Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors

Abstract

The molecular mechanisms by which Notch receptors induce diverse biological responses are not fully understood. We recently cloned a mammalian homologue of the Mastermind gene of Drosophila melanogaster, MAML1 (Mastermind-like-1 molecule) and determined that it functions as a transcriptional coactivator for Notch receptors. In this report, we characterize two additional genes in this Mastermind-like gene family: MAML2 and MAML3. The three MAML genes are widely expressed in adult tissues but exhibit distinct expression patterns in mouse early spinal cord development. All MAML proteins localize to nuclear bodies, share a conserved basic domain in their N termini that binds to the ankyrin repeat domain of Notch, and contain a transcriptional activation domain in their C termini. Moreover, as determined by using coimmunoprecipitation assays, each MAML protein was found to be capable of forming a multiprotein complex with the intracellular domain of each Notch receptor (ICN1 to -4) and CSL in vivo. However, MAML3 bound less efficiently to the ankyrin repeat domain of Notch1. Also, in U20S cells, whereas MAML1 and MAML2 functioned efficiently as coactivators with each of the Notch receptors to transactivate a Notch target HES1 promoter construct, MAML3 functioned more efficiently with ICN4 than with other forms of ICN. Similarly, MAML1 and MAML2 amplified Notch ligand (both Jagged2 and Delta1)-induced transcription of the HES-1 gene, whereas MAML3 displayed little effect. Thus, MAML proteins may modify Notch signaling in different cell types based on their own expression levels and differential activities and thereby contribute to the diversity of the biological effects resulting from Notch activation.

FIG. 1.

Sequence alignment and expression of the mammalian mastermind gene family. (A). Alignment of the conserved basic domains of three members of the mammalian Mastermind-like family—MAML1, MAML2, and MAML3—and Drosophila Mastermind by using CLUSTALW multiple sequence alignment software. The notations “✽”, “:”, and “.” indicate identical residues, conserved substitutions, and semiconserved substitutions in all sequences, respectively. The numbers after each sequence indicate the positions of the amino acids in each protein. (B) Northern blot analysis of MAML2 and MAML3 expression in human tissues. Lanes: 1, brain; 2, heart; 3, skeletal muscle; 4, colon; 5, thymus; 6, spleen; 7, kidney; 8, liver; 9, small intestine; 10, placenta; 11, lung; 12, peripheral blood leukocyte. A major 7.5-kb transcript was detected in these tissues for MAML2 and MAML3. (C) Expression of full-length MAML1, MAML2, and MAML3 proteins in COS7 cells. COS7 cells were transfected with vector alone (lane 1) or constructs encoding the full-length FLAG-tagged MAML1 (lane 2), MAML3 (lane 3), and MAML2 (lane 4) proteins for 44 h, and the cellular lysates were immunoblotted with anti-FLAG antibody. Numbers on the left are kilodaltons. (D). MAML2 and MAML3 are localized in nuclear dots. Transiently transfected COS7 cells expressing GFP-tagged MAML2 or MAML3 proteins (left) stained with DAPI (4′,6′-diamidino-2-phenylindole) (right) reveal the two proteins localize to the nucleus.

FIG. 2.

Formation of a ternary complex of MAML2 or MAML3, ICN1, and CSL in vivo. (A). MAML2, ICN1, and CSL form a ternary immunoprecipitate complex. COS7 cells were cotransfected with different combinations of three expression plasmids encoding FLAG-tagged MAML2, HA-tagged ICN1, and Myc-tagged CSL as indicated. Cellular lysates or anti-FLAG immunoprecipitates (IP) were immunoblotting with anti-FLAG, or anti-HA, or anti-Myc antibodies. (B) MAML3, ICN1, and CSL form a ternary immunoprecipitate complex. COS7 cells were cotransfected with different combinations of three expression plasmids encoding FLAG-tagged MAML3, HA-tagged ICN1, and Myc-tagged CSL. Analysis was performed as in panel A.

FIG. 3.

MAML1, MAML2, and MAML3 have differential binding to the ankyrin repeats of Notch1. U20S cells in a six-well plate were transfected with 25 ng of pRL-TK plasmid encoding Renilla luciferase, 0.5 μg of a firefly luciferase construct containing four GAL4-binding sites in the promoter (pG5luc), 0.5 μg of the plasmid encoding DB fused to the ankyrin repeats of Notch1, and 0.5 μg of pFLAG-CMV-2 empty vector or pFLAG-CMV-2 encoding MAML1, MAML2, or MAML3. Cells were harvested at 44 h posttransfection. Firefly luciferase activity, normalized to Renilla luciferase, was expressed as the fold activation (relative to the background level of firefly luciferase expression in the presence of an empty pFLAG-CMV-2 vector).

