C/EBP-beta drives expression of the nutritionally regulated promoter IA of the acetyl-CoA carboxylase-alpha gene in cattle

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Abstract

Acetyl-CoA carboxylase-alpha (ACC-α) is the rate-limiting enzyme for de novo fatty acid synthesis. Among the four promoters expressing the bovine gene, promoter IA (PIA) is dominantly active and nutritionally regulated in lipogenic tissues. CCAAT/enhancer binding proteins are crucially involved in regulating the activity of this promoter. We examine here, which member of this family of transcription factors is most important for promoter activation. To differentiate the individual contribution of different members of the C/EBP family transcription factors controlling the ACC-α gene expression in cattle, we established vectors expressing full length (FL) or N-terminally truncated (ΔN) variants of the C/EBP factors (α, -β, -δ, and -ε) in mammalian cells. Using nuclear extracts of cells expressing the ΔN-C/EBP factors we determined in electrophoretic mobility shift assays that C/EBPα, -β and -ε, but not C/EBPδ may directly bind to the cognate C/EBP-binding site in the immediate ACC-α PIA. Co-transfection analyses of the various FL-C/EBP expression vectors together with a reporter gene driven by the ACC-α-PIA promoter demonstrated that C/EBPβ has the strongest activation potential. Hence, activity of this factor may be a key regulator of ACC-α-expression in lipogenic tissues.

Introduction

Acetyl-CoA carboxylase-alpha (ACC-α, EC.6.4.1.2) catalyzes the conversion of acetyl-CoA into malonyl-CoA representing the basic unit for fatty acid synthesis [1], [2]. Activity of this enzyme is rate-limiting in fatty acid synthesis [2]. Expression of the mammalian ACC-α-encoding gene is controlled by several promoters of varying tissue specificity [3]. The bovine gene was previously known to be expressed by three promoters [4], [5], [6]. However, a fourth promoter located more upstream was recently identified (Shi, unpublished) representing an orthologue to the previously published fourth ACC-α expressing promoter in sheep [7]. Considering their high degree of DNA-sequence conservation between cattle and sheep, we also name these four promoters from cattle as PI, PIA, PII, and PIII, similar to designations in sheep [3]. Among them, PIA (previously known as PI [5]) is actively expressed and nutritionally regulated in lipogenic tissues [5], [8]. We have identified recently two C/EBP-binding sites involved in regulating PIA expression. While the distal site (at − 183/−175) functions as a repressor the proximal site (− 6/+3) was found to drive PIA activity (Shi, unpublished). Hence, C/EBP factors play an important role in regulating ACC-α expression.

C/EBP factors bind to DNA-sequence motifs known as CCAAT-boxes. These are found in the promoters of many genes involved in adipogenesis and inflammation [9], [10], [11], [12]. The family of C/EBP factors comprises six members (α, β, δ, γ, ε, and ζ). They all are leucine zipper transcription factors [13], eventually dimerizing via the interaction of their hydrophobic surfaces, and bind to DNA via a basic domain. The factors C/EBPα, -β, -δ and -ε, each contains a transactivation domain at the N-terminus, which is absent in both other factors [13]. C/EBP factors are sequentially expressed during development. The members C/EBPβ and -δ are expressed early during adipocyte differentiation and are eventually replaced by C/EBPα [9], [14], [15]. It is known that C/EBPα and -β are important regulators of ACC-α gene expression in rat [16], [17]. However, it remained unknown to date which member of C/EBP family transcription factors is involved in regulating bovine ACC-α gene expression.

It is difficult to experimentally discriminate between the different bovine C/EBP factors. While factor-specific antibodies are available for human and mouse, those antibodies cross reacting with the factors from Farm Animal species are all directed against some highly conserved epitopes commonly found in all C/EBP factors. Hence, we choose as an alternative to using antibodies for discrimination between the different factors to separately analyze the effect of the individually over-expressed factor on the regulation of the target promoter. The genomic sequences of the C/EBPα, -β and -δ-encoding genes from cattle have been established [18], [19], [20] and the mRNA sequence encoding the -ε factor was deduced in silico (Acc. No. XM_614092.1). Hence, we cloned full length (FL) versions of all those C/EBP factors featuring transactivation domains into mammalian expression vectors. In addition, we also established N-terminally truncated variants of these factors keeping the DNA-binding and zipper dimerizing domains (ΔN-C/EBP factors). The molecular weights of those truncated factors differ significantly from the respective native, full length wild type factors. Hence, the ΔN-factors do not only allow to unambiguously validate DNA binding of the target factors in EMSA analyses but may also be used to eventually functionally dissect C/EBP factor mediated gene regulatory mechanisms in cattle.

We applied those constructs to discriminate the roles of different C/EBP factors for the activation of the nutritionally regulated PIA of the bovine ACC-α. We show that C/EBPβ is a main activator of PIA and the regulation of ACC-α.

Section snippets

Construction of expression vectors

We used PCR techniques to clone vectors expressing the full length of the coding sequences (FL-vectors) as well as N-terminally truncated version of those factors (ΔN-vectors). All PCR reactions were done with a GC-rich PCR amplification system (Roche, Basel, Switzerland). All primers used are listed in Table 1.

Abrogation of the C/EBP-binding site attenuates the PIA promoter activity

PIA is the dominantly expressed and nutritionally regulated promoter of ACC-α in ruminants and rodents [5], [24], [25]. Sequence comparison indicates that more distal parts of this promoter are divergent between ruminant and rodent [5]. However, the proximal PIA promoter is conserved (Fig. 1A). Particularly conserved are binding sites for C/EBP factors, the carbohydrate response element (ChORE) and for the upstream stimulatory factor (USF). These factors are all known as important regulators

Acknowledgements

We are grateful to Angelika Deike and Bärbel Pletz for technical assistance and Dr. Wolfgang Tomek for help with the ECL detection system. X.S. and S.L. contributed equally to this work and the results are integral part of either of their doctoral thesis. This work is supported by the Deutsche Forschungsgemeinschaft through grant Se 326/13.

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