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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The protein biotin ligase, holocarboxylase synthetase (HLCS), is a chromatin protein that interacts physically with the DNA methyltransferase DNMT1, the methylated cytosine-binding protein MeCP2 and the histone H3 K9-methyltransferase EHMT1, all of which participate in folate-dependent gene repression. Here we tested the hypothesis that biotin and folate synergize in the repression of pro-inflammatory cytokines and long-terminal repeats (LTRs), mediated by interactions between HLCS and other chromatin proteins. Biotin and folate supplementation could compensate for each other's deficiency in the repression of LTRs in Jurkat and U937 cells. For example, when biotin-deficient Jurkat cells were supplemented with folate, the expression of LTRs decreased by >70%. Epigenetic synergies were more complex in the regulation of cytokines compared with LTRs. For example, the abundance of TNF-α was 100% greater in folate- and biotin-supplemented U937 cells compared with biotin-deficient and folate-supplemented cells. The NF-κB inhibitor curcumin abrogated the effects of folate and biotin in cytokine regulation, suggesting that transcription factor signalling adds an extra layer of complexity to the regulation of cytokine genes by epigenetic phenomena. We conclude that biotin and folate synergize in the repression of LTRs and that these interactions are probably mediated by HLCS-dependent epigenetic mechanisms. In contrast, synergies between biotin and folate in the regulation of cytokines need to be interpreted in the context of transcription factor signalling.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The roles of nutrients in immune function are undisputed, including the vitamins biotin and folate. For example, children with hereditary abnormalities of biotin metabolism developed candida dermatitis, had absent delayed-hypersensitivity skin test responses, IgA deficiency and subnormal percentages of T lymphocytes in peripheral blood [1]. In biotin-deficient rats, the synthesis of antibodies is reduced [2]. Biotin deficiency in mice decreases the number of spleen cells and the percentage of B lymphocytes in spleen [3], inhibits thymocyte maturation [4] and increases the production of pro-inflammatory cytokines [5]. Likewise, severe folate deficiency inhibits the proliferation of primary human CD8+ T lymphocytes in vitro, may cause atopy and impairs natural killer cell-mediated cytotoxicity in rats [6-8]. However, evidence also suggests that an intake of more than 400 μg/day folate may impair natural killer cell cytotoxicity in post-menopausal women [9], that is, both folate deficiency and supplementation of 400 μg/day can be detrimental to immune function.

The interpretation of the effects of nutrition on immune function is further complicated by the fact that recommendations for nutrient intake are largely based on considering nutrients in isolation as opposed to taking into account their synergies and interactions [10]. Notable exceptions include vitamins B6 and E and to some extent folate. In previous studies, we laid the groundwork for establishing synergistic mechanisms between biotin and folate in gene regulation (Fig. 1). In these previous studies, we demonstrated that the folate-dependent methylation of DNA is a prerequisite for the subsequent binding of the protein biotin ligase, holocarboxylase synthetase (HLCS), to chromatin, but that DNA methylation does not depend on HLCS-dependent events [11]. We further demonstrated that HLCS interacts physically with the DNA methyltransferase DNMT1 and the methylated cytosine-binding protein MeCP2 [12]. While histone biotinylation marks are over-represented in repressed loci, these marks are very rare in the epigenome and, therefore, can hardly explain the robust correlation between those marks and gene repression [11, 13, 14]. Importantly, HLCS also interacts physically with the histone H3 K9-methyltransferase (H3K9me) EHMT1 and catalyses the biotinylation of K161 in the HLCS-binding domain in EHMT1, thereby strengthening the interaction between the two proteins [15]. When the biotinylation site in EHMT1 is mutated or deleted, the physical interaction between the two proteins is reduced. Importantly, H3K9me marks are abundant in the epigenome and play an undisputed role in gene repression [16]. HLCS knockdown causes a depletion of H3K9me marks and, consequently, derepresses loci coding for the biotin transporter SMVT, long-terminal repeats (LTRs) and interleukin-2 [11, 13, 17].

image

Figure 1. Synergies between biotin, folate and chromatin proteins in gene repression. Methyl donors may include folate, methionine and perhaps choline and betaine. bio, biotin; me, methyl.

