and whose expression was significantly changed by HDAC4.
Results: Cluster analysis revealed 85 genes which were directly down-regulated by HDAC4. This cluster was highly enriched for sarcomeric genes (11.8% of genes, p<0.001), such as troponin and myosin heavy-chain. Similarly, calcium regulatory proteins (9.6%, p=0.01) such as the L-type calcium channel and phospolamban, were also reduced. HDAC4 is classically known to act as a repressor. However, this method revealed 40 direct target genes which were up-regulated by HDAC4. This cluster was highly enriched for cell cycle genes (29.4%, p=0.01) and angiogenesis genes, such as angiopoietin-like (11.7%, p=0.004). We then validated selected genes from the up- and down-regulated clusters using conventional ChiP and quantitative RT-PCR assays. Results from these analyses confirmed the data obtained from the microarray analysis.
Conclusions: Using a genome wide unbiased approach we have identified, for the first time, direct targets of HDAC4. Remarkably, this analysis identified a large number of genes which are upregulated by HDAC4. This result suggests HDAC4 may also function to activate genes, along with its known ability as a repressor. Thus, HDAC4 as an activator may represent a paradigm shift in the understanding of the function of these deacetylases.