Abstract
BackgroundSmall molecule inhibitors of histone deacetylases (HDACi) hold promise as anticancer agents for particular malignancies. However, clinical use is often confounded by toxicity, perhaps due to indiscriminate hyperacetylation of cellular proteins. Therefore, elucidating the mechanisms by which HDACi trigger differentiation, cell cycle arrest, or apoptosis of cancer cells could inform development of more targeted therapies. We used the myelogenous leukemia line K562 as a model of HDACi-induced differentiation to investigate chromatin accessibility (DNase-seq) and expression (RNA-seq) changes associated with this process.ResultsWe identified several thousand specific regulatory elements [~10 % of total DNase I-hypersensitive (DHS) sites] that become significantly more or less accessible with sodium butyrate or suberanilohydroxamic acid treatment. Most of the differential DHS sites display hallmarks of enhancers, including being enriched for non-promoter regions, associating with nearby gene expression changes, and increasing luciferase reporter expression in K562 cells. Differential DHS sites were enriched for key hematopoietic lineage transcription factor motifs, including SPI1 (PU.1), a known pioneer factor. We found PU.1 increases binding at opened DHS sites with HDACi treatment by ChIP-seq, but PU.1 knockdown by shRNA fails to block the chromatin accessibility and expression changes. A machine-learning approach indicates H3K27me3 initially marks PU.1-bound sites that open with HDACi treatment, suggesting these sites are epigenetically poised.ConclusionsWe find HDACi treatment of K562 cells results in site-specific chromatin remodeling at epigenetically poised regulatory elements. PU.1 shows evidence of a pioneer role in this process by marking poised enhancers but is not required for transcriptional activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0065-5) contains supplementary material, which is available to authorized users.
Highlights
Small molecule inhibitors of histone deacetylases (HDACi) hold promise as anticancer agents for particular malignancies
Histone deacetylase inhibitors (HDACi) drive site‐specific chromatin accessibility changes in K562 cells To assess the extent of chromatin accessibility changes cancer cells might undergo as a result of HDACi treatment, we performed DNase-seq on the myelogenous leukemia line K562 following 72-h incubations with the 0.5 mM sodium butyrate (NaBut) or 1 μM suberanilohydroxamic acid (SAHA)
Our analysis found roughly equal numbers of DNase I-hypersensitive (DHS) sites that significantly “open” and “close” in response to treatment: NaBut treatment resulted in 1151 opening DHS sites and 1132 closing DHS sites, while SAHA resulted in 7962 opening DHS sites and 10,349 closing (FDR < 0.05)
Summary
Small molecule inhibitors of histone deacetylases (HDACi) hold promise as anticancer agents for particular malignancies. Elucidating the mechanisms by which HDACi trigger differentiation, cell cycle arrest, or apoptosis of cancer cells could inform development of more targeted therapies. 76 % of urothelial carcinomas analyzed by TCGA were found to carry at least one inactivating mutation in a chromatin regulatory gene [1]. These types of mutations are thought to lead to aberrant epigenetic states at many loci in the genome that together. Multiple HDACi have been shown to have severe doselimiting toxicities [5], indicating that a more nuanced understanding of HDACi anticancer effects could be beneficial to the development of more specific and welltolerated therapies. A better understanding of how these drugs impact gene expression is necessary
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