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Here we tested the hypothesis that biotin and folate synergize in the regulation of pro-inflammatory cytokines and LTRs, using vitamin concentrations in cell cultures that are nutritionally relevant. We assessed the regulation of the following loci and genes. (1) LTR transcripts were tested, because their regulation depends on biotin, HLCS and methylation events [11, 18], and derepression of LTRs impairs genome stability [19, 20]. (2) Tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were tested because folate supplementation represses the lipopolysaccharide-induced transcription of TNF-α in RAW264.7 macrophages [21], whereas folate deficiency increases the expression of TNF-α [22]; both TNF-α and IL-6 are inducible by NF-κB [23] and play a central role in the pathogenesis of Crohn's disease [24] and fat-induced liver inflammation [25].

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Cell cultures

Human T lymphoma Jurkat cells and monocytic myeloid U937 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in biotin- and folate-defined media. Culture media were prepared using customized RPMI-1640 (Hyclone, Ogden, UT, USA), which was free of biotin, folate and the methyl donor L-methionine. RPMI-1640 was mixed with 10% of biotin-depleted foetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA, USA) prepared as described previously and antibiotics [26]. Chemically pure L-methionine was added back to produce a concentration of 10 μm methionine representing the levels in normal human plasma [27]. The endogenous levels of folate (22.7 nm) and methionine (2.7 μm) in FBS were quantified in the Physicians' Laboratories (Omaha, NE, USA) and the Metabolomics core facility at the University of Nebraska-Lincoln, respectively, and taken into account when adjusting nutrient levels in culture media. Biotin was added to media to produce the following concentrations (nm): 0.025, 0.25 and 10, representing plasma levels in biotin-deficient, biotin-sufficient and biotin-supplemented adults [28, 29]. Folate levels were adjusted to 4.9, 14 and 32 nm, representing plasma levels in folate-deficient, folate-sufficient and folate-supplemented women, respectively, living in regions without mandatory folate fortification [30]. Cells were cultured in defined media for 14 days and were resuspended in fresh medium every 48 h. Cell viability and proliferation rate were monitored at 2-day intervals using Trypan Blue staining and a haemocytometer. Note that for all treatments used in our studies, cell viability was 90–99%; the proliferation rate of cells decreased by about 20 and 15% in Jurkat and U937 cells, respectively, that were cultured in folate-deficient media compared with sufficient media.

The secretion of cytokines was induced by treatment with 50 μg/l phorbol 12-myristate 13-acetate (PMA) and 2 mg/l phytohaemagglutinin (PHA) [26]. Jurkat cells were treated for 4 h, while U937 cells were treated for 24 h; treatments were determined based on the preliminary time course experiments to determine times of peak secretion (data not shown).

In select experiments, NF-κB signalling was inhibited by treatment with curcumin (diferuloylmethane), which, at concentrations of 1–60 μm, is an effective inhibitor of the IκB alpha/beta kinase in human myelomonoblastic leukaemia cells and U937 cells [31-33]. Briefly, cells were treated with 5 μm curcumin (final concentration; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h before addition of PMA and PHA to culture media; this treatment did not measurably alter cell viability.

Determination of biotin and folate status

Biotinylated carboxylases are well-established markers for biotin status [34] and were assessed by streptavidin gel electrophoresis as described previously [26, 35, 36]. The ratio of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH), two metabolites of methyl donors, represented the methylation index [37], and the intracellular concentrations of SAM and SAH were measured by reversed-phase high-performance liquid chromatography (HPLC) and a photodiode array detector.

Quantitative real-time PCR

qRT-PCR analysis of mRNA coding for LTRs and cytokines was performed as described previously [35]. The following primers were used: LTR R/U5 transcripts: 5′-GCGGGCAGCAATACTGCTTTGTAA-3′ (forward) and 5′-ACCAGCGTTCAGCATATGGAGGAT-3′ (reverse); TNF-α: 5′–ACTTTGGAGTGATCGGCC-3′ (forward) and 5′-GCTTGAGGGTTTGCTACAAC-3′ (reverse); IL-6: 5′-CCACTCACCTCTTCAGAACG-3′ (forward) and 5′-CATCTTTGGAAGGTTCAGGTTG-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5′-TCCACTGGCGTCTTCACC-3′ (forward) and 5′-GGCAGAGATGATGACCCTTT-3′ (reverse). GAPDH was used to normalize for amplification efficiency. Note that the transcript sequences are nearly identical in the 54 transcriptionally active LTRs in the human genome [38]. Therefore, the LTR transcript abundance reported here represents the total of all transcriptionally active LTRs. Transcription in humans can be initiated in both the U5 region and the R/U5 boundary in LTRs. The epigenetic regulation is the same for both transcripts [11]; we quantified R/U5 transcripts because they mark active retrotranscription and retrotransposition events [38, 39].

