Differences in DNA‐binding efficiency of Sp1 to aldolase and pyruvate kinase promoter correlate with altered redox states in resting and proliferating rat thymocytes

Abstract

Thymocytes induce their glycolytie enzymes as they undergo transition from the resting to the proliferating state. Corresponding increases in mRNA levels point to a transcriptional regulation. Electrophoretic mobility shift assays revealed that the DNA-binding efficiency of Spl is increased when nuclear extracts from proliferating compared to resting rat thymocytes were used. Here we demonstrate that hydrogen peroxide, added to nuclear extract from proliferating cells, decreases the Spl DNA-binding activity, whereas in nuclear extracts from resting cells dithioerythritol fully restores DNA-binding efficiency. Moreover we show that in contrast to resting thymocytes, production of reactive peroxide anions upon priming with phorbol 12-myristate 13-acetate is nearly abolished in the proliferating cells. From these results we propose that reactive oxygen intermediates affect the interaction of the Spl transcription factor with its consensus sequence and subsequently regulate glycolytic gene expression.

Sp Family Members and Nuclear Factor-Y Cooperatively Stimulate Transcription from the Rat Pyruvate Kinase M Gene Distal Promoter Region via Their Direct Interactions

“…Sp1 stimulates cAMPdependent transcriptional stimulation from the CYP11A gene promoter in SL2 cells (52). However, it has been reported that the phosphorylated and the nonreduced form of Sp1 showed a decrease in binding to a DNA fragment that contained both GC boxes 1 and 2 of the PKM gene (51,53). On the other hand, cdc25C gene transcription, which occurs late in the S/G 2 phase, is regulated by a cell cycle-dependent repressor element and an upstream activating sequences which contains three CCAAT motifs and two Sp1-binding sites.…”

Redox‐regulated expression of glycolytic enzymes in resting and proliferating rat thymocytes

“…Furthermore, chemiluminescence measurements revealed that resting, but not proliferating thymocytes do produce peroxide anions. This could be the reason for the observed decrease in Spi-binding efficiency with nuclear extracts from resting thymocytes [5].…”

“…Previous experiments [5] with nuclear extracts from prolif- erating rat thymocytes revealed that hydrogen peroxide decreases the binding activity of Spi to the aldolase A and pyruvate kinase M 2 promoters. Addition of DTE fully restores DNA binding efficiency.…”

“…Resting thymocytes, which produce reactive oxygen species, cannot induce glycolytic enzyme expression due to oxidative inactivation of Spi. In contrast the presence of an effective scavenging system and/or a suppression of ROS formation in proliferating cells [5] enables them to induce glycolytic enzymes and progress orderly through the somatic cell division cycle [1,2].…”

“…The concomitant increase in the activity of glycolytic enzymes with the level of mRNA indicates that these enzymatic activities in proliferating cells are stimulated by activation of gene transcription [3]. The promoter regions of the aldolase A and pyruvate kinase M 2 genes contain various Spi-binding sites, and Spi has been shown to play a critical role in the transcriptional regulation of these glycolytic enzymes [4,5]. Recently we demonstrated with gel shift analyses that peroxide anions inhibit the interaction of Spi transcription factor with its consensus sequence, a process which can be reversed by addition of dithioerythritol (DTE).…”