The histone gene transcription factor HiNF-P stabilizes its cell cycle regulatory co-activator p220NPAT

Abstract

Orderly progression through the cell cycle requires the transcriptional activation of histone genes to support packaging of newly replicated DNA. Induction of human histone gene expression is mediated by a co-activation complex containing transcription factor HiNF-P and its cofactor p220NPAT. Here, using cells synchronized in S-phase and in mitosis, as well as serum-stimulated cells, we have investigated how HiNF-P is regulated during the cell cycle and examined its stability relative to p220NPAT. We find that while HiNF-P is maintained at steady-state levels throughout the cell cycle, both HiNF-P and p220NPAT are actively degraded by the proteasome pathway. Importantly, elevation of HiNF-P levels enhances the stability of its co-activator p220NPAT. The HiNF-P-dependent stabilization of p220NPAT may reinforce signaling through the cyclin E/CDK2/p220NPAT pathway and contribute to coordinate control of histone gene expression.

Figure 1. HiNF-P mRNA levels during the cell cycle and cell proliferation

HiNF-P mRNA levels were analyzed in either HeLa cells synchronized by the double thymidine block method (left panel) or in T98G released from quiescence by serum stimulation (right panel). Total RNA was isolated from samples taken at the indicated time points after release and analyzed by northern blot using specific probes shown on the left. Lower panels' shows band quantification by PhosphoImager and are expressed as fold change compared to 18S RNA (Asy: asynchronous).

Figure 2. HiNF-P protein levels during the cell cycle and cell proliferation

HiNF-P protein levels were analyzed for cell cycle distribution in HeLa cells synchronized by the double thymidine block (A), and SaOS cells blocked by nocodazole treatment (B), and for cell proliferation in T98G released from quiescence by serum stimulation (C). Whole cell extract was prepared from samples taken at the indicated time points after release from blockage and analyzed by western blot using specific antibodies shown on the left. To confirm synchrony in each cell line, cell cycle distribution was analyzed by FACS sorting and profiles along with the percentage in each cell cycle stage are shown underneath.

Figure 3. Degradation of HiNF-P in HeLa cells

Cells were incubated in presence of DMSO (control) or the protease inhibitor MG132 for 8 or 12 hr (A). Whole cell extracts were analyzed by western blot using specific antibodies shown on the left. MCM6 is shown to confirm equal loading as well as the Coomassie staining underneath. Specificity of the proteasome pathway was addressed by using a combination of protease inhibitors (B). HeLa cells were incubated in presence of DMSO (control), MG132 (proteasome and calpain inhibitor), Lactacystin (proteasome inhibitor) or ALLM (calpain inhibitor) for 12 hr. Whole cell extracts were analyzed by western blot using specific antibodies shown on the left. MCM6 is shown to confirm equal loading as well as the Coomassie staining underneath.

Figure 4. Ubiquitination of HiNF-P in vivo

Exponentially growing HeLa cells were transfected with pFLAG-HiNF-P. Whole cell extracts were prepared from cells treated with DMSO (control) or MG132 for 12 hr. HiNF-P was immunoprecipitated using a polyclonal anti-FLAG antibody and analyzed by western blot with a monoclonal anti-ubiquitin (clone P4D1) antibody (A). As a control for immunoprecipitations normal rabbit IgG was used. To confirm the presence of HiNF-P in the immunoprecipitated, the membrane was re-blotted using a monoclonal anti-FLAG antibody (B).

Figure 5. Stabilization of p220^NPAT by HiNF-P

Endogenous (control) or exogenously expressed HiNF-P turnover was analyzed in HeLa cells after treatment with 50 μg/ml cycloheximide (CHX) for the indicated times (A). Cells were harvested and whole cells extracts prepared for western blotting with the indicated antibodies on the left. MCM6 is shown to confirm equal loading as well as the Coomassie staining underneath. Cyclin E turnover was analyzed under conditions described in panel A. The lower panel shows quantification of cyclin E in control cells or cells expressing FLAG-HiNF-P. Results are plotted as the log of the percentage of protein remaining at each time point (B). Panel C shows quantification of the western blots shown in panel A and values are expressed as “log of percentage remaining” as described above. HiNF-P and FLAG-HiNF-P (left) or p220^NPAT (right) were analyzed in untransfected cells (control) or cells transfected with pFLAG-HiNF-P (exogenous).

Figure 5. Stabilization of p220^NPAT by HiNF-P

Endogenous (control) or exogenously expressed HiNF-P turnover was analyzed in HeLa cells after treatment with 50 μg/ml cycloheximide (CHX) for the indicated times (A). Cells were harvested and whole cells extracts prepared for western blotting with the indicated antibodies on the left. MCM6 is shown to confirm equal loading as well as the Coomassie staining underneath. Cyclin E turnover was analyzed under conditions described in panel A. The lower panel shows quantification of cyclin E in control cells or cells expressing FLAG-HiNF-P. Results are plotted as the log of the percentage of protein remaining at each time point (B). Panel C shows quantification of the western blots shown in panel A and values are expressed as “log of percentage remaining” as described above. HiNF-P and FLAG-HiNF-P (left) or p220^NPAT (right) were analyzed in untransfected cells (control) or cells transfected with pFLAG-HiNF-P (exogenous).

Figure 5. Stabilization of p220^NPAT by HiNF-P

Endogenous (control) or exogenously expressed HiNF-P turnover was analyzed in HeLa cells after treatment with 50 μg/ml cycloheximide (CHX) for the indicated times (A). Cells were harvested and whole cells extracts prepared for western blotting with the indicated antibodies on the left. MCM6 is shown to confirm equal loading as well as the Coomassie staining underneath. Cyclin E turnover was analyzed under conditions described in panel A. The lower panel shows quantification of cyclin E in control cells or cells expressing FLAG-HiNF-P. Results are plotted as the log of the percentage of protein remaining at each time point (B). Panel C shows quantification of the western blots shown in panel A and values are expressed as “log of percentage remaining” as described above. HiNF-P and FLAG-HiNF-P (left) or p220^NPAT (right) were analyzed in untransfected cells (control) or cells transfected with pFLAG-HiNF-P (exogenous).