Negative Regulation of the Mouse Aldolase A Gene

Abstract

The expression of aldolase A L-type mRNA is increased in growth-arrested mouse NIH3T3 cells and remarkably down-regulated in actively proliferating cells. Treatment of proliferating cells with cycloheximide abolished the down-regulation of L-type mRNA expression, thus indicating that a protein factor acts as repressor in proliferating cells. Transient transfection experiments in NIH3T3 cells showed that a negative regulatory cis-element (NRE) is involved in the modulation of the transcriptional activity of the distal L promoter. The repressor, which is a protein of ϳ97 kDa, binds the murine aldolase A NRE, revealing a much more intense DNA-protein complex in proliferating NIH3T3 cells than in serum-deprived cells. Mutations in the negative regulatory cis-element showed that the GArich motif is required for protein binding and silencer function. We conclude that the expression of L-type mRNA is modulated by the interaction between a cell cycle-dependent DNA-binding protein and the murine aldolase A NRE.Aldolase A gene expression is regulated by an intriguing mechanism that involves three alternative promoter regions (distal or pL, middle or pM, and proximal or pF). Transcription of multiple mRNA species (L, M, and F types), containing the same coding region but different untranslated 5Ј-ends, is driven by three autonomous promoters and splicing of alternative leader exons (L, M, and F) (1-3). In man, two of these mRNA species (L and F types) are ubiquitously expressed, whereas the third one (M type) is muscle-specific (4 -6).We have demonstrated that the human genomic regions upstream from exons F and L are able to drive autonomous transcription (7,8). We found that the basal transcriptional activity of pF is regulated by several binding sites for the ubiquitous factor Sp1 (7). Binding of nuclear trans-acting factors AP-1 and NF-1 in the Ϫ384/Ϫ262 region increases pF transcriptional activity (7, 9).The pL promoter is regulated by both positive and negative cis-elements (8). Within this promoter, we detected a negative regulatory element (hAldA-NRE) 1 and a protein factor from Hep3B cells that binds to this element. This complex downregulates to one-fifth the transcription of the distal promoter in several cell types and furthermore regulates transcription of other cellular genes (10).In rodent tissues, only two mRNAs, the ubiquitous AH type, corresponding to the human F type, and the muscle-specific type (M type), have been characterized (11,12). We found that a third species, which corresponds to human aldolase A L-type mRNA, is expressed and correctly processed in rodent cell lines and that its expression is increased during differentiation and is associated with cell-growth arrest (13).Here we report that the modulation of murine L-type mRNA expression is transcriptionally controlled and differentially regulated in proliferating and confluence-arrested cell populations. Within the murine distal promoter, we identified a negative regulatory element (mAldA-NRE), homologous to the human aldolase A silencer, that ...

The Kruppel-like Zinc Finger Protein ZNF224 Recruits the Arginine Methyltransferase PRMT5 on the Transcriptional Repressor Complex of the Aldolase A Gene

“…In fact, we found that the repressor complex down-regulates the transcription of the L-type promoter in proliferating cell types, whereas the binding activity of the complex to the DNA is inhibited in growth-arrested cells, and this correlates with an increased L-type gene transcription (29,30).…”

“…This prompted us to verify the recruitment of PRMT5 to the promoter regions of genes encoding proteins involved in cell cycle regulation using the cell cycle-dependent modulation of L-type aldolase A promoter activity as an experimental tool (29,30). Our findings indicate that PRMT5 is involved in the formation of the complex that negatively regulates aldolase A gene transcription during the cell cycle (Fig.…”

“…In proliferating cells, ZNF224 specifically binds to the negative regulatory element, AldA-NRE, located in the L-type aldolase A promoter, and acts as a transcriptional repressor. The interaction between ZNF224 and AldA-NRE is responsible for the modulation of aldolase A mRNA expression during the cell cycle (29,30). The NH 2 terminus of ZNF224 contains a well conserved KRAB domain, including a canonical box A and a degenerated box b, whereas the C terminus contains 19 C 2 H 2 zinc finger motifs (31).…”

PKC‐dependent phosphorylation of the p97 repressor regulates the transcription of aldolase A L‐type promoter

“…Nuclear protein extracts from NIH 3T3 and Caco‐2 cells were prepared according to Dignam et al [16] modified as previously described [11]. Southwestern experiments [11] and EMSA [1] were performed as described.…”

“…Nuclear protein extracts from NIH 3T3 and Caco‐2 cells were prepared according to Dignam et al [16] modified as previously described [11]. Southwestern experiments [11] and EMSA [1] were performed as described. In phosphatase assay nuclear extracts (4 μg) were incubated with or without potato acid phosphatase (PAP) 0.06 and 0.12 U. for 20 min at 37°C in Parker buffer 1× (glycerol 10%, HEPES 10 mM (pH 7.9), KCl 100 mM, EDTA 0.1 mM, DTT 0.25 mM) prior to the EMSA experiments.…”