The two mechanisms each enable a transcription factor called hypoxia-inducible factor (HIF) to increase expression of genes that the cell uses to respond to the stress of hypoxia. Transcription factors bind to a site on the gene called the promoter and trigger the process that decodes the gene and makes the protein for which that gene codes. HIF binds to and activates many genes that contribute to the survival response of tumors; for example, genes that control biochemical reactions that don't require oxygen to extract energy from glucose or genes needed to build new blood vessels that bring additional oxygen to hypoxic cells.
The St. Jude finding is important because it suggests that developing new therapies that interfere with both mechanisms instead of just one might enhance the efficacy of treatments designed for solid tumors that become hypoxic as they outgrow their oxygen supply, according to Paul Brindle, Ph.D., an associate member of the Department of Biochemistry. Brindle is senior author of a report on this work that appeared in November 16 issue of The EMBO Journal.
The St. Jude researchers showed that, in addition to a mechanism controlled by two proteins called CBP and p300 (CBP/p300 collectively), a second mechanism that appears to use an enzyme called a histone deacetylase (HDAC) contributes significantly to increasing the expression of hypoxia-sensitive genes. The investigators also found evidence that suggests HIF might activate genes by a third type of biochemical pathway. If true, this would further expand the range of potential strategies for treating solid tumors.
HIF is unstable and cannot work well when the cell contains a normal amount of oxygen. But when oxygen levels are so low they stress the cell, HIF becomes stable and binds to specific genes. Once on a target gene, HIF recruits CBP and p300, each of which contains a section called the CH1 domain. The CH1 domain of each protein binds to a section of HIF called the C-TAD. This binding of the CH1 domain to the C-TAD prompts HIF to turn on the gene. Because CBP and p300 each help HIF activate genes, they are called co-activators.
CBP and p300 belong to a group of coactivators called acetylases, and have long been thought to bind to HIF during the cell's response to hypoxia, but definitive evidence for this occurring in cells was previously lacking. In contrast, HDACs were thought to be proteins that interfere with the expression of genes. Unexpectedly, the St. Jude team discovered that a drug-like inhibitor of HDACs called TSA interferes with the ability of HIF to turn on a large number of genes during times of hypoxia. The study further suggests that HDACs appear to cooperate with CBP/p300 to help HIF trigger the expression of most of the approximately 40 HIF responsive genes tested. The study also showed that different HIF-targeted genes rely to various degrees on the CH1 domain and the mechanism sensitive to TSA.
"This finding was surprising because until now it was generally accepted that acetylases are involved in activating genes, while deacetylases were mostly thought to have the opposite effect," Brindle said. "That increases our appreciation for the complexity of the control of HIF-responsive genes. That is important for future studies on how to manipulate these mechanisms to treat diseases linked to hypoxia."
Brindle's team studied HIF activation in laboratory models that had mutations that eliminated the CH1 domain in either or both of the genes for CBP and p300. The investigators found that certain genes whose activity could be induced by hypoxia were moderately to strongly dependent on the CH1 domain. One of these genes, Vegf, is important for the growth of new blood vessels, while another gene, Slc2a1, is important in bringing glucose into the cell for energy.
In addition, the St. Jude team discovered that some genes continue to be expressed fairly well even when both the CH1 and HDAC mechanisms are disrupted. This suggests that there are other coactivators, or that other domains on CBP and p300 in addition to CH1 work with HIF to activate gene expression. Alternatively, transcription factors other than HIF may mediate part of the response to hypoxia.
"Our study clearly showed that there is more to activating HIF-responsive gene expression than just the previously recognized CBP/p300 mechanism," said Lawryn H. Kasper, Ph.D., a research laboratory specialist in Brindle's laboratory. "In fact, not only does a mechanism involving HDAC appear to play a major role; but there is also evidence for a completely different pathway." Kasper is the first author of the article and together with her co-worker Fayçal Boussouar, Ph.D., did most of the work on this study.
Other authors of the study are Kelli Boyd, Wu Xu, Michelle Biesen, Jerold Rehg, Troy A. Baudino and John L. Cleveland of St. Jude.
This work was supported in part by the National Institutes of Health, a Cancer Center (CORE) grant and ALSAC.
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