Abstract

Williams–Beuren syndrome (WBS) is a developmental disorder associated with haploinsufficiency of multiple genes at 7q11.23. Here, we report the functional characterization of WBS critical region gene 14 (WBSCR14), a gene contained in the WBS commonly deleted region. It encodes a basic-helix–loop–helix leucine zipper (bHLHZip) transcription factor of the Myc/Max/Mad superfamily. WBSCR14 is expressed in multiple tissues, including regions of the brain and the intestinal tract. WBSCR14 forms heterodimers with the bHLHZip protein Mlx to bind the DNA sequence CACGTG. Like Max, Mlx has no intrinsic transcriptional activity, but its association with Mad1, Mad4, Mnt or WBSCR14 can repress E-box-dependent transcription. Preliminary results suggest a possible role of WBSCR14 in growth control. Our data support the view that the Max-like bHLHZip protein, Mlx, is a key element of a transcription factor network. We thus suggest that WBSCR14 may contribute to some aspects of the WBS pathology.

Received 18 December 2000; Revised and Accepted 29 January 2001.

INTRODUCTION

Williams–Beuren syndrome (WBS, OMIM 194050) is a neurodevelopmental disorder characterized by congenital heart and vascular disease, hypertension, infantile hypercalcemia, dental abnormalities, dysmorphic facial features, mental retardation, premature aging of the skin and unique cognitive and personality profiles (1,2). This is a contiguous gene disorder caused by haploinsufficiency of multiple genes at 7q11.23. The cardiovascular aspects of the disorder are known to be caused by elastin haploinsufficiency (36), but it remains to be determined which of the remaining 15 or more genes (FKBP6, FZD3/FZD9, WSTF/WBSCR9, BCL7B, WS-βTRP/TBL2, WBS critical region gene 14 (WBSCR14)/WS-bHLH, CPETR1/CLDN4, STX1A, CPETR2/CLDN3/RVP1, LIMK1, WBSCR1/EIF4H, RFC2, CYLN2/CLIP-115/WBSCR4/WBSCR3, WBSCR11/GTF3/GTF2IRD1 and GTF2I/BAP135/SPIN) contribute to the other symptoms (7,8).

The WBSCR14/WS-bHLH gene encodes a polypeptide that contains a tripartite conserved motif called the basic-helix–loop–helix leucine zipper (bHLHZip), characteristic of a subclass of transcription factors (9,10). The basic region of these proteins binds a canonical CANNTG sequence called E-box (11) and the HLH and Zip motifs participate in protein dimerization, a prerequisite for DNA-binding (12,13). The bHLHZip transcription factors of the Myc/Max/Mad subclass are linked to diverse aspects of eukaryotic cell function, integral to proliferation, growth, apoptosis and differentiation (1420). Max forms heterodimers with the Mycs (c-Myc, N-Myc and L-Myc), the Mads (Mad1, Mxi1, Mad3 and Mad4), Mnt (also named Rox) and Mga (2127). The Mycs are involved in a wide range of neoplasias (28), whereas the Mads block proliferation and Myc-induced transformation and promote differentiation (29). These opposing features are consistent with the ability of Myc:Max and Mad:Max heterodimers to activate and repress E-box-dependent transcription, respectively (22,25,3032). Recently, we and others described the isolation of Mlx, a new Max-like bHLHZip protein (27,33). Like Max, Mlx has no intrinsic transcriptional activity but, when associated with Mad1, Mad4 or Mnt, it represses E-box transcription through an mSIN3/HDAC-dependent mechanism. In the present report we show that the bHLHZip WBSCR14 protein is interacting with Mlx. The WBSCR14:Mlx heterodimer binds canonical and non-canonical E-box DNA sequences and represses transcription. Our results suggest that WBSCR14 plays a role in cell proliferation and/or differentiation. Moreover, the identification of a new Mlx partner further supports the existence of a new subfamily of dimeric E-box binding factors that might contribute to some aspect of the WBS pathology.

