Genome binding/occupancy profiling by high throughput sequencing Expression profiling by high throughput sequencing
Summary
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. We identified several thousand specific regulatory elements (~10% of total DHS sites) that become significantly more or less accessible with sodium butyrate or suberanilohydroxamic acid (SAHA) 72-hour treatments. Most of the differential DNase-hypersensitive (DHS) sites display hallmarks of enhancers; including being enriched for non-promoter regions, associated with nearby gene expression changes, and capable of 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 becomes up-regulated and increases binding at opened DHS sites by ChIP-seq with HDACi treatment, but show that increased PU.1 protein levels alone are sufficient for only modest increases in chromatin accessibility. PU.1 knockdown by shRNA failed to block the HDACi-induced chromatin accessibility and expression changes in K562, suggesting factors other than PU.1 are responsible for establishment of active enhancers in the HDACi induced differentiation process.
Overall design
K562 +/- NaBut for 72 hr. contains 3 replicates for Dnase-seq and RNA-seq. K562 +/- SAHA for 72 hr. contains 3 replicates for Dnase-seq and 2 replicates for RNA-seq. PU.1 overexpression in K562 cells followed by 2 replicates of Dnase-seq. PU.1 depletion in K562 cells followed by 72 hr. SAHA treatment contains 2 replicates per shRNA (2 shRNAs used). PU.1 ChIP-seq in K562 contains 3 replicates for SAHA treatment plus input controls.
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