Janine Doly

Specific Binding of High-Mobility-Group I (HMGI) Protein and Histone H1 to the Upstream AT-Rich Region of the Murine Beta Interferon Promoter: HMGI Protein Acts as a Potential Antirepressor of the Promoter

ABSTRACT The high-mobility-group I (HMGI) protein is a nonhistone component of active chromatin. In this work, we demonstrate that HMGI protein specifically binds to the AT-rich region of the murine beta interferon (IFN-β) promoter localized upstream of the murine virus-responsive element (VRE). Contrary to what has been described for the human promoter, HMGI protein did not specifically bind to the VRE of the murine IFN-β promoter. Stably transfected promoters carrying mutations on this HMGI binding site displayed delayed virus-induced kinetics of transcription. When integrated into chromatin, the mutated promoter remained repressed and never reached normal transcriptional activity. Such a phenomenon was not observed with transiently transfected promoters upon which chromatin was only partially reconstituted. Using UV footprinting, we show that the upstream AT-rich sequences of the murine IFN-β promoter constitute a preferential binding region for histone H1. Transfection with a plasmid carrying scaffold attachment regions as well as incubation with distamycin led to the derepression of the IFN-β promoter stably integrated into chromatin. In vitro, HMGI protein was able to displace histone H1 from the upstream AT-rich region of the wild-type promoter but not from the promoter carrying mutations on the upstream high-affinity HMGI binding site. Our results suggest that the binding of histone H1 to the upstream AT-rich region of the promoter might be partly responsible for the constitutive repression of the promoter. The displacement by HMGI protein of histone H1 could help to convert the IFN-β promoter from a repressed to an active state.

Synergism between Multiple Virus-induced Factor-binding Elements Involved in the Differential Expression of Interferon A Genes

Comparative transfection analysis of murine interferon A4 and interferon A11 promoter constructs transiently transfected in mouse L929 and human HeLa S3 cells infected with Newcastle disease virus showed that the second positive regulatory domain I-like domain (D motif), located between nucleotides −57 and −46 upstream of the transcription start site, contributes to the activation of virus-induced transcription of the interferon (IFN)-A4 gene promoter by cooperating with the positive regulatory domain I-like and TG-like domains previously described. Electrophoretic mobility shift assay performed with the virus-inducible fragments containing these motifs indicated that the binding activity that we have denoted as virus-induced factor (Génin, P., Bragança, J., Darracq, N., Doly, J., and Civas, A. (1995) Nucleic Acids Res. 23, 5055–5063) is different from interferon-stimulated gene factor 3. It binds to the D motif but not to the virus-unresponsive form of the D motif disrupted by a G−57 → C substitution. We show that the low levels of IFN-A11 gene expression are caused essentially by the lack of two inducible enhancer domains disrupted by the A−78 → G and the G−57 → C substitutions. These data suggest a model taking account of the differential regulation of IFN-A gene family members. They also suggest that virus-induced factor may correspond to the primary transcription factor directly activated by virus that is involved in the initiation of IFN-A gene transcription.

Gene reorganization during serial divisions of normal human cells

We have followed during serial divisions of human fibroblasts the presence in chromosomal and extrachromosomal DNA, of two genes that are expressed in fibroblasts, actin and interferon, and of one that is not expressed, globin. The intensity of the blot hybridization of the actin and globin probes with chromosomal DNA diminished during serial divisions of diploid fibroblasts. The interferon gene remained constant throughout the human fibroblast life span. Chromosomal DNA sequences were present in extrachromosomal circular DNA which appeared at the end of the fibroblast life span. The results could explain some functional changes that occur in these cell populations when their division potential declines.

Autocrine regulation of terminal differentiation by interleukin-6 in the pluripotent KU812 cell line

The human KU812 leukemic cell line is a model for studying cell commitment towards different hematopoietic lineages. Indeed, this cell line is characterized by both a capacity for self-renewal and the ability to differentiate spontaneously along erythroid and basophilic cell lineages. In this study we show that interleukin-6 (IL-6) and its specific receptor (IL-6-R) are spontaneously expressed in the human KU812 cell line. Addition of antibody against IL-6 weakly inhibited its cell proliferation (20 to 30%) suggesting that the endogenous production of IL-6 was partially responsible for the growth of the cell line. In contrast, the spontaneous terminal differentiation of this cell line towards the erythroid and basophilic lineages was inhibited by an antibody against IL-6 and this effect was reversed by addition of recombinant human IL-6 (rIL-6). These results suggest that IL-6 is involved more in differentiation than in the proliferation of KU812 cells. After several passages, KU812 cells lose their capacity to differentiate spontaneously. In these cells, the IL-6-R was no more detectable. We therefore suggest that this loss of spontaneous differentiation is associated with an interruption of the IL-6 autocrine loop.