Rox, a novel bHLHZip protein expressed in quiescent cells that heterodimerizes with Max, binds a non-canonical E box and acts as a transcriptional repressor

Abstract

Proteins of the Myc and Mad family are involved in transcriptional regulation and mediate cell differentiation and proliferation. These molecules share a basic-helix-loop-helix leucine zipper domain (bHLHZip) and bind DNA at the E box (CANNTG) consensus by forming heterodimers with Max. We report the isolation, characterization and mapping of a human gene and its mouse homolog encoding a new member of this family of proteins, named Rox. Through interaction mating and immunoprecipitation techniques, we demonstrate that Rox heterodimerizes with Max and weakly homodimerizes. Interestingly, bandshift assays demonstrate that the Rox-Max heterodimer shows a novel DNA binding specificity, having a higher affinity for the CACGCG site compared with the canonical E box CACGTG site. Transcriptional studies indicate that Rox represses transcription in both human HEK293 cells and yeast. We demonstrate that repression in yeast is through interaction between the N-terminus of the protein and the Sin3 co-repressor, as previously shown for the other Mad family members. ROX is highly expressed in quiescent fibroblasts and expression markedly decreases when cells enter the cell cycle. Moreover, ROX expression appears to be induced in U937 myeloid leukemia cells stimulated to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The identification of a novel Max-interacting protein adds an important piece to the puzzle of Myc/Max/Mad coordinated action and function in normal and pathological situations. Furthermore, mapping of the human gene to chromosome 17p13.3 in a region that frequently undergoes loss of heterozygosity in a number of malignancies, together with the biochemical and expression features, suggest involvement of ROX in human neoplasia.

The miR-17-92 MicroRNA Cluster Is Regulated by Multiple Mechanisms in B-Cell Malignancies

“…55 MNT, however, has been reported to bind with higher affinity to this noncanonical sequence than to canonical E-boxes. 56 Thus, it is likely that in some cell types these upstream noncanonical sites are preferentially bound by MNT and perhaps other repressive MYC family members whose sequence specificity has not been analyzed in detail. Although deleting these sequences had little effect in the promoter-reporter assays ( Figure 4A), we cannot exclude the possibility that they are functional in their normal chromosomal context.…”

The basic region/helix – loop – helix/leucine zipper domain of Myc proto-oncoproteins: Function and regulation

“…Over recent years several other bHLHZip proteins, Mad1, Mxi1, Mad3, Mad4, and Mnt (or Rox) have been identi®ed that heterodimerize with Max Zervos et al, 1993;Hurlin et al, 1995aHurlin et al, , 1997Meroni et al, 1997). Thus Max is the central component of the Myc/Max/Mad network of transcriptional regulators.…”

Sequential expression of the MAD family of transcriptional repressors during differentiation and development

“…MAX is an obligatory heterodimeric partner for the MYC proteins in mediating their functions as sequence-speci®c DNAbinding transcriptional activators, that regulate cell cycle progression, transformation and apoptosis (Kretzner et al, 1992;Amin et al, 1993;Mukherjee et al, 1992;Amati et al, 1993a,b). MAX can also form heterodimers with at least two other types of bHLHZip proteins, the MAD proteins (MAD1, MXI1, MAD3 and MAD4) Zervos et al, 1993;Hurlin et al, 1995a) and the recently identi®ed MNT protein (Hurlin et al, 1997a;Meroni et al, 1997). Whereas MYC ± MAX, MAD ± MAX, and MNT ± MAX heterodimers recognize the same E-box sequence CACGTG (Blackwell et al, 1990(Blackwell et al, , 1993, their transcriptional activities are di erent: MYC ± MAX is a transcription activator while MAD ± MAX and MNT ± MAX are repressors of transcription (Kretzner et al, 1992;Amati et al, 1992;Amin et al, 1993;Zervos et al, 1993;Hurlin et al, 1995a;1997a).…”