RESULTS

The WBSCR14 gene

In order to find protein homologous to the newly identified Mlx protein we screened the nucleotide sequences deposited in GenBank (27,33). We identified a cDNA that encodes a new member of the bHLHZip protein family. This cDNA, named WS-bHLH, mapped on chromosome 7q11.23, in the region commonly altered in WBS (9). The sequences of clones isolated from a human liver cDNA library (Stratagene) revealed that the mRNA contains an open reading frame (ORF) of 3245 nucleotides and encodes a protein of 852 residues, rather than the previously reported 231 (10). The homology between WS-bHLH and Mlx extends beyond the DNA binding and dimerization domains to the C-terminal tail, a region we named WBSCR14-Mlx C-tail (WMC) (Fig.1) (9,10). To avoid confusion with the probably chimeric WS-bHLH, we adopted the committee-approved gene symbol WBSCR14. We characterized five alternatively spliced isoforms, WBSCR14-α, -β, -γ, -δ and -ε, and the genomic structure of the WBSCR14 gene (available upon request; note that partial exon/intron organization tables were already presented in references 9,10). All intron–exon junctions conform to the donor–acceptor rules. WBSCR14-β and -δ encode WBSCR14 proteins deleted for helix 2, whereas WBSCR14-ε lacks the bHLHZip and WMC domains. Hence, these three alternative splice forms probably encode non E-box-binding WBSCR14 proteins. We isolated the full-length murine WBSCR14 homolog and characterized five alternatively spliced forms (Wbscr14-ζ, -η, -θ, -ι and -κ) (Fig. 1) (10), the longest of which encodes a polypeptide of 864 residues. The Wbscr14 isoforms -ι and -κ lack Helix 2, leucine zipper and the WMC regions, and isoform -η lacks the last 320 residues and the entire bHLHZip domain. Homology searches in dbEST revealed WBSCR14-related genes in Drosophila melanogaster and Caenorhabditis elegans. To possibly identify functionally important WBSCR14 regions we cloned and sequenced three fruitfly isoforms and the nematode full-length cDNA (dWBSCR14-α, -β and -γ, and cWBSCR14, respectively) (Fig. 1).

Wbscr14 expression during development

To investigate WBSCR14 expression during embryonic development, we performed in situ hybridization of E10.5 through E16.5 mouse embryo sections. Although Wbscr14 is expressed in both the ventricular zone (VZ) and the intermediate zone (IZ) of the developing spinal cord of E12.5 murine embryos, the signal is slightly stronger in the differentiating compartment (Fig. 2J). The VZ is composed of actively dividing neurons and glia precursors. The IZ consists of precursors that have exited the cell cycle and have begun to differentiate. Later in embryonic development Wbscr14 is expressed in the central nervous system (CNS), olfactory epithelium, lung, liver, kidney, adrenal gland, midgut within the physiological hernia, villi of the duodenum, thymus, trigeminal and facial ganglia, wall of the heart, cartilage primordia of the ribs and vibrisse follicles (Fig. 2A–I and data not shown).

Previous work indicated that human WBSCR14 expression was detected in the liver, heart and kidney (9,10) (S. Cairo and A. Reymond, unpublished data). Although not demonstrated, the pattern of expression of WBSCR14 seems not to be restricted to the above tissues, as WBSCR14-corresponding expressed sequence tags (ESTs) were isolated from brain, cerebellum, breast, lung, colon, testis and numerous tumors type cDNA libraries. Consistently, we show expression of WBSCR14 in the adult human brain cerebral cortex area (frontal, temporal, parietal and occipital lobes) and cerebellum by northern blot analysis on multi-tissue filters (Fig. 2K). Strong signals were also detected in intestinal tissues (jejunum, ileum and colon).

WBSCR14, a new Mlx partner

Members of the bHLHZip family must form dimers to bind DNA. We tested proteins for interaction with WBSCR14 using the interaction-mating technology (34). A panel of bHLHZip proteins expressed as fusion proteins with the LexA-DNA binding domain (bait) were tested for interaction with WBSCR14-α and Mlx-β fused to the B42-acidic moiety (prey). WBSCR14-α forms heterodimers with the three Mlx isoforms, -α, -β and -γ (Fig. 3A). We found no interaction between WBSCR14 and Mnt, Max1, Max2, Mad1, Mxi1, Mad3, USF and MITF. No indication of WBSCR14 homodimerization was detected in these assays. As previously reported, Mlx interacts with Mad1, Mnt and itself, but not with Max1, Max2, Mxi1, Mad3, MITF or USF (27,33).

We then determined that WBSCR14 forms heterodimers with Mlx in vivo. COS-7 cells were transiently transfected with plasmids that expressed the HA-Wbscr14-ζ (864 residues), HA-Wbscr14-θ (842 residues) and HA-Wbscr14-θ-Δbasic fusion proteins. These fusion proteins are 879, 857 and 842 residues long, respectively (the tag is 15 residues long). Expression was assessed by immunoprecipitation with anti-HA antibodies (Fig. 3B, lanes 1–4). The anti-HA mAb reactive band paralleled the predicted molecular weight of the transfected Wbscr14 isoforms and deletion mutant (compare lanes 1–3). To determine whether the Wbscr14 isoforms bind to the COS-7 endogenous Mlx (S. Cairo and A. Reymond, unpublished data) and Max proteins, cell lysates were immunoprecipitated with two different immune sera raised against Mlx (Fig. 3B, lanes 5–13) or two different anti-Max antibodies (lanes 14–15). The precipitated proteins were separated by gel electrophoresis and subjected to immunoblot analysis using an anti-HA (lanes 5–7 and 11–15) or anti-Wbscr14 P53 antibody (lanes 8–10). The HA-tagged Wbscr14 proteins were found in the anti-Mlx R5852 and anti-Mlx R4432 immunoprecipitates (lanes 5–11 and 13), but not in the control (lane 12) or the anti-Max C17 and anti-Max C124 immunoprecipitates. These findings confirmed that WBSCR14 forms heterodimers with Mlx, but not with Max. Max and Mlx are relatively stable proteins, whereas their dimerization partners are known to have short half-lives (22,24,33,35,36). To determine the stability of Wbscr14 we measured its half-life in transfected COS-7 cells treated with cycloheximide to block new protein synthesis. Following 0, 30, 60, 120 and 240 min of cycloheximide regimen, Wbscr14 was immunoprecipitated and detected by western blot. Wbscr14 has a half-life of ∼30 min (Fig. 3C).

