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. 2011 Apr;22(4):328-33.
doi: 10.1016/j.jnutbio.2010.02.011. Epub 2010 Aug 6.

Novel histone biotinylation marks are enriched in repeat regions and participate in repression of transcriptionally competent genes

Valerie Pestinger  1 , Subhashinee S K Wijeratne, Rocio Rodriguez-Melendez, Janos Zempleni
Affiliations

Novel histone biotinylation marks are enriched in repeat regions and participate in repression of transcriptionally competent genes

Valerie Pestinger et al. J Nutr Biochem. 2011 Apr.

Abstract

Covalent histone modifications play crucial roles in chromatin structure and genome stability. We previously reported biotinylation of lysine (K) residues in histones H2A, H3 and H4 by holocarboxylase synthetase and demonstrated that K12-biotinylated histone H4 (H4K12bio) is enriched in repeat regions and participates in gene repression. The biological functions of biotinylation marks other than H4K12bio are poorly understood. Here, novel biotinylation site-specific antibodies against H3K9bio, H3K18bio and H4K8bio were used in chromatin immunoprecipitation studies to obtain first insights into possible biological functions of these marks. Chromatin immunoprecipitation assays were conducted in human primary fibroblasts and Jurkat lymphoblastoma cells, and revealed that H3K9bio, H3K18bio and H4K8bio are enriched in repeat regions such as pericentromeric alpha satellite repeats and long-terminal repeats while being depleted in transcriptionally active promoters in euchromatin. Transcriptional stimulation of the repressed interleukin-2 promoter triggered a rapid depletion of histone biotinylation marks at this locus in Jurkat cells, which was paralleled by an increase in interleukin-2 mRNA. Importantly, the enrichment of H3K9bio, H3K18bio and H4K8bio at genomic loci depended on the concentration of biotin in culture media at nutritionally relevant levels, suggesting a novel mechanism of gene regulation by biotin.

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Figures

Fig. 1
Fig. 1
Relative enrichment of histone biotinylation marks in heterochromatin and euchromatin in Jurkat cells. Chromatin was immunoprecipitated using antibodies against H3K9bio (panel A), H3K18bio (B), H4K8bio (C), H4K12bio (D), H3K9me2 (E), and H3K9ac (F). qRT-PCR was used to quantify sequences from alpha satellite repeats on chromosome 4 (Chr4alpha), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and aldehyde dehydrogenase 5 (ADH5) in the precipitated DNA. Bars denote the percent of input DNA from a given locus that was precipitated with antibodies (mean ± S.D., n = 4). a, bBars not sharing the same letter are significantly different (P < 0.05).
Fig. 2
Fig. 2
Relative enrichment of histone biotinylation marks in the transcriptionally inactive and active interleukin-2 promoter in Jurkat cells. Jurkat cells were collected before (“−PMA/PHA” = inactive IL-2 promoter) and after (“+PMA/PHA” = active promoter) stimulation with 50 μg/l PMA and 2 mg/l PHA for 2 h. Chromatin was immunoprecipitated using antibodies against H3K9bio (panel A), H3K18bio (B), H4K8bio (C), H4K12bio (D), H3K9me2 (E), and H3K9ac (F). qRT-PCR was used to quantify IL-2 promoter sequences in the precipitated DNA. Bars denote the percent of input DNA from the IL-2 promoter locus that was precipitated with antibodies (mean ± S.D., n = 4). a, bBars not sharing the same letter are significantly different (P < 0.05).
Fig. 3
Fig. 3
The enrichment of H3K9bio, H3K18bio, and H4K8bio at LTR22 depends on biotin availability in Jurkat cells. Cells were cultured in biotin-defined media for 12 d, and the relative enrichment of H3K9bio (panel A), H3K18bio (B), H4K8bio (C), and H3K9me2 (D) at the LTR22 locus was quantified by μChIP and qRT-PCR. Bars denote the percent of input DNA from the LTR22 locus that was precipitated with antibodies (mean ± S.D., n = 4). a, bBars not sharing the same letter are significantly different (P < 0.05).
Fig. 4
Fig. 4
Abundance of biotinylated carboxylases and LTR transcripts in biotin-defined Jurkat cells. Cells were cultured in biotin-defined media for 12 d. Panel A: Streptavidin was used as a probe for biotinylated pyruvate carboxylase (PC), 3-methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PC). Panel B: mRNA originating in the U5 region of LTRs was quantified by qRT-PCR. Values are means ± SD, n = 3. a, b, cBars not sharing the same letter are significantly different (P < 0.05).
Fig. 5
Fig. 5
The enrichment of H3K9bio, H3K18bio, and H4K8bio at LTR22 depends on HCS activity in human fibroblasts. HCS mutant (“WG2215”) and wild-type (“IMR-90”) human fibroblasts were cultured in medium containing 10 nmol/l biotin. The relative enrichment of H3K9bio (panel A), H3K18bio (B), and H4K8bio (C) at the LTR22 locus was quantified by μChIP and qRT-PCR. Bars denote the percent of input DNA from the LTR22 locus that was precipitated with antibodies (mean ± S.D., n = 4). a, bBars not sharing the same letter are significantly different (P < 0.05). Panel D: Streptavidin was used as a probe for biotinylated pyruvate carboxylase (PC), 3-methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PC).
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