Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.
The Ca2+ sensor protein calmodulin can interact with the DNA binding basic helix-loop-helix (bHLH) domain of E12, E47, and SEF2-1 (E2-2), which belong to the E-protein subclass of bHLH transcription factors. This interaction inhibits the DNA binding of these bHLH proteins in vitro, and an ionophore that increases intracellular Ca2+ can inhibit transcriptional activation by the E-proteins. Here we have attempted to determine if these phenomena reflect a direct calmodulin-dependent inhibition of DNA binding by E-proteins in vivo. We show that calmodulin overexpression inhibits the transcriptional activity of E12, E47, and SEF2-1. We have compared calmodulin effects on DNA binding in vitro and on activation of transcription in vivo using a series of E12 mutants harboring defined alterations within the basic sequence of the bHLH domain that reduce their ability to bind calmodulin to varying degrees. We find a striking direct correlation between the ability of calmodulin to inhibit their DNA binding in vitro and the ability of overexpressed calmodulin or cellular Ca2+ mobilization to inhibit their transcriptional activity in vivo. Furthermore, E12 and overexpressed calmodulin were co-localized in the nucleus, and calmodulin pull-down experiments with cell extracts showed a Ca2+-dependent interaction between calmodulin and E12 but not with a calmodulin inhibition-deficient E12 mutant. Chromatin immunoprecipitation showed that calmodulin overexpression leads to decreased binding of E12 and E47, but not a calmodulin inhibition-deficient E12 mutant, to the DNA recognition sequence in vivo. The data suggest that Ca2+ signaling can inhibit the transcriptional activities of E-proteins through direct binding of Ca2+/calmodulin to the basic sequence of E-proteins, resulting in inhibition of their DNA binding.
Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden.
Juha
J
Saarikettu
Natalia
N
Sveshnikova
Thomas
T
Grundström
D000595:Amino Acid Sequence; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012685:Sepharose / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051820:TCF Transcription Factors; D019284:Thapsigargin / Q000494:pharmacology; D000073940:Transcription Factor 4; D058125:Transcription Factor 7-Like 1 Protein; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002147:Calmodulin; D002843:Chromatin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D051820:TCF Transcription Factors; C532096:TCF4 protein, human; C544912:TCF7L1 protein, human; C544894:TCF7L2 protein, human; C090743:Tcf4 protein, mouse; C544913:Tcf7l1 protein, mouse; C544895:Tcf7l2 protein, mouse; D000073940:Transcription Factor 4; D058125:Transcription Factor 7-Like 1 Protein; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D019284:Thapsigargin; D004247:DNA; D012685:Sepharose; D002118:Calcium