The results of the interaction-mating and in vivo co-immunoprecipitation experiments suggest that WBSCR14 forms heterodimers with Mlx but not with Max. Therefore, WBSCR14 is a member of the emerging Mlx network that specifically dimerizes with Mlx (27,33).

WBSCR14:Mlx dimer binds DNA

We used electrophoretic mobility shift assays (EMSAs) to determine whether the WBSCR14:Mlx dimer binds E-box DNA sequences. Two specific protein–DNA complexes are formed by mixing reticulocyte lysates containing HA-Wbscr14-θ residues 412–842 and FL-Mlx-γ, and an end-labeled oligonucleotide containing the CACGTG E-box sequence (Fig. 4A, lanes 4, 7, 11 and 18). The faster migrating complex corresponds to FL-Mlx:FL-Mlx homodimers bound to the probe as a similar complex is present in mixes between probe and reticulocyte expressing only FL-Mlx (lanes 4 and 10). Moreover, this complex can be supershifted in the presence of anti-FLAG, but not with anti-HA mAb (lanes 4, 5, 6, 9 and 10). These results confirm previous studies, which demonstrated that Mlx homodimers bind to CACGTG sequences (27). The slower migrating complex contains FL-Mlx:HA-Wbscr14 heterodimers, as this DNA–protein complex is competed with both anti-FLAG and anti-HA mAbs (lanes 4–6). No DNA binding was detected when HA-Wbscr14 was tested alone (lanes 1–3).

Competition experiments show that the Wbscr14:Mlx dimer also recognizes the non-canonical CACGCG DNA sequences (Fig. 4A, lanes 11–18). As Mlx:Mlx dimers were shown to prefer CACGTG sequences (27,33), we can postulate that Mlx binds the CAC, and WBSCR14 the GYG half-site. The EMSA experiments confirm that WBSCR14 is able to heterodimerize with the Max-like protein, Mlx.

WBSCR14 represses transcription

Previous experiments demonstrated that Mlx has no intrinsic transcriptional properties. However , Mlx:Mad1, Mlx:Mad4 and Mlx:Mnt repress transcription, probably by recruiting mSIN3/HDAC-containing complexes, via the SIN3 interacting domain (SID)of Mad1, Mad4 and Mnt (33). The influence of WBSCR14 on transcription was studied using a luciferase reporter construct containing four reiterations of the E-box sequence upstream of the thymidine kinase (TK) minimal promoter (27,33). Expression of Wbscr14-θ alone slightly represses transcription (1.5-fold) (Fig. 4B), whereas co-transfection of Wbscr14-θ with Mlx-α enhances repression (2.5-fold) (Fig. 4C). Similar results were obtained after co-transfection with the Mlx-γ isoform (data not shown). These synergies of action suggest that the WBSCR14 and Mlx proteins are members of the same pathway, and that WBSCR14:Mlx complexes bind DNA in vivo.

To map the WBSCR14 repression domains we fused multiple portions of the Wbscr14-θ protein to the DNA binding domain of GAL4 (Fig. 5A). Fusions of Wbscr14-θ residues 1–400 and 1–178 diminish transcription from a reporter gene containing GAL4 binding sites, (2.4- and 3.0-fold decrease, respectively) (Fig. 5B). This observation suggests that the Wbscr14 repression domain is located in the N‐terminal portion of the protein, which we named PADRE (protein amino-terminal domain of repression). Interestingly, the two portions of this domain, PADRE1 and PADRE2, were conserved among the four species examined (Fig. 1). To our surprise, fusion of Wbscr14-θ residues 348–595 positively influenced transcription and the reporter was activated 7.2-fold (Fig. 5C). These results indicate that the Wbscr14-θ proline-rich region spanning residues 361–540 likely binds co-activator proteins. We named this domain MADRE (middle activation domain as in RelB) based on its weak homology with the activation domain of the RelB protein (37). Again, this functionally important domain was evolutionary conserved (Fig. 1).

WBSCR14 overexpression suppresses growth

To determine whether WBSCR14 suppresses cell growth we tested the capacity of U2OS cells to form G-418 resistant colonies when WBSCR14 was overexpressed (colony formation assay). Cells were transfected with a HA-Wbscr14-ζ or -θ expression vector or an empty plasmid. We observed a dramatic drop in the ability of U2OS cell to form colonies in the presence of both Wbscr14 isoforms (Fig. 6A). These results suggest that WBSCR14 has growth suppressive activities.

