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. 2010 Sep;140(9):1546-51.
doi: 10.3945/jn.110.126359. Epub 2010 Jun 30.

Biotin regulates the expression of holocarboxylase synthetase in the miR-539 pathway in HEK-293 cells

Baolong Bao  1 , Rocio Rodriguez-Melendez, Subhashinee S K Wijeratne, Janos Zempleni
Affiliations

Biotin regulates the expression of holocarboxylase synthetase in the miR-539 pathway in HEK-293 cells

Baolong Bao et al. J Nutr. 2010 Sep.

Abstract

Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to carboxylases and histones. In mammals, the expression of HCS depends on biotin, but the mechanism of regulation is unknown. Here we tested the hypothesis that microRNA (miR) plays a role in the regulation of the HCS gene. Human embryonic kidney cells were used as the primary model, but cell lines from other tissues and primary human cells were also tested. In silico searches revealed an evolutionary conserved binding site for miR-539 in the 3 prime -untranslated region (3 prime -UTR) of HCS mRNA. Transgenic cells and reporter gene constructs were used to demonstrate that miR-539 decreases the expression of HCS at the level of transcription rather than translation; these findings were corroborated in nontransgenic cells. When miR-539 was overexpressed in transgenic cells, the abundance of both HCS and biotinylated histones decreased. The abundance of miR-539 was tissue dependent: fibroblasts gt kidney cells gt intestinal cells gt lymphoid cells. Dose-response studies revealed that the abundance of miR-539 was significantly higher at physiological concentrations of biotin than both biotin-deficient and biotin-supplemented media in all cell lines tested. In kidney cells, the expression of HCS was lower in cells in physiological medium than in deficient and supplemented medium. In contrast, in fibroblasts, lymphoid cells, and intestinal cells, there was no apparent link between miR-539 abundance and HCS expression, suggesting that factors other than miR-539 also contribute to the regulation of HCS expression in some tissues. Collectively, the results of this study suggest that miR-539 is among the factors sensing biotin and regulating HCS.

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Conflict of interest statement

Author disclosures: B. Bao, R. Rodriguez-Melendez, S. S. K. Wijeratne and J. Zempleni, no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
miR-539 represses HCS expression by binding to the HCS 3′-UTR in HEK-293 cells. (A) Cells were stably transformed with Ecd-mir539-CMV-Luc-HCS3UTR and pVgRXR, and miR-539 was induced with PonA for 48 h. (B) Cells from panel A were used to quantify miR-539 by stem-loop qRT-PCR. (C) Same as panel B, but miR-539 was visualized by northern blot analysis. (D) Same as panel B, but luciferase mRNA was quantified by qRT-PCR. (E) Same as panel B, but luciferase protein was visualized by western blot analysis; GAPDH served as loading control. (F) Same as panel B, but the abundance of endogenous HCS mRNA was quantified by qRT-PCR. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.
FIGURE 2
FIGURE 2
The 3′UTR of HCS mRNA contains a miR-593 binding site. (A) Sequence alignment between miR-593 and its putative binding site in the 3′UTR of human HCS mRNA and other species. The box denotes the most highly conserved region. (B) Schematic presentation of plasmids CMV-MCS1-CMV-Luci-539T (upper panel) and CMV-miR539-CMV-Luci-539T (lower panel). HEK-293 cells were stably transformed with CMV-MCS1-CMV-Luci-539T or CMV-miR539-CMV-Luci-539T, and the abundance of miR-539 (C), luciferase mRNA (D), and luciferase activity (E) were quantified by stem-loop qRT-PCR, qRT-PCR, and chemiluminescence, respectively. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.
FIGURE 3
FIGURE 3
Overexpression of miR-539 in HEK-293 cells causes low HCS expression and low abundance of biotinylated histones. (A) Schematic presentation of plasmids CMV-MCS1-CMV-GFP (upper panel) and CMV-miR539-CMV-GFP (lower panel). HEK-293 cells were stably transformed with CMV-MCS1-CMV-GFP or CMV-miR539-CMV-GFP, and the abundance of miR-539 (B), HCS mRNA (panel C), and biotinylated histone H4 (D, upper gel) were quantified. In Western blots, equal loading was confirmed by using anti-H3 (D, lower gel). Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.
FIGURE 4
FIGURE 4
The abundance of miR-539 depends on tissue (A) and biotin concentration (B–F) in IMR-90 fibroblasts, HEK-293 cells, Caco-2 cells, and Jurkat cells. Cells were cultured in biotin-defined media and miR-539 was quantified in IMR-90 fibroblasts (B), HEK-293 (C), Jurkat cells (D), and Caco-2 cells (E); in control experiments, HEK-293 cells were stably transformed with plasmid miR539Pro-GFP and GFP mRNA was quantified (F). Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.
FIGURE 5
FIGURE 5
The expression of HCS depends on biotin concentration in HEK-293 cells, Jurkat cells, and Caco-2 cells. Cells were cultured in biotin-defined media and HCS mRNA was quantified in HEK-293 cells (A), IMR-90 fibroblasts (B), Jurkat cells (C), and Caco-2 cells (D). Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.
FIGURE 6
FIGURE 6
The abundance of miR-539 depends on HCS in HEK-293. Cells were stably transformed with plasmid HCS-GFP; wild-type HEK-293 cells were used as controls. HCS mRNA (A) and miR-539 mRNA (B) were quantified by qRT-PCR. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.
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