Statistical analysis

Normality was tested by normal quantile plot [40], and homogeneity of variance was analysed by F-test [41]. Data sets with a P-value <0.05 in the F-test were log-transformed before subsequent statistical analysis. Student's t-test was used when comparing two groups. When more than two groups were compared, one-way anova and Fisher's least significant difference (LSD) post hoc test were used. Means and standard deviations from independent samples are reported. Different letters indicated significant difference with P-value <0.05. All calculations were based on the three independent experimental repeats. Error bars represented standard deviations.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Efficacy of treatment

The abundance of biotinylated carboxylases in Jurkat cells responded well to changes in biotin concentrations in culture media (Fig. 2). In U937 cells, the abundance of biotinylated carboxylases was not different in cells cultured in biotin-sufficient and biotin-supplemented levels of biotin, suggesting that carboxylases were saturated with coenzyme at physiological concentrations of biotin in this macrophage cell line, while we cannot formally exclude the possibility that other biotin-related pathways and proteins were affected. Propionyl-CoA carboxylase was barely detectable in U937 cells. Likewise, acetyl-CoA carboxylases 1 and 2 were barely detectable in both cell lines, consistent with previous observations in immune cells [26, 35, 36]. Importantly, the abundance of biotinylated carboxylases is an excellent, if not the sole, marker that reliably discriminates among states of biotin deficiency, sufficiency and supplementation in human feeding studies [42]. Collectively, these observations are consistent with the notion that changes in biotin supply affected cellular biotin and that the biotinylation of carboxylases reaches peak levels at lower levels of biotin in U937 cells compared with Jurkat cells.

image

Figure 2. The abundance of biotinylated carboxylases depends on biotin in culture media in Jurkat and U937 cells. The gel depicts a representative example. ACC1&2, acetyl-CoA carboxylases 1 and 2; PC, pyruvate carboxylase; MCC, methylcrotonyl-CoA carboxylase; PCC, propionyl-CoA carboxylase.

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The levels of SAM and SAH in cell cultures were below the detection limit of the reversed-phase HPLC system (about 93.3 and 9.6 mm for SAM and SAH, respectively) and were not quantified.

LTR transcripts

Supplementation with folate rescues the repression of LTRs in biotin-depleted Jurkat and U937 cells. When biotin-deficient Jurkat and U937 cells were supplemented with folate, the expression of LTRs decreased by >70 and >30%, respectively (Fig. 3A). Likewise, when folate-deficient Jurkat cells were supplemented with biotin, the expression of LTRs decreased by >50% (Fig. 3B). In contrast, biotin supplementation had no significant effect on folate-deficient U937 cells, possibly because cellular biotin status cannot be increased in biotin-supplemented compared with biotin-sufficient macrophages. Collectively, these observations suggest that biotin can rescue folate-deficient cells and vice versa, consistent with the theory of synergies between the two micronutrients.

image

Figure 3. Folate and biotin can compensate for each other's deficiency in the repression of LTRs in Jurkat and U937 cells. (A) Biotin-deficient cells were supplemented with folate. (B) Folate-deficient cells were supplemented with biotin. a,bColumns not sharing the same letters are significantly different in the same cell line (P < 0.05, n = 3). DEF, deficient; SUP, supplemented.

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Pro-inflammatory cytokines

The effects of biotin and folate on the expression of TNF-α and IL-6 presented a much more complex and less coherent picture than that of LTRs; the cytokine data can be summarized as follows. The expression of TNF-α increased significantly in response to biotin supplementation when U937 cells in folate-supplemented medium were cultured in biotin-defined media (Fig. 4A). In contrast, the expression of TNF-α decreased significantly in response to high levels of folate when biotin-supplemented U937 cells were cultured in folate-defined media (Fig. 4B). However, the expression of TNF-α was not affected by biotin in folate-supplemented Jurkat cells (data not shown), consistent with the notion that cells of the T cell lineage play a minor role in TNF-α production. Subsequent studies of cytokines focused on the myeloid U937 cells rather than T cells, because innate phagocytic cells, that is, macrophages, are the primary source of TNF-α in immune defence [43].

image

Figure 4. Biotin and folate have opposing effects in the regulation of TNF-α transcription in U937 cells. (A) Folate-supplemented cells were cultured in biotin-defined medium and stimulated with PMA/PHA. (B) Biotin-supplemented cells were cultured in folate-defined medium and stimulated with PMA/PHA. a,b,cColumns not sharing the same letters are significantly different in the same data set (P < 0.05, n = 3). DEF, deficient; SUF, sufficient; SUP, supplemented.