FIG. 4.

Like MAML1, MAML2, and MAML3 are transcriptional coactivators. U20S cells were transfected with 0.5 μg of a firefly luciferase construct containing four GAL4-binding sites in the promoter (pG5luc) and 0.5 μg of pBIND plasmid encoding either the GAL4 DB only or the DB fused to full-length MAML1, MAML2, or MAML3. Firefly luciferase activity, normalized to Renilla luciferase expressed from the pBIND plasmid, was expressed as the fold activation (relative to the background level of firefly luciferase expression in the presence of an empty pBIND vector).

FIG. 5.

MAML family members exhibit differential effects on HES-1 activation upon stimulation with ligands of Notch receptors, Jagged2, or Delta1. (A) U20S cells were transfected with 25 ng of a pRL-TK control plasmid expressing Renilla luciferase, 0.5 μg of HES-1-luc, and increasing amounts of expression plasmids encoding either MAML1, MAML2, or MAML3. At 20 h posttransfection, 10⁵ NIH 3T3 cells expressing Jagged2 or 10⁵ NIH 3T3 cells infected with empty pBABE virus as control were added to each well. Cell extracts were prepared 44 h posttransfection. HES-1 reporter firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as the fold activation relative to cells not expressing MAML family members that were cocultured with control NIH 3T3 cells. (B). U20S cells in the 60-mm plates were transfected with 75 ng of pRL-TK control plasmid encoding Renilla luciferase, 1.5 μg of HES-1-luc, and different amounts of expression plasmids encoding either MAML1, MAML2, or MAML3. At 24 h posttransfection, cells were split into four wells in the 24-well plates, two wells coated with human IgG and two wells coated with Delta ligand, and cultured for another 20 h. Cellular extracts were then prepared, and the luciferase activity was measured. HES-1 reporter firefly luciferase activity, corrected for Renilla luciferase activity, was expressed as the fold activation relative to cells transfected with control vector plasmid and cultured in the wells coated with human IgG.

FIG. 6.

MAML family members exhibit differential effects on HES-1 activation induced by ICN1 to -4. U20S cells were transfected with 25 ng of pRL-TK plasmid encoding Renilla luciferase, 0.5 μg of HES-1-luc, and increasing amounts of pFLAG-CMV-2 plasmid encoding MAML1, MAML2, or MAML3 (each at 0, 0.25, and 0.5 μg) in the presence of 0 to 50 ng of pcDNA3 plasmids encoding human ICN1, human ICN2, murine ICN3, or human ICN4. Cellular extracts were prepared 44 h posttransfection, and the luciferase activity was measured. HES-1 reporter firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as the fold activation relative to cells not expressing MAML1, MAML2, MAML3, and ICN.

FIG. 7.

MAML1, MAML2, and MAML3 have differential effects on HES-1 activation induced by the ankyrin repeats of Notch1. U20S cells were transfected with 25 ng of pRL-TK plasmid encoding Renilla luciferase, 0.5 μg of HES-1-luc, and increasing amounts of pFLAG-CMV-2 plasmid encoding MAML1, MAML2, or MAML3 in the presence of 0, 5, or 25 ng of pcDNA3 plasmid encoding the ankyrin repeats (ANK) of human ICN1. Cellular extracts were prepared 44 h posttransfection, and the luciferase activity was measured. HES-1 reporter firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as the fold activation relative to cells not expressing MAML and ANK.

FIG. 8.

MM3/1 and MM1/3 were able to form complexes with ICN1 and CSL, exhibited transcriptional activities, and showed differential binding determined by the type of the basic domain. (A) Diagram showing two chimeric proteins, MM3/1 and MM1/3, in which MAML1 and MAML3 have their basic domains swapped. (B) COS7 cells were cotransfected with different combinations of three expression plasmids encoding FLAG-tagged MAML1, MAML3, MM3/1, MM1/3, HA-tagged ICN1, and Myc-tagged CSL as indicated. Cellular lysates or anti-FLAG immunoprecipitates (IP) were immunoblotted with anti-FLAG, anti-HA, or anti-Myc antibodies. (C). U20S cells were transfected with 0.5 μg of a firefly luciferase construct containing four GAL4-binding sites in the promoter (pG5luc) and 0.5 μg of pBIND plasmid encoding either the GAL4 DNA- binding domain (DB) only or DB fused to full-length MAML1, MAML2, MAML3, MM3/1, or MM1/3. Firefly luciferase activity, normalized to Renilla luciferase expressed from the pBIND plasmid, was expressed as the fold activation (relative to the background level of firefly luciferase expression in the presence of an empty pBIND vector). (D) U20S cells were transfected with 25 ng of pRL-TK plasmid encoding Renilla luciferase, 0.5 μg of a firefly luciferase construct containing four GAL4-binding sites in the promoter (pG5luc), 0.5 μg of the plasmid encoding DB fused to the ankyrin repeats of Notch1, and 0.5 μg of pFLAG-CMV-2 empty vector or pFLAG-CMV-2 encoding MAML1, MAML2, MAML3, MM3/1, or MM1/3. Cells were harvested at 44 h posttransfection. Firefly luciferase activity, normalized to Renilla luciferase, was expressed as the fold activation (relative to the background level of firefly luciferase expression in the presence of an empty pFLAG-CMV-2 vector).

