Relief of cyclin A gene transcriptional inhibition during activation of human primary T lymphocytes via CD2 and CD28 adhesion molecules

Abstract

Cyclin A transcription is cell cycle regulated and induced by cell proliferative signals. To understand the mechanisms underlined in this regulation in normal human cells, we have analysed in vivo protein-DNA interactions at the Cyclin A locus in primary T lymphocytes. Stimulation of puri®ed T lymphocytes by a combination of monoclonal antibodies directed at CD2 and CD28 adhesion molecules gives rise to a long lasting proliferation in the absence of accessory cells. Cyclin A was observed after 4 days of costimulation with anti CD2+CD28 whereas stimulation by anti CD2 or anti CD28 alone was not e ective. In vivo genomic DMS footprinting revealed upstream of the major transcription initiation sites, the presence of at least three protein binding sites, two of which were constitutively occupied. They bind in vitro respectively ATF-1 and NF-Y proteins. The third site was occupied in quiescent cells or in cells stimulated by anti CD2 or anti CD28 alone. The mitogenic combination of anti CD2+ anti CD28 released the footprint as cells were committed to proliferation. Consistent with theses results, nuclear extracts prepared from quiescent cells formed a speci®c complex with this element, whereas extracts prepared from cells treated with anti CD2+ anti CD28 failed to do so after cells entered a proliferative state.

Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb

“…We thus performed an in vivo genomic footprint analysis of the region surrounding the CCRE element in both adherent and non adherent cells. As previously described, the CCRE was quantitatively occupied in Go-early G1 adherent cells at the exclusion of the other phases of the cell cycle (Zwicker et al, 1995;Huet et al, 1996;Plet et al, 1997). The protection vanished as cells transited through G1/S ( Figure 2).…”

“…The protection vanished as cells transited through G1/S ( Figure 2). Next to the CCRE, two other previously characterized sites, binding respectively proteins from the CREB/ ATF (CRE) and NF-Y (NFY) families, harbored a constitutive protection (Barlat et al, 1995;Huet et al, 1996;Plet et al, 1997;Philips et al, 1998). Whereas reproducibly observed, the latter site harbored a partial protection probably due to a weak accessibility to DMS even in the presence of the protein.…”

“…Transcription from the cyclin A promoter is active in proliferating cells and inhibited in quiescent cells through a negative regulatory element (Cell Cycle Responsive Element, CCRE/cell Cycle Dependent Element, CDE) whose mutation renders the promoter constitutively active (Huet et al, 1996;Schulze et al, 1995;Zwicker et al, 1995;Plet et al, 1997). We thus performed an in vivo genomic footprint analysis of the region surrounding the CCRE element in both adherent and non adherent cells.…”

“…In adherent cells, transcription of the gene coding for cyclin A is cell cycleregulated and the absence of the transcript in quiescent or in early G1 cells results in a large part from the presence of an inhibitory complex on its promoter (Barlat et al, 1993(Barlat et al, , 1995Henglein et al, 1994;Schulze et al, 1995;Zwicker et al, 1995;Huet et al, 1996;Plet et al, 1997;Philips et al, 1998). Relief from this negative control gives rise to the activation of cyclin A gene transcription which takes place at the G1/S boundary.…”

“…Repression is mediated by a bipartite cell cycle-dependent repressor DNA element comprised of a GC-rich region (Cell Cycle Responsive Element, or cell Cycle Dependent Element: CCRE/CDE) and a directly adjacent motif (cell Cycle gene Homology Region: CHR) shared by several other cell cycleresponsive genes (Schulze et al, 1995;Zwicker et al, 1995;Huet et al, 1996). E2F and CDF-1 have been proposed to interact with this region (Schulze et al, 1995;Zwicker et al, 1995;Liu et al, 1997;ZerfassThome et al, 1997) and interfere with the transactivation potential of factors binding upstream such as NF-Y and ATF (Desdouets et al, 1995;Nakamura et al, 1995;Yoshizumi et al, 1995;Zwicker et al, 1995;Huet et al, 1996;Plet et al, 1997).…”