Ph.D. student Keith Earley in the group characterized HDA6 biochemically and demonstrated that it was, in fact, a histone deacetylase, as predicted, and that the protein would remove acetyl groups from several different histones. A collaboration with mass spectrometry expert Michael Gross, Ph.D., Washington University professor of chemistry, helped define the precise locations of the acetyl groups that HDA6 can remove, down to which acetylated amino acids are involved.
"The bottom line is that HDA6 has very broad specificity. It can remove the acetyl groups from multiple histones and from multiple lysines of those histones" said Pikaard.
When multiple acetyl groups are on the histones, the genes are turned on, Pikaard explained. When they are removed by HDA6, it contributes to gene silencing. Using antibodies that recognize specific histone modifications that occur on the genes when they switch from off to on, the group was able to confirm that the deacetylation specificities they observed for HDA6 in the test tube fit with the changes in acetylation that occur on ribosomal RNA genes in living cells.
They also found that the mechanism behind the silencing involves both modifications of histones and changes in DNA methylation, and that HDA6 affects both.
Circular pathway to silence
"Somehow these modifications are linked together," Pikaard said. "We know that they work together and that HDA6 is a key player. They are intimately linked in a circular, self-reinforcing pathway. Each specifies the other. For instance, in modifying the histones a pathway is set in motion to recruit enzymes to perform DNA methylation. Likewise, changing DNA methylation leads to cha
Source:Washington University in St. Louis