Three distinct sub-nuclear populations of HMG-I protein of different properties revealed by co-localization image analysis

We have studied the nuclear distribution of the non-histone HMG-I protein b y indirect immunofluorescence in several human and murine somatic cell line s and in growing mouse oocytes. We show that HMG-I, a high mobility-group p rotein which interacts in vitro with the minor groove of AT-rich B-DNA, is found exclusively in the nucleus and that this localization corresponds to a complex distribution. By comparing the HMG-I-dependent fluorescence signa l with the chromatin density determined by Hoechst 33342 or propidium iodid e staining, we present evidence for the existence of three HMG-I sub-popula tions whose contribution to the total fluorescence can be determined using a newly developed quantitative co-localization image analysis program: foci that correspond to regions of heterochromatin, intense dots located within decondensed chromatin, and a more diffuse component extending throughout t he nucleoplasm. In addition, we show that these sub-populations differ in t heir sensitivity to nuclease digestion and in vivo displacement by the mino r-groove binder Hoechst 33342. Finally, double immunolabeling of RNA polyme rase II-dependent transcription and HMG-I shows that the intense dots are n ot correlated with sites of high transcriptional activity, We discuss the p ossibility that these three sub-populations reflect distinct and separable biological functions of the HMG-I protein.