Cooperation between phosphorylation and acetylation processes in transcriptional control

Area of Science:

• Molecular Biology
• Gene Regulation
• Epigenetics

Background:

• Histone deacetylase (HDAC) inhibitors, such as butyrate, are known to modulate gene expression.
• Previous studies indicated that mitogen-activated protein kinase kinase (MEK) inhibitors block HDAC inhibitor-induced activation of the choline acetyltransferase (ChAT) promoter.

Purpose of the Study:

• To elucidate the signaling pathways mediating the transcriptional effects of HDAC inhibitors.
• To identify the specific kinases and phosphatases involved in the regulation of ChAT and other gene promoters by HDAC inhibitors.

Main Methods:

• Transient and stable transfection assays using reporter constructs for ChAT, Rous sarcoma virus (RSV), and simian virus 40 (SV40) promoters.
• Inhibition studies using specific kinase (H7, PD98059) and phosphatase (okadaic acid) inhibitors.
• Overexpression of coactivator protein p300 and adenovirus E1A 12S protein.
• Western blotting and in vivo metabolic labeling to assess histone H3 phosphorylation.

Main Results:

• Activation of the ChAT promoter by butyrate and trapoxin was inhibited by H7, a serine/threonine protein kinase inhibitor.
• Overexpression of p300 enhanced ChAT promoter activity, an effect blocked by H7.
• Both H7 and PD98059 (MEK inhibitor) suppressed butyrate-induced activation of ChAT, RSV, and SV40 promoters.
• Butyrate's induction of the histone H1(0) gene was blocked by H7 and PD98059, and also by okadaic acid (phosphatase inhibitor).
• Butyrate exhibited a biphasic effect on histone H3 phosphorylation: initial depression followed by stimulation.

Conclusions:

• The transcriptional effects of HDAC inhibitors are mediated by the activation of MEK1 and an H7-sensitive protein kinase.
• Protein phosphatases also play a role in this signaling pathway.
• These findings reveal a complex signaling network involving kinases and phosphatases in HDAC inhibitor-mediated gene regulation.