WBSCR14 expression is tightly regulated in differentiating tissue cultures

As expression of multiple members of the Myc/Max/Mad family is tightly regulated during differentiation and cell proliferation (2225,27,3845), we investigated expression of WBSCR14 as quiescent fibroblast re-entered the cell cycle and during monoblastic differentiation. Quiescent serum-starved WI-38 cells were stimulated to enter the cell cycle synchronously. Total RNA samples were collected at defined times after serum addition and the expression of WBSCR14 was monitored by northern blot analysis. Synchronization was assessed by hybridization with the S-phase-specific Cyclin A. As shown in Figure 6B, the expression of WBSCR14 persisted throughout the experiment. U-937 human leukemia cells were induced by a phorbol ester (TPA) to differentiate along the monocyte/macrophage pathway (41). WBSCR14 mRNA steady-state levels were measured by northern blot of cells treated for increasing amounts of time. Under these conditions, WBSCR14 was induced as early as 1 h after TPA treatment (Fig. 6C) and was maintained throughout the induction. Monitoring Mxi1 mRNA levels assessed correct differentiation. Together, our findings suggest that expression of WBSCR14 is under complex regulation, as previously shown for the other Mlx partners, Mad1 and Mad4 (25,44,46).

DISCUSSION

A novel Mlx partner

The Max-like protein, Mlx, was previously isolated in screens for Mad1 and Mnt interactors (27,33). It encodes a new member of the bHLHZip Myc/Max/Mad superfamily. We describe in this report the functional characterization of the bHLHZip protein WBSCR14 that forms functional heterodimers with Mlx, but not with Max, and represses the expression of E-box driven reporters. Our results are corroborated by the recent description of MondoA, a WBSCR14 paralog and novel Mlx partner (47).

WBSCR14 pattern of expression

Because WBSCR14, Mnt, Mad1 and Mad4 all dimerize with Mlx to repress transcription (27,33 and this work), it is crucial to understand both their unique functions and redundant activities. During differentiation along the monocyte/macrophage pathway, expression of Mad1, Mxi1 and WBSCR14 is induced rapidly (23,45 and this work). During C.elegans development, the expression of cWBSCR14 (ORF T20B12.6) was high in the egg and at 36 h, but extremely rare during other developmental phases, an expression pattern typical of worm transcription factors and/or rare transcripts (25,44,48). In the mouse, a surrogate for the human, we sought to identify tissues and biological settings where Wbscr14 may function. A clear example of cell compartmentalization is found in the developing CNS with a population of proliferating and differentiating cells. Mlx and Wbscr14 expression is not restricted to one cellular population, but Wbscr14 expression signal is stronger in the IZ (G. Meroni and S. Messali, unpublished data, and this work). Similarly the other Mlx-interactors, Mad1 and Mad4, are expressed in the IZ, where the N-Myc signal is low (25). These observations suggest that the expression of the new Mlx-interactor WBSCR14 is under complex regulation, as previously shown for the other Mlx partners Mad1 and Mad4 (25,44,46). In addition, Mad1, Mnt and WBSCR14 proteins are short lived, whereas Mlx is relatively stable (22,24,33,36 and this work), inferring that the function of Mlx-containing dimers is under the control of their unstable partners.

WBSCR14 maps within the region commonly deleted in WBS and may contribute to certain of its phenotypical features. The comparison of chromosomal deletions in certain unusual patients suggests that genes responsible for WBS classical facial features and mental retardation map between STX1A and GTF2I. More centromeric genes might contribute to other phenotypic aspects, in particular subtle defects in cognition, transient hypercalcemia and possibly the variety of gastrointestinal problems experienced by WBS patients (7,8,4951). In this regard, the expression of Wbscr14 in the murine developing CNS and intestinal tract and in adult human brain and intestine is intriguing. Interestingly, transcription factors are implicated in dominant developmental disorders. For example, haploinsufficiency at the MITF locus, a gene encoding a bHLHZip protein of the TFE3 subfamily, causes Waardenburg’s syndrome type II. In addition, semi-dominant or recessive mutations in the murine homologous gene are the source of the microphthalmia phenotype (52).

To understand the unique spectrum of the WBS phenotype it will be important to identify the genes for which expression is affected by the transcription factors already identified in the WBS commonly deleted region. Previous work has demonstrated that the DNA targets recognized by bHLHZip heterodimers might depend on slight differences in the basic region and/or loop sequence and length (12,53,54). In this regard, the longer loop of the Mlx protein relative to Max and the WBSCR14 unique features in the basic region amongst Myc superfamily members might probably both contribute to the targets selected by the WBSCR14:Mlx dimers. Further investigation, combining inducible expression and cDNA microarray technologies, for example, should reveal the functional targets of the WBSCR14 proteins. Colony formation assays suggest that WBSCR14 may regulate the expression of genes involved in growth control. The Myc genes have been shown to positively regulate the expression of genes important in cellular growth and metabolism (17,53,55,56). In particular, mutations in the fruitfly Myc gene delay the accumulation of cell mass and reduce cell and animal size (57,58).

