MondoA, a novel basic helix-loop-helix-leucine zipper transcriptional activator that constitutes a positive branch of a max-like network

Abstract

Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix-leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

FIG. 1

Identification and cloning of an Mlx-binding protein. (A) Mlx and associated proteins were immunoprecipitated from whole-cell extract with Mlx antibodies and probed in a far-Western blot with [³⁵S]Met-labeled Mlx. The arrow indicates an ∼130-kDa Mlx-binding protein. Block, incubation (+) of the antibody with cognate antigen as a control for the specificity of the immunoprecipitation reaction. (B) Alignment of the BHLHZip domain of MondoA with Mlx and members of the Max network. Conserved residues in the basic region predicted to dictate binding to CACGTG are marked by dots, as are the hydrophobic amino acids that make up the leucine zipper. The dashes indicate gaps in the aligned sequences. (C) Expression of mondoA in adult human tissues as determined by Northern blot analysis (Sk. muscle, skeletal muscle; PBL, peripheral blood lymphocytes). (D) Domain diagram of the MondoA protein. The percentages of amino acid identity between MondoA and D. melanogaster Mondo (dMondo), C. elegans Mondo (cMondo), and H. sapiens Mlx are shown. The serine-threonine-proline (STP) rich domain is shaded. The TAD is within the STP domain.

FIG. 2

Mlx and MondoA associate in the cytoplasm. (A) Mlx immunoprecipitates were resolved on SDS gels and transferred to a PVDF membrane, and the blot was sequentially probed with ³⁵S-labeled Mlx (left) or anti-MondoA (right) antiserum. Block, incubation of the antibody (+) with cognate antigen as a control for the specificity of the immunoprecipitation reaction. (B) Nuclear (N) and cytoplasmic (C) compartments were prepared by hypotonic lysis, immunoprecipitated with Mlx antibodies, and probed in a far-Western blot with [³⁵S]Met-labeled Mlx (left) or anti-MondoA (right) antiserum. An ∼130-kDa band is detected in the cytoplasmic fraction (arrow). (C and D) Nuclear and cytoplasmic fractions were immunoprecipitated with Mlx (C) or mSin3 (D) antibodies, and the immunoprecipitates were examined by Western blotting with Mlx or mSin3A antibodies.

FIG. 3

Mlx and MondoA interact in vivo and bind CACGTG sequences as heterodimers. (A) Cytoplasmic extracts of P19 cells were incubated with either CACGTG E-box (W.T. CM1) or mutant E-box CACCTG (Mut CM1) oligonucleotides immobilized on agarose beads. As a control, cell extracts were also precipitated with α-Mlx (IP Mlx). The arrow indicates MondoA (left) or Mlx (right). (B) EMSA was used to show specific dimerization and DNA binding by MondoA and Mlx. Proteins in the indicated combinations were bound to a ³²P-labeled oligonucleotide containing a single CACGTG binding site. ΔLZMlx lacks the leucine zipper and cannot heterodimerize with Mondo. RL, reticulocyte lysate alone. On the right, MondoA-Mlx heterocomplexes were incubated with either preimmune or antiMlx/GST serum. The asterisk indicates the supershifted heterocomplex. +, present.

FIG. 4

Mlx and Mondo shuttle between the cytoplasmic and nuclear compartments. Expression vectors encoding FLAG-tagged Mlx and MondoA were transfected into NIH 3T3 cells. Their subcellular localization was determined by indirect immunofluorescence using anti-FLAG and anti-MondoA antibodies. (A and B) Two representative cells coexpressing Mlx and Mondo, respectively. (C) The subcellular distribution of Mlx and MondoA was quantified in the absence or presence (+) of leptomycin B. Nuc, nuclear; Cyto, cytoplasmic. The error bars indicate standard deviations.

FIG. 5

Nuclear-targeted MondoA-Mlx is a transcriptional activator. (A) V5 epitope-tagged Mondo and NLSMlx were transfected into NIH 3T3 cells, and their subcellular localization was determined and quantified by immunofluorescence. MondoA and Mlx were detected using anti-V5 and anti-Mlx, respectively, as the primary antibodies. (B) Transient-transfection assays were performed to determine the transcriptional activities of NLSMlx and MondoA on CACGTG E-boxes in NIH 3T3 cells. The cells were transfected with 0.5 μg of Mlx, NLSMlx, or MondoA in the combinations shown, along with a luciferase reporter containing four CACGTG binding sites. The results are shown as fold activation, and the error is expressed as the standard error of the mean. +, present; Nuc, nuclear; Cyto, cytoplasmic.

FIG. 6

The amino terminus of MondoA contains an autonomous transcriptional domain. (A) Diagram illustrating the amino-terminal MondoA deletions. hMondoA, human MondoA. (B) The amino-terminal deletion series of MondoA was tested for transcriptional activation when coexpressed with Mlx. The subcellular localization of coexpressed Mlx and MondoA is indicated. (C) Amino acids 322 to 445 of MondoA were fused to the Gal4DBD and tested for activity in NIH 3T3 cells using a luciferase reporter gene containing the thymidine kinase promoter and four Gal4 binding sites. Gal4DBD (450 ng), Gal4–c-Jun (450 ng), or increasing amounts (50, 150, and 450 ng) of Gal4DBD(332-445)MondoA were transfected. The results are shown as fold activation, and the error is expressed as the standard error of the mean. +, present; −, absent.