MRG15 activates the cdc2 promoter via histone acetylation in human cells

Abstract

Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.

Fig. 1

(A) MRG15 occupancy of the cell cycle promoters MCM4, PCNA, Cyclin A2, Cyclin B1, and cdc2, as quiescent HCA2 cells are released into serum and progress through one cell cycle. Chromatin immunoprecipitation revealed that MRG15 occupancy of the cdc2 promoter increased most substantially between the 12 and 24 hour time points as the cells entered late S phase. Values shown are the average percent of total input of three independent immunoprecipitations and are representative of at least two separate experiments; (B) Chromatin immunoprecipitation also revealed an increase in H4 acetylation and decreases in both HDAC1 and HDAC2 at the same locus in the cdc2 promoter region with acetylation of lysine 12 of H4 exhibiting a very significant at 24 hours as compared to quiescent cells (C). The time period between 12 and 24 hours corresponds to the progression of the majority of this population of fibroblasts through S phase and to the time period when cdc2 mRNA expression shows the largest increase.

Fig. 2

MRG15 exerts control over the cdc2 promoter in HeLa cervical carcinoma cells. HeLa cells were cotransfected with a promoter-reporter construct containing the full length cdc2 promoter followed by a luciferase reporter gene and (A) wild type MRG15, (B and C) siRNA directed against MRG15, (D and E) or MRG15 mutants lacking the chromodomain or leucine zipper domain. (A) Full-length MRG15 caused an increase in cdc2 promoter-driven luciferase expression in a dose-dependent manner while (B) siRNA (100 nM) directed against MRG15 resulted in a statistically significant loss (p value 0.0026 as determined by ANOVA) of cdc2 promoter-driven luciferase expression. Values expressed are relative luciferase units normalized to total protein. (D and E) Compared to transfection with MRG15, removal of the chromodomain (aa 26–62) caused little change in promoter firing whereas deletion of the leucine zipper (aa 284–305), which is part of the MRG domain, caused a decrease in induction of the promoter at both 0.5 and 1µg of DNA.

Fig.3

MRG15 interacts with the HAT Tip60 to cooperatively modulate the cdc2 promoter. (A) cdc2 mRNA expression requires HAT activity. HCA2 fibroblasts were cultured in low serum (1%) conditions for one week until a semi-heterogeneous quiescent population was achieved. Cells were then either treated with 50uM anacardic acid, a HAT inhibitor, in DMSO or with DMSO alone in media supplemented with 10% FBS. Cells were harvested 12 and 24 hours post-stimulation and mRNA isolated. cdc2 mRNA was quantitated by RT-PCR. Fibroblasts treated with vehicle alone showed a robust increase in cdc2 mRNA compared to 0h while cells treated with the HAT inhibitor showed no increase; (B) Tip60 localizes to the cdc2 promoter within 110 bp of MRG15. HCA2 fibroblasts were synchronized in low serum conditions for one week and then released to serum for 24 hours. Crosslinked cells were then immunoprecipitated with an antibody against Tip60. 24 hours post serum stimulation Tip60 was localized to a region within 110 bp of MRG15; (C) Tip60 and MRG15 act cooperatively at the cdc2 promoter. HeLa cells were co-transfected with the cdc2 promoter-reporter plasmid and empty vector, MRG15, Tip60, or MRG15 and Tip60 together. Cells were harvested 24 hours post-transfection and luciferase activity was assayed. Activity was normalized to total protein. Both MRG15 and Tip60 alone activated the promoter, but together had a cooperative effect.