Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

Abstract

Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase II (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP II or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominantnegative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined subnuclear structures may also be advantageous in maintenance of genome stability.The Ku protein is part of the DNA-dependent protein kinase (DNA-PK), which is required for the nonhomologous end-joining pathway of DNA double-strand break (DSB) repair (53, 59). When a break occurs, Ku binds avidly to the DNA ends, translocates inward, and recruits the 470-kDa DNA-PK catalytic subunit (DNA-PKcs) to form an active protein kinase complex (19,26,74). Proteins in this complex cooperate with XRCC4, DNA ligase IV, and other factors to carry out DSB repair (reviewed in reference 22). Ku-deficient cells are sensitive to ionizing radiation and are unable to complete the process of V(D)J recombination, which involves a DSB intermediate.In addition to its well-documented role in DSB repair, Ku appears to have some interaction with the RNA polymerase II (RNAP II) transcription apparatus. Several reports describe the binding of Ku to promoter regions or its ability to regulate transcription of individual genes (reviewed in reference 21 and also see references 11, 25, 31, and 72). Other reports describe interaction of Ku with the general transcription machinery. DNA-PK efficiently phosphorylates RNAP II in vitro (50), and a fraction of Ku resides in RNAP II-containing complexes (41). The importance of these interactions is suggested by the finding that nuclear extracts of Ku-deficient cells exhibit a characteristic transcription defect (70). Transcription of several different promoters is decreased two-to fivefold relative to that of extracts from a matched Ku-containing cell line (70). The defect is entirely at the level of reinitiation and is not seen in assays in which transcription is limited to a ...

MINT, the Msx2 Interacting Nuclear Matrix Target, Enhances Runx2-dependent Activation of the Osteocalcin Fibroblast Growth Factor Response Element

“…Although capable of expressing OPN, Ku70 Ϫ/Ϫ calvarial osteoblasts have a profound deficiency in OC gene expression (18). Dynan and colleagues have clearly established a crucial role for Ku antigen in transcriptional activity (32), separate from its DNA-dependent protein kinase catalytic subunit-regulated role in DNA repair. Ku supports multiround transcriptional re-initiation processes (41) and selectively associates with RNAP IIo, the actively phosphorylated and processive form of RNA polymerase II, but not with general transcription factors associated with the pre-initiation complex complex (32).…”

“…Dynan and colleagues have clearly established a crucial role for Ku antigen in transcriptional activity (32), separate from its DNA-dependent protein kinase catalytic subunit-regulated role in DNA repair. Ku supports multiround transcriptional re-initiation processes (41) and selectively associates with RNAP IIo, the actively phosphorylated and processive form of RNA polymerase II, but not with general transcription factors associated with the pre-initiation complex complex (32). DNA-dependent protein kinase catalytic subunit is not associated with Ku and RNAP IIo (32), confirming unique localizing and roles for the Ku-RNAP IIo complexes in transcription (32) and the Ku-DNA-dependent protein kinase catalytic subunit complexes in DNA repair (42).…”

“…Ku supports multiround transcriptional re-initiation processes (41) and selectively associates with RNAP IIo, the actively phosphorylated and processive form of RNA polymerase II, but not with general transcription factors associated with the pre-initiation complex complex (32). DNA-dependent protein kinase catalytic subunit is not associated with Ku and RNAP IIo (32), confirming unique localizing and roles for the Ku-RNAP IIo complexes in transcription (32) and the Ku-DNA-dependent protein kinase catalytic subunit complexes in DNA repair (42). Because our novel Ku antigen complex binds Runx2 and the OCFRE (18), it is tempting to speculate that these interactions may provide a mechanism for ensuring the efficient association of Ku with RNAP IIo on the OC gene after promoter clearance of the pre-initiation complex (32,41).…”

“…The hyperphosphorylated C-terminal domain of RNA polymerase II, RNAP IIo, demarcates RNA synthesis in transcriptionally active chromatin (31) and can be specifically immunolocalized by the phospho-PolII monoclonal antibody H5 (32). Therefore, we evaluated the subnuclear localization of MINT with respect to RNAP IIo, using dual-color confocal immunofluorescence microscopy (see "Experimental Procedures").…”

The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage

“…This factor has been found to be associated with RNA polymerase II elongation sites without a sequence-specific DNA binding. 22 On the other hand, there are several reports indicating that Ku acts as a transcription factor: for example, in the repression of the human a-myosin heavy-chain promoter during heart failure; 23 or in contributing the oncogene ERBB2 overexpression in breast cancer cells by interacting with activator protein-2 transcription factors. 24 In this report we analyse the transcriptional regulation of murine Apaf1 and we demonstrate a functional interaction between Ku70/86 heterodimer and a specific region of Apaf1 promoter.…”