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. 2006 Nov;136(11):2735-42.
doi: 10.1093/jn/136.11.2735.

Drosophila melanogaster holocarboxylase synthetase is a chromosomal protein required for normal histone biotinylation, gene transcription patterns, lifespan, and heat tolerance

Gabriela Camporeale  1 , Ennio Giordano, Rosaria Rendina, Janos Zempleni, Joel C Eissenberg
Affiliations

Drosophila melanogaster holocarboxylase synthetase is a chromosomal protein required for normal histone biotinylation, gene transcription patterns, lifespan, and heat tolerance

Gabriela Camporeale et al. J Nutr. 2006 Nov.

Abstract

Post-translational modifications of histones play important roles in chromatin structure and genomic stability. Distinct lysine residues in histones are targets for covalent binding of biotin, catalyzed by holocarboxylase synthetase (HCS) and biotinidase (BTD). Histone biotinylation has been implicated in heterochromatin structures, DNA repair, and mitotic chromosome condensation. To test whether HCS and BTD deficiency alters histone biotinylation and to characterize phenotypes associated with HCS and BTD deficiency, HCS- and BTD-deficient flies were generated by RNA interference (RNAi). Expression of HCS and BTD decreased by 65-90% in RNAi-treated flies, as judged by mRNA abundance, BTD activity, and abundance of HCS protein. Decreased expression of HCS and BTD caused decreased biotinylation of K9 and K18 in histone H3. This was associated with altered expression of 201 genes in HCS-deficient flies. Lifespan of HCS- and BTD-deficient flies decreased by up to 32% compared to wild-type controls. Heat tolerance decreased by up to 55% in HCS-deficient flies compared to controls, as judged by survival times; effects of BTD deficiency were minor. Consistent with this observation, HCS deficiency was associated with altered expression of 285 heat-responsive genes. HCS and BTD deficiency did not affect cold tolerance, suggesting stress-specific effects of chromatin remodeling by histone biotinylation. To our knowledge, this is the first study to provide evidence that HCS-dependent histone biotinylation affects gene function and phenotype, suggesting that the complex phenotypes of HCS- and BTD-deficiency disorders may reflect chromatin structure changes.

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Figures

FIGURE 1
FIGURE 1
Holocarboxylase synthetase (HCS) binds to distinct chromosomal sites in wild-type Drosophila melanogaster. Panel A: Immunostaining of fixed polytene chromosomes with anti-HCS serum. Panel B: Phase contrast image of chromosomes in A. Panel C: Immunostaining of fixed polytene chromosomes with pre-immune serum. Panel D: Phase contrast image of chromosomes in C. Legend: filled arrowheads = puffs that stain for HCS; open arrowheads = puffs that do not stain for HCS; line = non-puffed site that stains for HCS; t = staining telomere; N = nucleolus; cc = heterochromatic chromocenter; X = X chromosome; 2L and 2R = left and right arms of chromosome 2; 3L and 3R = left and right arms of chromosome 3.
FIGURE 2
FIGURE 2
RNAi to HCS and biotinidase (BTD) decreases the expression of HCS and BTD, respectively, in Drosophila melanogaster compared to control flies. Panel A: mRNA levels of HCS and BTD in HCS-RNAi flies, BTD-RNAi flies, and Act5C-Gal4 control flies. Panel B: Quantification of HCS protein by Western blot analysis; β-actin was used as a loading control. Panel C: Quantification of BTD activity in fly homogenates. PABA = para-amino benzoic acid. a,bColumns not sharing the same letter are significantly different (P < 0.05). Values are means ± SD (n = 3).
FIGURE 3
FIGURE 3
RNAi knockdown of HCS and BTD is associated with decreased biotinylation of carboxylases in Drosophila melanogaster. Panel A: Abundance of biotinylated carboxylases in HCS-deficient (HCS-RNAi) and BTD-deficient (BTD-RNAi) flies compared with Act5C-Gal4 control flies. PCC = Propionyl-CoA carboxylase. Panel B: Gel densitometric quantification of pyruvate carboxylase (PC). Panel C: PCC activity in fly homogenates. a,b,cColumns not sharing the same letter are significantly different from other groups (P < 0.05). Values are means ± SD (n = 3).
FIGURE 4
FIGURE 4
RNAi knockdown of HCS and BTD is associated with decreased biotinylation of histones in Drosophila melanogaster. Panel A: Abundance of biotinylated histones in HCS-RNAi and BTD-RNAi flies compared with Act5C-Gal4 control flies, as judged by streptavidin blotting. Panel B: Transblots probed with anti-K9BioH3. Panel C: Transblots probed with anti-K18BioH3. Note that all lanes shown were from the same blot, although the order presented is changed in order to facilitate comparisons.
FIGURE 5
FIGURE 5
Effects of HCS and BTD RNAi knockdown on heat tolerance in HCS-deficient (HCS-RNAi) and BTD-deficient (BTD-RNAi) flies compared with Act5C-Gal4 control flies. Panel A: HCS-RNAi and BTD-RNAi male flies exhibited decreased heat tolerance compared with control flies. Panel B: HCS deficiency but not BTD deficiency decreased the heat tolerance in female knockdown flies.
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References

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