Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
Identification of the C/EBPα C-terminal tail residues involved in the protein interaction with GABP and their potency in myeloid differentiation of K562 cells
Introduction
The CCAAT/enhancer-binding protein α (C/EBPα) is the first transcription factor described to have a basic leucine zipper (bZIP) domain, whose characteristic is shared by several families of transcription factors [1]. At the C-terminal region of the C/EBPα molecule, the bZIP domain forms a DNA-binding domain (DBD), consisting of a basic amino-acid-rich DNA-binding region [basic region (BR)] followed by a heptad repeat of leucine residues [leucine zipper (LZ)]. The LZ forms a domain mediating homo- and hetero-dimerization with other members of the C/EBP family (C/EBPβ, C/EBPγ, C/EBPδ, C/EBPε, and CHOP) [2], which is a prerequisite to DNA binding via its adjacent BR [3]. On the other hand, early studies identified domains responsible for transcriptional activation and/or repression at the N-terminus [4], [5].
C/EBPα is expressed in a number of different tissues, and regulates the proliferation and differentiation of various cell types. In human hematopoietic cells, it is expressed in myeloid progenitors and granulocytes but not monocytes, and regulates a number of myeloid-specific genes, including FCAR encoding the Fc receptor for IgA (FcαR, CD89). C/EBPα levels increase during granulocytic but not monocytic differentiation [6], and its critical role in granulopoiesis has been demonstrated by many studies: Disruption of the Cebpa gene encoding C/EBPα in mice results in an absence of granulocytes without affecting monocytes [7], and conversely, enforced expression of C/EBPα in bipotential myeloid cell lines such as U937 and K562 cells induces granulocytic differentiation and inhibits monocytic differentiation [8], [9]. Consistent with its role in granulopoiesis, mutation in CEBPA and/or loss of C/EBPα expression have been implicated in the pathogenesis of acute myeloid leukemia (AML), a disease characterized by an early block in granulocytic differentiation [10], [11], [12], [13]. C/EBPα also has an anti-proliferation activity, which appears to be associated with its ability to promote differentiation [14].
C/EBPα plays a necessary role in the regulation of the myeloid-specific FCAR promoter by cooperating with GABP, a widely expressed ETS family member [15], [16]. GABP is the only known multimeric ETS family member, which consists of GABPα and GABPβ [17], [18]. GABPα includes an ETS DBD, and GABPβ contains ankyrin repeats and the transactivation domain (TAD). We previously demonstrated that the C/EBPα-GABP cooperativeness is derived by a physical interaction between the DBDs of C/EBPα and GABPα and the two composite DNA-binding sites in the FCAR promoter [15]. In addition, chromatin immunoprecipitation (ChIP) analysis using a GABPα mutant defective in the interaction with C/EBPα showed that one of the mechanisms underlying transcriptional synergy mediated by the C/EBPα-GABPα interaction is to facilitate the recruitment of C/EBPα to the FCAR promoter [19].
Different regions in the C/EBPα molecule have been identified as domains important for protein–protein interactions that lead to the initiation of transcription and/or potentially mediate its biological functions. Early studies identified TADs in the N terminus that bind TBP and TFIIB (basal transcription initiation factors) [20]. In relation to anti-proliferation activity of C/EBPα, each N-terminal TAD has been demonstrated to interact with different binding partners, including the SWI/SNF complexes (chromatin remodeling complexes) [21], Rb (tumor suppressor protein) [22], Cdk2 and Cdk4 (cyclin-dependent kinases), E2F (transcription regulator of cell cycle genes) [23], and p21 (cyclin-dependent kinase inhibitor) [24]. The C/EBPα C-terminal region including the bZIP region has also been reported to interact with cell cycle regulators and transcription factors including GABPα [19]. However, the precise sites of the C-terminus making most of these contacts remain to be defined to relate them to biological effects of C/EBPα.
In this study, we have identified GABPα-interacting sites within the last 18 amino acid residues of the C/EBPα C-terminus that is localized further downstream of the bZIP DNA-binding and dimerization region, and demonstrated that its C-terminal deletion mutant incapable of interacting with GABPα is also defective in C/EBPα-dependent granulopoiesis as well as growth arrest of K562 cells. Previous studies identified two critical regions in the C/EBPα molecule for granulopoiesis: the N-terminal part of the TAD and residues residing on the none-DNA binding face of the BR [25], [26], [27]. Our results implicate the C/EBPα C-terminal end as an additional critical region.
Section snippets
Plasmids
The human C/EBPα expression vector pD3C/EBPαKoz contains the CEBPA gene from hCMV-C/EBPα [28] with the perfect Kozak sequence (CCACCATGG) [29] in pcDNA3.1(+) (Invitrogen) [15]. The bZIP358 expression vector (previous pD3C/EBPα273–358 [15]) contains the C/EBPα C-terminal bZIP domain (amino acids 273–358) with the perfect Kozak sequence (CCACCATGG) in pcDNA3.1(+). C/EBPα bZIP C-terminal and internal deletion mutant plasmids were constructed by replacing the C/EBPα bZIP C-terminal region (the BlpI-
Residues 341–358 at the C-terminal end of C/EBPα are required for binding to the GABPα ETS domain but not to DNA
The C/EBPα molecule contains TADs at the N-terminus and a bZIP structure at the C terminus. We previously demonstrated that the C-terminal region including the bZIP domain (amino acids 273–358) interacts with the GABPα ETS domain (amino acids 318–399) [19]. To define the precise sites of C/EBPα allowing the recruitment of the GABPα ETS domain, we prepared a set of differentially C-terminus-truncated polypeptides of the C/EBPα bZIP region and a bZIP fragment lacking the BR (Fig. S1A), and
Discussion
In this study, we have defined the GABPα-interacting region of C/EBPα and its possible functions. We have demonstrated that the C-terminus of C/EBPα is required to interact with GABPα but not to bind to DNA, which thereby localizes it further downstream of the bZIP DNA-binding and dimerization region. This C-terminus is also required to synergize with GABP to activate the FCAR promoter, and can also confer the ability to synergize with GABP on a heterologous TAD when fused to the C terminus of
Acknowledgments
We thank Dr. Gretchen J. Darlington (Bayer College of Medicine, Houston, TX) for the C/EBPα expression vector. This work was supported in part by a grant for the High-Tech Research Center Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology, Japan (grant number: S0801033), grants from JSPS (KAKENHI, grant numbers 15390163 and 25461462), and a grant from Nihon University School of Medicine.
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Cited by (0)
- 1
Present address: Division of Anatomical Science, Department of Functional Morphology, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan.
- 2
Present address: Allergy and Immunology Group, Research Institute of Medical Science, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan.
- 3
Present address: Research Institute of Medical Science, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan.