Although levels of metallothionein gene expres sion vary in different cell lines, constitutively selleck chemicals CHIR99021 high levels are often observed in cancer cell lines. Metal lothionein expression can be induced in response to metal exposure, interleukins, cytokines, and stresses including ionizing radiation. Metallothionein genes are known to be regulated by many transcription factors, such as Metal responsive Transcription Factor 1, which is essential for inducing all metallothio neins. Other studies, however, have shown that metallothionein gene expression can be modulated by histone modifiers. The position of this gene family on human chromosome 16q13, which contains the 17 out of 18 metallothionein 1 gene iso forms, in addition to MT2, MT3 and MT4 genes, further substantiates a potential epigenetic control mechanism for MT gene expression.
Our network analysis of the genes in Cluster 3 suggested that epigenetic regulation may also play a role in metallothionein gene expression, specifically through the histone modifiers KDM5B, HDAC1 and HDAC2. KDM5B, which can act as a transcriptional repressor by de methylating histone H3 lysine residues bound to promoters, has been shown to be up regulated by hypoxia stress in a HIF1a dependent man ner, although there are no previous reports of its response to ionizing radiation. Scibetta et al. carried out extensive functional analyses of KDM5B and its effects on gene expression, and found MT1E, MT1F, MT1 H and MT1L mRNAs to be highly responsive to levels of KDM5B. Overexpression of KDM5B was shown to repress gene expression and RNAi mediated knockdown of KDM5B increased expression of all the above metallothionein genes.
Histone deacetylases, have also been shown to regulate metallothionein gene expression. The HDAC proteins act as transcriptional repressors by de acetylating histones and silencing chro matin. The direct effects of ionizing radiation on HDAC levels are not clearly known, but HDAC inhibitors are widely studied as radio sensitizers of cancer cells. Also, HDAC1 has been shown to interact directly with the KDM5B protein, raising the possibility that both proteins may act in concert to modulate the early response to radiation. Using western blot analysis, we found that protein levels of KDM5B, HDAC1 and HDAC2 were all decreased an hour after exposure, preceding the 4 hour peak in metallothionein gene expression.
These findings support the possible involvement of chromatin level modifications in regulating gene expres sion in both directly irradiated and bystander cells. His tone deacetylation and histone lysine demethylation activities could also poten tially contribute to the responses observed Cilengitide for other genes in addition to the metallothioneins, suggesting coordinate epigenetic control of gene expression as an important component of the cellular radiation response.