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 by indirect immunofluorescence in several human and murine somatic cell lines and in growing mouse oocytes. We show that HMG-I, a high mobility-group protein 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 signal with the chromatin density determined by Hoechst 33342 or propidium iodide staining, we present evidence for the existence of three HMG-I sub-populations 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 the nucleoplasm. In addition, we show that these sub-populations differ in their sensitivity to nuclease digestion and in vivo displacement by the minor-groove binder Hoechst 33342. Finally, double immunolabeling of RNA polymerase II-dependent transcription and HMG-I shows that the intense dots are not correlated with sites of high transcriptional activity. We discuss the possibility that these three sub-populations reflect distinct and separable biological functions of the HMG-I protein.
Amirand
C
C
Laboratoire de Physicochimie Biomoléculaire et Cellulaire, ESA 7033 CNRS, Université Pierre et Marie Curie, Paris, France.
Viari
A
A
Ballini
J P
JP
Rezaei
H
H
Beaujean
N
N
Jullien
D
D
Käs
E
E
Debey
P
P
D016475:3T3 Cells; D000818:Animals; D001562:Benzimidazoles; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008836:Micrococcal Nuclease; D018613:Microscopy, Confocal; D011419:Propidium; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic
D001562:Benzimidazoles; D002843:Chromatin; D005456:Fluorescent Dyes; D006609:High Mobility Group Proteins; D011419:Propidium; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease; C017807:bisbenzimide ethoxide trihydrochloride