Study of expression analysis of SIRT4 and the coordinate regulation of bovine adipocyte differentiation by SIRT4 and its transcription factors

Abstract

Sirtuins, NAD⁺-dependent deacylases and ADP-ribosyltransferases, are critical regulators of metabolism involved in many biological processes, and are involved in mediating adaptive responses to the cellular environment. SIRT4 is a mitochondrial sirtuin and has been shown to play a critical role in maintaining insulin secretion and glucose homeostasis. As a regulator of lipid homeostasis, SIRT4 can repress fatty acid oxidation and promote lipid anabolism in nutrient-replete conditions. Using real-time quantitative PCR (qPCR) to explore the molecular mechanisms of transcriptional regulation of bovine SIRT4 during adipocyte differentiation, we found that bovine SIRT4 is expressed at high levels in bovine subcutaneous adipose tissue. SIRT4 knockdown led to decreased expression of adipogenic differentiation marker genes during adipocyte differentiation. The core promoter of bovine SIRT4 was identified in the -402/-60 bp region of the cloned 2-kb fragment containing the 5'-regulatory region. Binding sites were identified in this region for E2F transcription factor-1 (E2F1), CCAAT/enhancer-binding protein β (CEBPβ), homeobox A5 (HOXA5), interferon regulatory factor 4 (IRF4), paired box 4 (PAX4), and cAMP responsive element-binding protein 1 (CREB1) by using Electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay. We also found that E2F1, CEBPβ, and HOXA5 transcriptionally activate SIRT4 expression, whereas, IRF4, PAX4, and CREB1 transcriptionally repress SIRT4 expression. We further verified that SIRT4 knockdown could affect the ability of these transcription factors (TFs) to regulate the differentiation of bovine adipocytes. In conclusion, our results shed light on the mechanisms underlying the transcriptional regulation of SIRT4 expression in bovine adipocytes.

Keywords: SIRT4; adipocyte; bovine; promoter region; transcription factor; transcription regulation.

Figure 1. Gene expression pattern and subcellular localization of SIRT4

(A) Analysis of the bovine SIRT4 spatial expression pattern in tissues and organs. (B) Relative mRNA expression levels of SIRT4 during different stages of bovine adipocyte differentiation. (C) During adipocyte differentiation, the expression of marker genes that promote bovine adipocyte differentiation was significantly inhibited after SIRT4 knockdown. (D) SIRT4 immunofluorescence (red) in bovine adipocytes; nuclei were visualized by DAPI staining (blue). A merged figure is shown in the right panel. The top left photograph provides magnified images as indicated by the arrows. Values are represented as mean ± S.D. * indicates significance at P<0.05 and ** indicates significance at P<0.01 compared with the control group. Error bars represent the S.D.

Figure 2. Isolation and sequence analysis of the core promoter of SIRT4

(A,B) Depiction and reporter assay results for the series of plasmids containing 5′ unidirectional deletions in the promoter region of SIRT4 fused in-frame to the luciferase gene. These plasmids were transfected into 3T3-L1 or bovine adipocytes. (C) Schematic representation of the relative loci of the potential motif binding sites in the SIRT4 promoter. Dashed lines indicate the GC percentage as represented by the y-axis; the x-axis denotes the bp position in the 5′ UTR. (D) Sequence of the 5′ regulatory region of bovine SIRT4. Arrows mark the transcription initiation sites. The translational start site is shown in red letters. The TF binding sites are boxed and primer sequences are underlined with respective names shown below the line. * indicates significance at P<0.05 and ** indicates significance at P<0.01 compared with the control group. Error bars represent the S.D.

Figure 3. Identification of the E2F1, CEBPβ, HOXA5, IRF4, PAX4, and CREB1 as transcriptional activators or repressors in the core promoter region of SIRT4

(A) Luciferase assays after transfection of constructs generated by site-directed mutagenesis of TF binding sites. White and black fill in sequence depictions represent wild and mutant types, respectively. (B) Knockdown efficiency of siE2F1, siCEBPβ, siHOXA5, siIRF4, siPAX4, and siCREB1. (C) Relative SIRT4 mRNA expression in cells transfected with siRNAs targetting TFs. (D) Luciferase reporter assays after E2F1, CEBPβ, HOXA5, IRF4, PAX4, and CREB1 knockdown by siRNA and co-transfection with pGL−402/+44 and pGL−160/+44 in bovine adipocytes. si-NC was used as a negative control. * indicates significance at P<0.05 compared with the control group. Error bars represent the S.D.

Figure 4. Identification of TFs binding to the core promoter of SIRT4 using EMSAs

EMSAs showing direct binding of E2F1, CEBPβ, HOXA5, IRF4, PAX4, and CREB1 to the SIRT4 promoter in vitro; nuclear protein extracts were incubated with 5′-biotin labeled probes for (A) E2F1, (B) CEBPβ, (C) HOXA5, (D) IRF4, (E) PAX4, or (F) CREB1 binding sites in the presence or absence of competitor (lane 2), a 50× concentration of mutated probe (lane 3), and a 50× concentration of unlabeled probes (lane 4). The super-shift assay was conducted using 10 μg of anti-E2F1, anti-CEBPβ, anti-HOXA5, anti-IRF4, anti-PAX4, or anti-CREB1 antibodies (lane 5). The arrows indicate the main complexes. Abbreviation: adipocyte NE, adipocyte nuclear protein extract.

Figure 5. TFs and SIRT4 cooperatively regulated the relative expression of adipocyte differentiation marker genes

(A) The relative expression levels of marker genes in undifferentiated and differentiated stages of bovine adipocytes. (B) The differences in expression of TFs in undifferentiated and differentiated bovine adipocytes. (C–H) E2F1, CEBPβ, and HOXA5 knockdown significantly down-regulated the expressions of marker genes that promote adipocyte differentiation; while the ablation of IRF4, PAX4, and CREB1 significantly up-regulated the expressions of marker genes. E2F1, CEBPβ, HOXA5, and SIRT4 had the same regulatory effect on the expression of adipogenic marker genes, and SIRT4 coordinated with E2F1, CEBPβ, and HOXA5 in promoting adipocyte differentiation. On the contrary, the regulation of IRF4, PAX4, and CREB1 on the expression of adipogenic marker genes was opposite to the effect of SIRT4, and SIRT4 was able to counteract the negative regulation of IRF4, PAX4, and CREB1 on the adipocyte differentiation.

Figure 6. Proposed schematic summary of the regulation of differentiation of bovine adipocytes by SIRT4 and its TFs

In short, SIRT4 promotes the differentiation of bovine adipocytes, E2F1, CEBPβ, and HOXA5 increase the SIRT4 promoter activity, while IRF4, PAX4, and CREB1 decrease the SIRT4 promoter activity.