The present report provides the first functional characterization of the WBSCR14 bHLHZip protein. A definitive proof of the contribution of this gene to WBS pathology awaits the identification of patients with intragenic mutations and specific manifestations of the syndrome. WBSCR14, like Mad1, Mad4 and Mnt, heterodimerizes with Mlx to form CACGTG-binding complexes and repress transcription. The presented data support the assumption that Mlx functions as a common dimerization partner of a new transcription network (27,33).

MATERIALS AND METHODS

Accession numbers, cDNA cloning and reconstruction

Overlapping clones, isolated from a murine liver library (Stratagene) and identified in dbEST (IMAGE clones 832580 and 2192619), allowed us to reconstruct the full-length murine Wbscr14 cDNA and to identify alternatively spliced forms. The Wbscr14-θ-Δbasic mutants were generated using the Quick-Change mutagenesis kit (Stratagene) and appropriate oligonucleotides.

Homology searches in dbEST revealed WBSCR14-related genes in D.melanogaster (BDGP/HHMI EST Project clones LD24192, LD27794 and SD03013) and C.elegans (Yuji Kohara, National Institute of Genetics, Japan, clones yk27f10, yk249b7, yk324h7, yk653f10, yk495h8 and yk612d4). The full-length fruitfly gene (dWBSCR14-α) was isolated from an embryo cDNA library (Clontech) and its nematode counterpart (cWBSCR14) was sequenced directly from overlapping ESTs. Two other Drosophila isoforms were sequenced (dWBSCR14-β and -γ).

The sequences of the cDNAs discussed in this report were all submitted to GenBank under the following accession numbers: WBSCR14-α, AF245470; WBSCR14-β, AF245471; WBSCR14-γ, AF245472; WBSCR14-δ, AF245473; WBSCR14-ε, AF245474; Wbscr14-ζ, AF245475; Wbscr14-η, AF245476; Wbscr14-θ, AF245477; Wbscr14-ι, AF245478; Wbscr14-κ, AF245479; dWBSCR14-α, AF264754; dWBSCR14-β, AF264755; dWBSCR14-γ, AF264756; and cWBSCR14, AF264757.

Interaction-mating, co-immunoprecipitation and antibody production

Interaction-mating and in vivo co-immunoprecipitation have previously been described (34,45,59). As judged by western blot analysis of bait and prey fusions with anti-LexA and anti-HA mAb, all fusions were expressed to similar levels. The production of R5852 and R4432 anti-Mlx polyclonal Ab was described previously (27). The anti-Max C17 and C124 antibodies were purchased from Santa Cruz Biotechnology. The first 178 residues of Wbscr14-θ fused to GST were expressed in bacteria and purified as published (34). For each antigen, two rabbits were injected intramuscularly at monthly intervals to generate O53 and P53 Wbscr14 antisera. As our antibodies do not allow identification of the endogenous Wbscr14 proteins, half-life experiments were performed as previously detailed (33). The efficacy of treatment with cycloheximide to inhibit new protein synthesis was measured by monitoring disappearance of transfected Myc protein.

EMSA

The binding reaction was achieved as described by Meroni et al. (45) using 1 ng of end-labeled probe (5′-GGAAGCAGACCACGTGGTCTGCTTCC-3′). When specified, unlabeled probe or specific competitors were added at the same time as the labeled probe. Specific sequences of competitors are described in detail (45). For supershift experiments, 1 µg of purified anti-FLAG M2 antibody (Sigma) or 1 µg of purified anti-HA 12CA5 (Boehringer Mannheim) was added to the reaction mix after protein–DNA complex formation.

Transfection and transactivation experiments

HEK293 cells were transfected by calcium phosphate and COS-7 cells were transfected with lipofectamine (Gibco BRL). For the transactivation experiments, 300 000 cells/35 mm plate were transfected with 1 µg of pTK81 4× [E-box]-luciferase reporter (45) or pTK81 5× [Gal site]-luciferase reporter, 1 µg of the expression vector(s) (pCDNA3 modified to include an HA-Tag; Invitrogen) and 100 ng of pCH110 (SV40 promoter driven β-galactosidase; Clontech) to monitor transfection efficiency. Luciferase and β-galactosidase were assayed according to the manufacturer’s instructions (Promega) 48 h post-transfection. Transactivation assays were performed in triplicate and repeated at least three times.

Colony assays

Colony assays were performed as described by Fagioli et al. (60). In summary, 5 × 105 U2OS cells were transfected with 8 µg of clones or control DNAs (HA-pCDNA3; Invitrogen) and 0.5 µg of pEGFP-C3 (Clontech) by the calcium phosphate method. After 48 h, cells were collected and split at various dilutions. Transfection efficiency was monitored by flow cytometry (FACScan; Becton Dickinson). Stable transfectants were selected for 12 days in medium containing 500 µg/ml G418 (Gibco BRL). Colonies were visualized with crystal violet staining and counted. The experiments were performed three times and the standard deviation was calculated.

