A HIC-5- and KLF4-dependent mechanism transactivates p21(Cip1) in response to anchorage loss.

Anchorage loss elicits a set of responses in cells, such as transcriptional changes, in order to prevent inappropriate cell growth in ectopic environments. However, the mechanisms underlying these responses are poorly understood. In this study, we investigated the transcriptional up-regulation of cyclin-dependent kinase inhibitor p21(Cip1) during anchorage loss, which is important for cell cycle arrest of nonadherent cells in the G1 phase. Up-regulation was mediated by an upstream element, designated as the detachment-responsive element (DRE), that contained Kruppel-like factor 4 (KLF4) and runt-related transcription factor 1 (RUNX1) recognition sites; both of these together were necessary for transactivation, as individually they were insufficient. RNAi experiments revealed that KLF4 and a multidomain adaptor protein, hydrogen peroxide-inducible clone 5 (HIC-5), were critically involved in DRE transactivation. The role of HIC-5 in this mechanism was to tether KLF4 to DNA sites in response to cellular detachment. In addition, further analysis suggested that oligomerization and subsequent nuclear matrix localization of HIC-5, which was accelerated spontaneously in cells during anchorage loss, was assumed to potentiate the scaffolding function of HIC-5 in the nucleus and consequently regulate p21(Cip1) transcription in a manner responding to anchorage loss. At the RUNX1 site, a LIM-only protein, CRP2, imposed negative regulation on transcription, which appeared to be removed by anchorage loss and contributed to increased transcriptional activity of DRE together with regulation at the KLF4 sites. In conclusion, this study revealed a novel transcriptional mechanism that regulated gene expression in a detachment-dependent manner, thereby contributing to anchorage-dependent cell growth.