The NuRD complex, which contains both the CHD4 ATPase and the HDAC2 deacetylase, is required for cells to repair and survive IR-induced DNA damage. Importantly, expression of the MTA1 sub-unit of NuRD is induced under hypoxic conditions, 
and both MTA1 and histone deacetylases contribute to Hif1a SCH727965 stability. This indicates that components of the NuRD complex may be upregulated by Hif1a. Therefore, we examined if components of the NuRD complex were increased by hypoxia mimics and if this induction contributed to the observed radioprotection by DMOG. To examine the transcriptional activity of Hif1a, we first reduced expression of Hif1a using shRNA. shRNA targeting Hif1a blocked the accumulation of Hif1a after treatment with either DMOG or CoCl2and reduced the levels of Hif1a mRNA. Silencing of Hif1a also inhibited the accumulation of VEGF mRNA, a key target of Hif1a, demonstrating that Hif1a function is abolished in the shRNAHif1a cells. Next, we determined if Hif1a regulates expression of the CHD4 and MTA3 genes. Figure 2D and 2E demonstrate that activation of Hif1a by CoCl2 increased the levels of CHD4 and MTA3 mRNA, and this increase was abolished when Hif1a was silenced with shRNA. This demonstrates that Hif1a is required for the accumulation of CHD4 and MTA3 mRNA after exposure to CoCl2. Further, CoCl2 increased the levels of Hif1a, CHD4 and MTA3 protein with similar kinetics, consistent with the Hif1adependent increase in their mRNA levels. Significantly, suppression of Hif1a with shRNA also reduced the basal levels of CHD4 protein, whereas basal levels of MTA3 were unaffected by loss of Hif1a. Further, loss of Hif1a greatly attenuated the accumulation of CHD4 after exposure to CoCl2, but had only a small impact on the accumulation of MTA3 protein. Figure 2 therefore demonstrates that increased levels of Hif1a lead to increased levels of MTA3 and CHD4 mRNA, potentially identifying CHD4 and MTA3 as transcriptional targets for Hif1a. Significantly, loss of Hif1a decreased both the basal and stimulated levels of CHD4 protein, indicating that Hif1a plays a critical role in maintaining basal and stimulated levels of CHD4. Previous work indicates that CHD4 can participate in the cells response to IR-induced DNA damage. This suggests that, because cells expressing shRNA to Hif1a have decreased levels of CHD4, they should be more sensitive to IR. Figure 3A demonstrates that cells lacking Hif1a exhibit a small but significant increase in radiosensitivity, consistent with a key role for Hif1a in regulating radiosensitivity. However, when shRNA was used to deplete CHD4 protein levels to a level similar to those detected in Hif1a depleted cellsno significant impact on radiosensitivity was seen. Similarly, silencing MTA3 expression with shRNA did not alter cellular radiosensitivity. We interpret this to mean that, while Hif1a contributes to cell survival after exposure to IR, this regulation of radiosensitivity is not mediated Adriamycin through the ability of Hif1a to regulate the expression of either CHD4 or MTA3. Therefore, although we have identified CHD4 and MTA3 as potential transcriptional targets for Hif1a, the ability of Hif1a to protect cells from radiation damage does not require either CHD4 or MTA3. It is more likely that upregulating CHD4 and MTA3, which are components of NuRD deacetylase complex, plays a key role in other processes, such as transcriptional repression, which are a feature of the hypoxia response. To further explore how stabilization of Hif1a regulates radiosensitivity, we examined a second group of proteins, the histone demethylases, which are transcriptionally activated by Hif1a.