Expression studies

Human multiple tissues, brain and intestine northern blots (Clontech and Invitrogen) were hybridized with partial WBSCR14-α ORF cDNA following the manufacturers’ recommendations. Sample loading was assessed with an actin probe (data not shown). Mouse embryo sections were hybridized with 35S-UTP-labeled Wbscr14 3′-UTR riboprobes as described by Rugarli et al. (61). Specimens were viewed and photographed using Hoechst epifluorescence optics combined with darkfield illumination provided by a red light source. WI-38 human embryo lung fibroblasts were cultured at early passages in Dulbecco’s modified Eagle’s medium/10% fetal calf serum (FCS) and growth was arrested for three days in media with 5% bovine calf serum. They were stimulated to re-enter the cell cycle by addition of 20% FCS. U-937 human leukemia cells were induced to differentiate by the addition of 5 nM TPA (45).

ACKNOWLEDGEMENTS

We thank S.E. Antonarakis, P. Byers, G. Meroni, V. Orlando and E. Zanaria for their helpful suggestions and/or critical reading of the manuscript. We would like to thank C. Gattuso, M. Ghiani, M. Quarto and M. Zollo for core assistance, and A. Simon and M. Smith for preparation of the manuscript. We are grateful to M. Eilers, R.N. Eisenman, Y. Kohara and T. Nagase for plasmids and reagents. This work was supported by a Fondazione Italiana per la Ricerca sul Cancro (FIRC) fellowship to G.M., and a Telethon Foundation fellowship and Swiss Cancer League grant to A.R.

+

These authors contributed equally to this work

§

Present address: Telethon Institute of Genetics and Medicine (TIGEM), Via Pietro Castellino 111, 80131 Naples, Italy

Present address: Division of Medical Genetics, University of Geneva Medical School, CMU, Geneva, Switzerland

To whom correspondence should be addressed at: Division of Medical Genetics, University of Geneva Medical School, CMU, 1 rue Michel Servet, 1211 Geneva 4, Switzerland. Tel: +41 22 702 5719; Fax: +41 22 702 5706; Email: alexandre.reymond@medecine.unige.ch

Figure 1. Opposite WBSCR14 was conserved through evolution. (A) Alignment of murine (Mm), human (Hs), fruitfly (Dm) and nematode (Ce) WBSCR14 proteins. Conserved and similar amino acids are boxed. The consensus sequence is indicated below. The PADRE and MADRE domains, which are able to repress and activate transcription, respectively, when fused to the DNA binding domain of GAL4, are indicated (see text for details). The positions of the bHLHZip domain regions and the WMC portion are shown. (B) Conservation of the different WBSCR14 domains through evolution. Amino acids positions of the different WBSCR14 domains are indicated on top. Percentage of identical and, in parentheses, similar residues are given.

Figure 1. Opposite WBSCR14 was conserved through evolution. (A) Alignment of murine (Mm), human (Hs), fruitfly (Dm) and nematode (Ce) WBSCR14 proteins. Conserved and similar amino acids are boxed. The consensus sequence is indicated below. The PADRE and MADRE domains, which are able to repress and activate transcription, respectively, when fused to the DNA binding domain of GAL4, are indicated (see text for details). The positions of the bHLHZip domain regions and the WMC portion are shown. (B) Conservation of the different WBSCR14 domains through evolution. Amino acids positions of the different WBSCR14 domains are indicated on top. Percentage of identical and, in parentheses, similar residues are given.

Figure 2.WBSCR14 expression pattern. The Wbscr14 embryonic pattern of expression was examined using in situ hybridization sagittal sections at E12.5 (A and J), E14.5 (BF) and E16.5 (GI). The white signal, indicating expression, is obvious at the level of the CNS, olfactory epithelium, heart, lung, liver, thymus, midgut (within the physiological hernia) and villi of the duodenum (A–C). High magnification fields show expression (red signal) in the olfactory epithelium (D), the wall of the heart (E), midgut (F), vibrissa follicles (G), villi of the large intestine (H) and cartilage primordia of the ribs (I). (J) Wbscr14 expression in the E12.5 murine developing spinal cord. The VZ and IZ define populations of proliferating and differentiating cells, respectively. (K) Northern blot analysis of human adult brain portions (left) and intestine sections (right) probed with WBSCR14-α cDNA. Size markers are shown on the left in kb. Arrowheads on the right indicate the position of the WBSCR14 mRNAs with their relative size. CP, cartilage primordia of the rib; IZ, intermediate zone, L, lung; LI, liver; Li, large intestine; M, mesencephalon; Mg, midgut; NC, nasal cavity; OE, olfactory epithelium; T, telencephalon; Th, thymus; V, heart ventricle; VF, vibrissa follicle; VZ, ventricular zone; W, heart wall.