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Similar to the effects described for TNF-α, the expression of IL-6 increased in response to biotin supplementation when U937 cells in a high folate environment were cultured in biotin-defined media, although the differences between biotin-sufficient and biotin-supplemented media did not reach statistical significance (Fig. 5).

image

Figure 5. Curcumin abrogates the biotin-dependent expression of IL-6 in U937 cells. a,b,c,dColumns not sharing the same letters are significantly different (P < 0.05, n = 3). DEF, deficient; SUF, sufficient; SUP, supplemented.

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Biotin-dependent cell signals include the transcription factors Sp1/Sp3, NF-κB and Fos/Jun [44-46]. Curcumin blocks NF-κB signalling through inhibiting the IκB kinase alpha/beta [31, 32]. Curcumin (diferuloylmethane) abolished effects of biotin and folate in the regulation of TNF-α (data not shown) and IL-6 (Fig. 5), suggesting that the activation of biotin-dependent transcription factors precedes the epigenetic events that regulate cytokine expression, which indicates that investigation into the synergies between biotin and folate in gene regulation has to take into consideration the variation in biotin- and folate-dependent transcription factors. Jurkat cells do not express IL-6 and were not considered for analysis.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Here we report novel synergies between the vitamins folate and biotin in gene regulation, including genes coding for pro-inflammatory cytokines. For LTRs, the data are unambiguous and suggest that folate and biotin supplementation can compensate for each other's deficiency in mediating LTR repression to a quantitatively meaningful extent. This observation is firmly grounded in our previous reports that the binding of HLCS to human chromatin depends on prior folate-dependent DNA methylation and that folate-dependent creation of H3K9me histone repression marks depends on HLCS and perhaps HLCS-dependent biotinylation events [12, 15]. The unambiguous nature of the LTR data is probably due to the tight regulation of these repeats through epigenetic mechanisms including various methylation marks [18].

Previous studies suggest that the expression of TNF-α increases and decreases in folate-free and folate-supplemented media, respectively, in macrophages [21, 22]. However, the levels of folate used in these studies lacked physiological relevance, because zero folate is not consistent with survival and millimolar concentrations greatly exceed levels observed in human plasma [30, 47]. Here we demonstrated for the first time that biologically relevant concentrations of folate and biotin decrease and increase, respectively, the expression of TNF-α. We propose that cytokines, unlike LTRs, are regulated by a complex network involving numerous transcription factors and that changes in biotin- and folate-dependent transcription factors need to be considered when investigating synergies between the two vitamins in gene regulation. For example, biotin deficiency – and nutrient deficiency, cell stress in general – causes the activation of NF-κB [45]. In this study, curcumin (diferuloylmethane) abrogated the effects of biotin and folate on cytokine expression, consistent with the theory that transcription factor signalling is an important component when evaluating epigenetic synergies between vitamins.

Synergies between biotin and folate are important for human health, based on the following rationale. LTRs make up about 8% of the human genome, and at least 51 LTRs are transcriptionally competent [38]. Repetitive elements such as LTRs pose a burden to genome stability, as their mobilization facilitates recombination between non-homologous loci, leading to chromosomal deletions and translocations [18, 19]. Mobilization of LTR transposons is associated with 10% of all spontaneous mutations in mice [20]. Derepression of LTRs may impair genome stability through insertional mutagenesis, recombination events that cause translocations and other rearrangements, deregulation of genes in the host genome mediated by LTR promoter activity and antisense effects if transcription extends into exon sequence downstream of the transposon [48]. Likewise, the tight regulation of pro-inflammatory cytokines is important to ensure an appropriate response of the immune system to external challenges such as infection [49]. Previous studies suggest that changes in the levels of cytokines similar to those reported here are sufficient to relieve collagen-induced arthritis [50]. An unregulated continued expression of pro-inflammatory cytokines is associated with diseases such as rheumatoid arthritis [51] and inflammatory bowel disease [52], which affect a significant portion of the population in Western societies [53, 54].

We conclude that biotin and folate synergize in the repression of LTRs and that these interactions are probably mediated by HLCS-dependent epigenetic mechanisms. In contrast, synergies between biotin, folate and HLCS in the regulation of cytokines need to be interpreted in the context of transcription factor signalling. We are currently creating a conditional HLCS knockout mouse model, so that future studies of epigenetic synergies can be conducted in a whole animal model at a mechanistic level.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This work was supported in part by the University of Nebraska Agricultural Research Division with funds provided through the Hatch Act. Additional support was provided by NIH DK063945 and DK077816.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
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