No abstract

The interaction between the negative cis-element (AldA-NRE) and p97 repressor nuclear protein is a key step in modulating transcription of the human and mouse aldolase A (AldA) gene during the cell cycle and di¡erentiation. In an attempt to clarify the role of transcriptional repression in regulating gene expression, we puri¢ed, from HeLa cells, the nuclear protein that speci¢cally binds to the AldA negative regulatory element (NRE). Matrix-assisted laser desorption ionization-time of £ight analysis and examination of protein pro¢les from the SwissProt database revealed that the previously de¢ned p97 repressor is ZNF224, a zinc ¢nger protein.We demonstrate that ZNF224, a Kruppel-like zinc ¢nger transcription factor, is the repressor protein that speci¢cally binds to the negative cis-element AldA-NRE and a¡ects the AldA-NREmediated transcription. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Gene transcription in eukaryotes is modulated by the coordinated recruitment of specific transcription factors and chromatin-modulating proteins. Indeed, gene activation and/or repression is/are regulated by histone methylation status at specific arginine or lysine residues. In this work, by co-immunoprecipitation experiments, we demonstrate that PRMT5, a type II protein arginine methyltransferase that monomethylates and symmetrically dimethylates arginine residues, is physically associated with the Kruppel-like associated box-zinc finger protein ZNF224, the aldolase A gene repressor. Moreover, chromatin immunoprecipitation assays show that PRMT5 is recruited to the L-type aldolase A promoter and that methylation of the nucleosomes that surround the L-type promoter region occurs in vivo on the arginine 3 of histone H4. Consistent with its association to the ZNF224 repressor complex, the decrease of PRMT5 expression produced by RNA interference positively affects L-type aldolase A promoter transcription. Finally, the alternating occupancy of the L-type aldolase A promoter by the ZNF224-PRMT5 repression complex in proliferating and growth-arrested cells suggests that these regulatory proteins play a significant role during the cell cycle modulation of human aldolase A gene expression. Our data represent the first experimental evidence that protein arginine methylation plays a role in ZNF224-mediated transcriptional repression and provide novel insight into the chromatin modifications required for repression of gene transcription by Kruppel-like associated box-zinc finger proteins.Gene transcription is controlled by the interplay of several transacting factors and chromatin-modifying activities that are sequentially recruited to the promoter region. Post-translational modifications of histone and non-histone chromosomal proteins are considered to be additional mechanisms that contribute to the epigenetic inheritance of phenotypic alterations. The temporal and combinatorial recruitment of the histonemodifying activities can determine differential outcomes in gene expression (1-3).Histone methylation, which usually occurs on arginine or lysine residues, is involved in regulation of chromatin structure, which in turn either stimulates or inhibits gene transcription. In fact, methylation of Lys-4 and Arg-17 of histone H3 and Arg-3 of histone H4 has been associated with transcriptional activation, whereas methylation of Lys-9 and Lys-27 of histone H3 has been related to gene repression (4, 5). Arginine methylation of nucleosomal histones is catalyzed by a homogenous class of enzymes that are known as "protein arginine methyltransferases" (PRMTs). 4 In this reaction the methyl group, which is provided by the S-adenosyl-L-methionine, is transferred to one of the guanidinium nitrogens of arginine residues. PRMTs are divided into type I PRMTs, which catalyze monomethylation and asymmetric dimethylation of arginine residues, and type II PRMTs, which also catalyze monomethylation and, in addition, symmetric dimethylation of a...

No 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 ...