Figure 2.WBSCR14 expression pattern. The Wbscr14 embryonic pattern of expression was examined using in situ hybridization sagittal sections at E12.5 (A and J), E14.5 (BF) and E16.5 (GI). The white signal, indicating expression, is obvious at the level of the CNS, olfactory epithelium, heart, lung, liver, thymus, midgut (within the physiological hernia) and villi of the duodenum (A–C). High magnification fields show expression (red signal) in the olfactory epithelium (D), the wall of the heart (E), midgut (F), vibrissa follicles (G), villi of the large intestine (H) and cartilage primordia of the ribs (I). (J) Wbscr14 expression in the E12.5 murine developing spinal cord. The VZ and IZ define populations of proliferating and differentiating cells, respectively. (K) Northern blot analysis of human adult brain portions (left) and intestine sections (right) probed with WBSCR14-α cDNA. Size markers are shown on the left in kb. Arrowheads on the right indicate the position of the WBSCR14 mRNAs with their relative size. CP, cartilage primordia of the rib; IZ, intermediate zone, L, lung; LI, liver; Li, large intestine; M, mesencephalon; Mg, midgut; NC, nasal cavity; OE, olfactory epithelium; T, telencephalon; Th, thymus; V, heart ventricle; VF, vibrissa follicle; VZ, ventricular zone; W, heart wall.

Figure 3. WBSCR14:Mlx heterodimers. (A) Interaction-mating assays between strains carrying bHLHZip family members (34). EGY42 bait strains containing plasmids that expressed LexA fusions to human Mlx-α, Mlx-β, Mlx-γ, WBSCR14-α residues 620–852, Mnt 60–582, Max1, Max2, Mad1 ΔSID, Mxi1 ΔSID, MITF and USF, and murine Mad3 ΔSID, were mated to EGY48 derivatives that contained B42 fusions to human Mlx-β and WBSCR14-α residues 620–852. Numbers (1–3) indicate the degree of β-galactosidase activity on GAL plates. No activity was detected for the interaction represented by empty squares. (B) WBSCR14 interacts with endogenous Mlx in vivo. COS-7 cells were transfected with plasmids expressing HA-Wbscr14-ζ, HA-Wbscr14-θ or HA-Wbscr14-θ Δbasic as indicated above the lanes. The cell lysates were immunoprecipitated with two different anti-Mlx antisera or a preimmune counterpart (27), two anti-Max antibodies or an anti-HA mAb as shown. The immunoblot was performed with anti-HA mAb or anti-WBSCR14 P53 immune sera as marked on top. The arrowheads on the left indicate, from top to bottom, the position of HA-Wbscr14-ζ, HA-Wbscr14-θ or HA-Wbscr14-θ Δbasic, respectively. The N-terminal region of HA-Wbscr14-θ, used to produce the Wbscr14 antigen, is not conserved in HA-Wbscr14-ζ, explaining the weakness of the signal in lane 9. (C) Wbscr14-θ has a short half-life. COS-7 cells transfected with HA-Wbscr14-θ were treated with cycloheximide, immunoprecipitated and subjected to western blot. Co-immunoprecipitated HA-Wbscr14-θ was quantitated and plotted against the cycloheximide treatment time. Standard deviations of three experiments are indicated by vertical bars.

Figure 3. WBSCR14:Mlx heterodimers. (A) Interaction-mating assays between strains carrying bHLHZip family members (34). EGY42 bait strains containing plasmids that expressed LexA fusions to human Mlx-α, Mlx-β, Mlx-γ, WBSCR14-α residues 620–852, Mnt 60–582, Max1, Max2, Mad1 ΔSID, Mxi1 ΔSID, MITF and USF, and murine Mad3 ΔSID, were mated to EGY48 derivatives that contained B42 fusions to human Mlx-β and WBSCR14-α residues 620–852. Numbers (1–3) indicate the degree of β-galactosidase activity on GAL plates. No activity was detected for the interaction represented by empty squares. (B) WBSCR14 interacts with endogenous Mlx in vivo. COS-7 cells were transfected with plasmids expressing HA-Wbscr14-ζ, HA-Wbscr14-θ or HA-Wbscr14-θ Δbasic as indicated above the lanes. The cell lysates were immunoprecipitated with two different anti-Mlx antisera or a preimmune counterpart (27), two anti-Max antibodies or an anti-HA mAb as shown. The immunoblot was performed with anti-HA mAb or anti-WBSCR14 P53 immune sera as marked on top. The arrowheads on the left indicate, from top to bottom, the position of HA-Wbscr14-ζ, HA-Wbscr14-θ or HA-Wbscr14-θ Δbasic, respectively. The N-terminal region of HA-Wbscr14-θ, used to produce the Wbscr14 antigen, is not conserved in HA-Wbscr14-ζ, explaining the weakness of the signal in lane 9. (C) Wbscr14-θ has a short half-life. COS-7 cells transfected with HA-Wbscr14-θ were treated with cycloheximide, immunoprecipitated and subjected to western blot. Co-immunoprecipitated HA-Wbscr14-θ was quantitated and plotted against the cycloheximide treatment time. Standard deviations of three experiments are indicated by vertical bars.

