Abstract
Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.
Highlights
In the nucleus of eukaryotes, the genomic DNA is packaged into a complex nucleoprotein structure, known as chromatin
We examined the roles of human Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex using respective KO cells
While analyzing the results obtained from the cells without oxidative stress (Figure 2A, left panel) and cells induced with oxidative stress (Figure 2A, right panel), we found a correlation between the degree of misregulation observed in Arp5 KO and Arp8 KO cells (R2 = 0.64 and 0.77 in the absence and presence of oxidative stress, respectively); more than 90% of the misregulated genes were found either in zone I or in zone IV in the zone plot of transcript expression (Figure 2B)
Summary
In the nucleus of eukaryotes, the genomic DNA is packaged into a complex nucleoprotein structure, known as chromatin. It is known that some of the chromatin remodeling complexes contain actin family proteins as essential subcomponents (Oma and Harata, 2011). Whereas some of the Arps are predominantly localized in the cytoplasm, several Arps (namely Arp 4, Arp 5, Arp 6, Arp 7, Arp 8, and Arp 9) are accumulated in the nucleus (Dion et al, 2010; Oma and Harata, 2011) Consistent with these results, all of these nuclear Arps were found to be parts of chromatin remodeling complexes of which actin is a component
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