DREAM-alphaCREM interaction via leucine-charged domains derepresses downstream regulatory element-dependent transcription

Abstract

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, alphaCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of alphaCREM is not required for the interaction, but phospho-alphaCREM shows higher affinity for DREAM. The interaction with alphaCREM is independent of the Ca(2+)-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both alphaCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca(2+).

FIG. 1

Effect of αCREM on DRE-dependent transcription. (a) Scheme showing the modular structure of the CREM and CREB genes and the different isoforms used. DBD, DNA-binding domain. (b) Transactivation by αCREM of the DRE-containing reporter pHD3CAT after transient transfection in NB69 cells. For comparison, the effect of forskolin treatment is shown (hatched bar), as well as the lack of effect of other CREM isoforms, ICER-I, or CREB. (c) Repression by DREAM (black bars) of the pHD3CAT reporter in HEK293 cells is relieved after cotransfection with αCREM or ɛCREM. For comparison, the lack of effect of αCREMΔLZ, βCREM, ICER-I, or CREB is shown. White bars represent the corresponding control transfections in the absence of DREAM.

FIG. 2

In vitro analysis of the DREAM-αCREM interaction. (a) EMSA with a DRE probe showing the interaction between recombinant DREAM and αCREM proteins at different ratios and its modulation by PKA phosphorylation. (b) EMSA with a DRE probe showing the interaction between DREAM and αCREM and the lack of effect of other CREM isoforms, ICER, and CREB. A DREAM/CRE-binding protein ratio of 1:1 was used. For comparison, the interaction between αCREM and DREAM is shown. The lack of binding of αCREM and ɛCREM to the DRE probe is shown. (c) Pull-down experiments showing that the DREAM-αCREM interaction is increased after PKA phosphorylation and not affected by calcium. The lack of effect of PKA on the phosphorylation mutant αCREMS68A is shown.

FIG. 3

Two LCDs in αCREM are necessary for the interaction with DREAM. (a) Alignment of LZ domains I and II from αCREM and βCREM, respectively, showing the 13-amino-acid difference and the positions of the different mutations and the truncated form αCREMΔ223. The putative LCD is boxed. Transient transfections in HEK293 cells (b) and EMSA with recombinant proteins (c) show the lack of interaction of αCREMβ212,218, αCREML217F, and αCREML73,76V with DREAM. The gain of function in mutant βCREMα212,218 is also shown. Values of CAT activity after transfection with wild-type or mutant αCREM, in the absence (open bars) or presence (black bars) of DREAM, are relative to basal acetylation of reporter pHD3CAT in cotransfection with empty reporter vector. Equal amounts of DREAM and αCREM proteins were used in the EMSA.

FIG. 4

Two LCDs in DREAM are necessary for the interaction with αCREM. Transient transfection in HEK293 cells (a) and EMSA with recombinant proteins (b) show that DREAM LCD mutants DREAML47,52V and DREAML155V are no longer able to interact with αCREM. On the other hand, truncated DREAM43-256 missing a putative LCD at position 15 still interacts with αCREM. Values of CAT activity after transfection with wild-type or mutant DREAM, with (gray bars) or without (open bars) αCREM, are relative to basal acetylation of reporter pHD3CAT in cotransfection with empty reporter vector. Equal amounts of αCREM and DREAM proteins were used for the EMSA. (c) EMSA with a DRE probe showing the unaffected sensitivity to Ca²⁺ of the DREAM LCD mutants. For comparison, the lack of Ca²⁺ sensitivity of the dominant negative mutant EFmDREAM is shown.

FIG. 5

The αCREM-DREAM interaction directs prodynorphin gene expression in human neuroblastoma cells after forskolin treatment. (a) Western blot analysis of the accumulation of CREM repressor proteins in SK-NMC cells at different times following forskolin treatment. The lack of effect on the levels of τCREM and CREB proteins is also shown. (b) Northern blot showing the induction of prodynorphin mRNA in SK-NMC cells after αCREM overexpression and the lack of effect of mutant αCREML73,76V. Levels of β-actin are shown as a control of the loading of each lane. (C) Western blot analysis showing similar levels of overexpressed wild-type or mutant αCREM in SK-NMC cells.