Figure 4. WBSCR14:Mlx dimer binds DNA and represses transcription. The ability of WBSCR14:Mlx dimers to bind DNA was tested by EMSA. Reticulocyte lysates expressing FL-Mlx-γ and/or HA-Wbscr14-θ 412–842 (A), as specified above, were mixed together with labeled 5′-GGAAGCAGACCACGTGGTCTGCTTCC-3′ DNA. The following additions were made to the reaction mixtures: 200 ng of unlabeled probe, lanes 1, 7, 8 and 12; buffer, lanes 2, 4, 10, 11 and 18; 1 µg of anti-HA mAb, lanes 3 and 5; 1 µg of anti-FLAG mAb, lanes 6 and 9; and 200 ng of the above-specified cold competitors, lanes 13–17. The black arrowheads show the position of the Mlx:Mlx–DNA (bottom) and WBSCR14:Mlx–DNA complexes (top). The gray arrow indicates the Mlx:Mlx-DNA-mAb supershifted band. (B and C) To determine the transcriptional activity of WBSCR14:Mlx heterodimers, HEK293 cells were transiently transfected with expression vector encoding the indicated proteins, together with luciferase and β-galactosidase reporters. The transitional activities of the WMC-containing bHLHZip proteins were tested singularly (B) or in combination (C).

Figure 4. WBSCR14:Mlx dimer binds DNA and represses transcription. The ability of WBSCR14:Mlx dimers to bind DNA was tested by EMSA. Reticulocyte lysates expressing FL-Mlx-γ and/or HA-Wbscr14-θ 412–842 (A), as specified above, were mixed together with labeled 5′-GGAAGCAGACCACGTGGTCTGCTTCC-3′ DNA. The following additions were made to the reaction mixtures: 200 ng of unlabeled probe, lanes 1, 7, 8 and 12; buffer, lanes 2, 4, 10, 11 and 18; 1 µg of anti-HA mAb, lanes 3 and 5; 1 µg of anti-FLAG mAb, lanes 6 and 9; and 200 ng of the above-specified cold competitors, lanes 13–17. The black arrowheads show the position of the Mlx:Mlx–DNA (bottom) and WBSCR14:Mlx–DNA complexes (top). The gray arrow indicates the Mlx:Mlx-DNA-mAb supershifted band. (B and C) To determine the transcriptional activity of WBSCR14:Mlx heterodimers, HEK293 cells were transiently transfected with expression vector encoding the indicated proteins, together with luciferase and β-galactosidase reporters. The transitional activities of the WMC-containing bHLHZip proteins were tested singularly (B) or in combination (C).

Figure 5. Fold repression (B) and activation (C) of the GAL4-Wbscr14-θ fusion proteins depicted in (A), transiently transfected in HEK293 cells.

Figure 5. Fold repression (B) and activation (C) of the GAL4-Wbscr14-θ fusion proteins depicted in (A), transiently transfected in HEK293 cells.

Figure 6. (A) Effects of Wbscr14-θ and -ζ expression on growth of U2OS cells. (B) WBSCR14 and Cyclin A mRNA steady state levels during normal quiescent fibroblast cell cycle re-entry stimulation. RNA was isolated from WI-38 cells at different times after serum addition. Twenty micrograms of total RNA was blotted successively with WBSCR14, Cyclin A and actin probes. WBSCR14 (closed squares) and Cyclin A (open squares) mRNA levels are normalized to actin mRNA. (C) WBSCR14, Mxi1 and actin mRNA steady state levels during monoblasts differentiation. RNA was isolated from U-937 cells at different times after TPA treatment. Twenty micrograms of total RNA was blotted successively with WBSCR14, Mxi1 and actin. WBSCR14 (squares) and Mxi1 (circles) mRNA levels are normalized to actin mRNA.

Figure 6. (A) Effects of Wbscr14-θ and -ζ expression on growth of U2OS cells. (B) WBSCR14 and Cyclin A mRNA steady state levels during normal quiescent fibroblast cell cycle re-entry stimulation. RNA was isolated from WI-38 cells at different times after serum addition. Twenty micrograms of total RNA was blotted successively with WBSCR14, Cyclin A and actin probes. WBSCR14 (closed squares) and Cyclin A (open squares) mRNA levels are normalized to actin mRNA. (C) WBSCR14, Mxi1 and actin mRNA steady state levels during monoblasts differentiation. RNA was isolated from U-937 cells at different times after TPA treatment. Twenty micrograms of total RNA was blotted successively with WBSCR14, Mxi1 and actin. WBSCR14 (squares) and Mxi1 (circles) mRNA levels are normalized to actin mRNA.

DDBJ/EMBL/GenBank accession nos AF245470–AF245479 and AF264754–AF264757.

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