FIG. 9.

Effect of MM3/1 and MM1/3 on ANK-induced (A) or Jagged2-induced (B) HES-1 promoter activation. (A). U20S cells were transfected with 25 ng of pRL-TK plasmid encoding Renilla luciferase, 0.5 μg of HES-1-luc, and increasing amounts of pFLAG-CMV-2 plasmid encoding MAML1, MAML2, MAML3, MM3/1, or MM1/3 in the presence of 0, 5, or 25 ng of pcDNA3 plasmid encoding the ankyrin repeats (ANK) of human ICN1. Cellular extracts were prepared 44 h posttransfection, and the luciferase activity was measured. HES-1 reporter firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as the fold activation relative to cells not expressing MAM and ANK. (B). U20S cells were transfected with 25 ng of a pRL-TK control plasmid expressing Renilla luciferase, 0.5 μg of HES-1-luc, and increasing amounts of expression plasmids encoding either MAML1, MAML3, MM3/1, or MM1/3. At 24 h posttransfection, 10⁵ NIH 3T3 cells expressing Jagged2 or 10⁵ NIH 3T3 cells infected with empty pBABE virus as control were added to each well. Cell extracts were prepared 44 h posttransfection. HES-1 reporter firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as the fold activation relative to cells not expressing MAML family members that were cocultured with control NIH 3T3 cells.

FIG. 9.

Effect of MM3/1 and MM1/3 on ANK-induced (A) or Jagged2-induced (B) HES-1 promoter activation. (A). U20S cells were transfected with 25 ng of pRL-TK plasmid encoding Renilla luciferase, 0.5 μg of HES-1-luc, and increasing amounts of pFLAG-CMV-2 plasmid encoding MAML1, MAML2, MAML3, MM3/1, or MM1/3 in the presence of 0, 5, or 25 ng of pcDNA3 plasmid encoding the ankyrin repeats (ANK) of human ICN1. Cellular extracts were prepared 44 h posttransfection, and the luciferase activity was measured. HES-1 reporter firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as the fold activation relative to cells not expressing MAM and ANK. (B). U20S cells were transfected with 25 ng of a pRL-TK control plasmid expressing Renilla luciferase, 0.5 μg of HES-1-luc, and increasing amounts of expression plasmids encoding either MAML1, MAML3, MM3/1, or MM1/3. At 24 h posttransfection, 10⁵ NIH 3T3 cells expressing Jagged2 or 10⁵ NIH 3T3 cells infected with empty pBABE virus as control were added to each well. Cell extracts were prepared 44 h posttransfection. HES-1 reporter firefly luciferase activity, corrected for Renilla luciferase activity, is expressed as the fold activation relative to cells not expressing MAML family members that were cocultured with control NIH 3T3 cells.

FIG. 10.

Maml genes, Hes1 and Notch1 expression in early developing mouse spinal cord. In situ hybridization of cross sections through the cervical spinal cord from E9.5 and E11.5 Swiss Webster mouse embryos was performed with mouse Maml1, human MAML2 and MAML3, and mouse Hes1 and Notch1 mRNA antisense probes and sense probes (not shown). Maml1 and Hes1 are expressed in the dorsal spinal cord at E9.5, whereas Maml2 and Notch1 are expressed in the ventral spinal cord. Hes1 expression also was observed in the floor plate at this age. At E11.5, ventral expression of Maml2 is maintained, whereas Maml1 and Notch1 expression is evenly distributed in the VZ in the spinal cord. At this stage, Hes1 is strongly expressed in both dorsal and ventral VZ but not in the medial region. Maml3 expression was not detected in either E9.5 or E11.5 spinal cords (not shown).