Structure and Function of the Transcription Factor CP2c Complexes on Murine Definitive Erythropoiesis

Abstract

In cells, gene expression is regulated by transcription factors (TFs) that recognize short DNA sequence motifs. DNA binding activity of TFs can be modulated by formation of homo- or hetero-meric complexes, or by post-translational modification, such as phosphorylation. Therefore, identifying DNA binding regions and homo- or hetero-oligomerization states can establish the basis for defining biological regulatory pathways. The purpose of this study was 1) to establish the models in which the transcription factor CP2c forms the different complexes with partner proteins and binds to the target DNA, and 2) to defined a mechanism by which the CP2c complexes cross talk with the Mbd2-NuRD chromatin remodeling complex containing p66α, one of the partner proteins, to regulate the globin expression and the erythropoiesis. Many methods for detecting DNA–protein interactions, such as chromatin immunoprecipitation (ChIP), DNA electrophoretic mobility shift assay (EMSA), DNA pull-down, and microplate capture and detection, have been developed to determine the proteins present in DNA–protein complexes. Each of these methods has a unique history and utility as well as distinct strengths and weaknesses. In this study, we established an in vitro assay based on DNA immunoprecipitation (DIP) method for the specific and sensitive detection of DNA-protein complex interactions. We verified the accuracy and efficiency of the DIP analysis in quantitatively measuring DNA–protein binding using transcription factor CP2c as a model, and showed the newly established method was very useful in quantitatively detecting the binding dynamics of DNA–protein complex. Transcription factor CP2c (also known as TFCP2, CP2, α-CP2, LSF, and LBP-1c), is involved in many biological events, including hematopoiesis, immune response, the cell cycle and neural development, by regulating expression of specific target genes. Classically, transcription factor CP2c binds primarily to the CNRG-N(5-6)-CNRG DNA motif present in diverse cellular and viral promoters in the form of homotetramer, thereby activating or repressing transcriptional activity of the target gene. In addition to homotetrameric interactions, CP2c interacts with various partner proteins to generate novel DNA-binding complexes. In previous study, we also showed that CP2c interacts with CP2b and Pias1 in the erythroid cell line to form a ternary heterohexamer (CBP complex) and bind to the α-globin promoter DNA. Since different CP2c complexes activate various genes in cells, the differential binding specificity of the complexes was exemplified as one mechanism that contributes to this selectivity. First, we analyzed the correlation between CP2c complexes-forming ability and DNA binding ability using a series of point mutants for amino acids in the CP2c functional domain and CP2c binding peptide identified in the previous study. As a result, the four specific amino acids in the tetramerization domain of CP2c were important not only for the CP2c complexes formation, but also for the CP2c-DNA binding activity, whereas the nine specific amino acids in the DNA binding domain of CP2c did not affect CP2c complex formation and directly inhibited CP2c-DNA binding activity. In addition, we investigated the binding affinity of CP2c complexes using DNA probes containing various pattern of the CP2c binding sites in order to analyze the parameters of DNA binding sites in different CP2c complexes. As a result, the CBP heterohexamer bind to the sequences containing two or more consecutive or overlapping CP2c binding motifs were able to efficiently, and sequences containing only one CP2c binding motif were found to be the preferred binding sites for the CP2c homotetramer. Based on these results, we defined the parameters of the target DNA binding site for different CP2c complexes and suggest that they require various patterns of CP2c binding motifs. There are many erythropoiesis regulators, and at each erythropoiesis stage, intracellular signal transduction proteins and pioneer TFs interact with DNA-binding proteins, other TFs, chromatin modifiers, and multiple noncoding regulatory RNA. So far, the regulation of erythropoiesis and globin expression has been intensively studied, but the underlying mechanisms are not fully understood. We investigated cross talk between CP2 and its partner protein (p66α) in order to analyze the mechanisms of regulation of erythropoiesis and globin expression mediated by the CP2c transcription factor complex. The CP2c partner protein, p66a, identified by the yeast two-hybrid method, is a component of the Mbd2-NuRD chromatin remodeling complex, which regulates expression of the target gene through the coiled-coil interaction with the Mbd2, another component of the Mbd2-NuRD complex. Especially, the NuRD complex mediates activating and repressive functions of Gata1 complexed with Fog1 during hematopoiesis. However, the details of such interactions are unknown. In this work, we show that an axis of Gata1/Fog1, Mbd2-NuRD and CP2c complexes at the adjacent Gata1 and CP2c binding sites of erythroid related genes charged for globin gene expression and definitive erythropoiesis. In uninduced MEL cells, the Mbd2-containing NuRD complex, which is recruited to the globin locus by Gata1/Fog1, directly interacts with the CP2c family via the Mbd2-associated p66α to repress the transcriptional activity of CP2c leading to the suppression of the globin gene expression. In differentiation induced MEL cells, where Mbd2 is down regulated, the Mbd2-free NuRD complex does not dissociate from the chromatin and, rather, recruits the CP2c complexes to the chromatin, promotes transcription of active globin and erythroid related genes, and evokes active hemoglobin synthesis and terminal erythroid differentiation. Therefore, we suggest that the Mbd2-CP2c loop exerts a crucial role in the fate of proerythroblasts by altering the function of coactivator in corepressor by down-regulation of Mbd2 during erythroid differentiation. Taken together, this study showed that the selective DNA-binding specificity of the different CP2c contained complexes depends on a variety of CP2c binding site patterns, and suggest that a new mechanism of cross talk of CP2c complexes and Mbd2-NuRD complexes for erythropoiesis and globin gene expression.