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. 2014 Aug 1;461(3):477-86.
doi: 10.1042/BJ20131208.

Holocarboxylase synthetase interacts physically with nuclear receptor co-repressor, histone deacetylase 1 and a novel splicing variant of histone deacetylase 1 to repress repeats

Dandan Liu  1 , Janos Zempleni  1
Affiliations

Holocarboxylase synthetase interacts physically with nuclear receptor co-repressor, histone deacetylase 1 and a novel splicing variant of histone deacetylase 1 to repress repeats

Dandan Liu et al. Biochem J. .

Abstract

HLCS (holocarboxylase synthetase) is a nuclear protein that catalyses the binding of biotin to distinct lysine residues in chromatin proteins. HLCS-dependent epigenetic marks are over-represented in repressed genomic loci, particularly in repeats. Evidence is mounting that HLCS is a member of a multi-protein gene repression complex, which determines its localization in chromatin. In the present study we tested the hypothesis that HLCS interacts physically with N-CoR (nuclear receptor co-repressor) and HDAC1 (histone deacetylase 1), thereby contributing toward the removal of H3K9ac (Lys⁹-acetylated histone H3) gene activation marks and the repression of repeats. Physical interactions between HLCS and N-CoR, HDAC1 and a novel splicing variant of HDAC1 were confirmed by co-immunoprecipitation, limited proteolysis and split luciferase complementation assays. When HLCS was overexpressed, the abundance of H3K9ac marks decreased by 50% and 68% in LTRs (long terminal repeats) 15 and 22 respectively in HEK (human embryonic kidney)-293 cells compared with the controls. This loss of H3K9ac marks was linked with an 83% decrease in mRNA coding for LTRs. Similar patterns were seen in pericentromeric alpha satellite repeats in chromosomes 1 and 4. We conclude that interactions of HLCS with N-CoR and HDACs contribute towards the transcriptional repression of repeats, presumably increasing genome stability.

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Figures

Figure 1
Figure 1. HLCS-containing multi-protein gene repression complex
The binding of HLCS to chromatin depends on physical interactions of HLCS with the maintenance DNA methyltransferase DNMT1 and MeCP2. Chromatin-bound HLCS recruits the eukaryotic histone H3 methyltransferase EHMT-1, N-CoR and HDAC. ac, acetylation; bio, biotinylation; HP1, heterochromatin protein 1; me, methylation.
Figure 2
Figure 2. Schematic diagrams of domains in N-CoR and luciferase constructs used in the split luciferase complementation assays
(A) The N-terminus of N-CoR contains transcriptional repression domains (RDs) responsible for the recruitment of additional components of the co-repressor complex such as HDAC, mSin3 and GPS2 (G-protein-pathway suppressor 2). A pair of potent repressor motifs known as SANT motifs (SWI3, ADA2, N-CoR and TFIIIB) is positioned between the repression domains. SANT motifs recruit HDAC3 and histones to the repressor complex in order to enhance HDAC3 activity. The C-terminus of N-CoR includes a nuclear receptor interaction domain (NID), which binds ligand-free nuclear receptors. In order to assign putative interactions with HLCS to distinct domains in N-CoR, three overlapping fragments of N-CoR were cloned, N-terminal domain (NT), central domain (CD) and C-terminal domain (CT). (B) In split luciferase complementation assays, N-terminal and C-terminal fragments are fused to interacting proteins. Physical interactions between the two proteins reconstitute luciferase activity.
Figure 3
Figure 3. HLCS interacts physically with N-CoR
(A) In co-immunoprecipitation assays, Myc–HLCS and HA–N-CoR-CT were overexpressed in HEK-293 cells and cell lysates were precipitated with anti-Myc or anti-HA antibodies. Proteins were resolved by electrophoresis and probed with anti-HA or anti-HLCS antibodies. Empty vectors in various permutations and non-transfected cells were used as negative controls. (B) As for (A), but tags were swapped. (C) HA-tagged HLCS and Myc-tagged wild-type or mutant N-CoR C-terminus were overexpressed in HEK-293 cells and cell lysates were precipitated with anti-Myc or anti-HA antibodies. Proteins were resolved by electrophoresis and probed with anti-HLCS or anti-Myc antibodies. Non-transfected cells were used as negative controls. (D) Limited proteolysis assays: recombinant (r) HLCS and N-CoR C-terminus were pre-incubated before trypsin digestion (bottom panel). Negative controls were generated by omission of HLCS or N-CoR C-terminus (top and middle panels). (E) Extracts from normal non-transfected HEK-293 cells were precipitated using anti-HLCS antibody and probed using anti-N-CoR antibody. An anti-IgG antibody was used as a control for the anti-HLCS antibody. Gels depict representative examples from three biological repeats.
Figure 4
Figure 4. Split luciferase complementation assays for detection of HLCS and N-CoR interaction in HEK-293 cells
Fusion constructs of TP53 and MDM2 were used as positive controls and TP53 and CDK3 were used as negative controls. Values are means ± S.D. of independent experiments (n = 3). **P < 0.01 compared with the background control for self-association and negative control.
Figure 5
Figure 5. HLCS interacts physically with HDAC1
(A) In co-immunoprecipitation assays, Myc–HLCS and HA–HDAC1 were overexpressed in HEK-293 cells and cell lysates were precipitated with anti-Myc or anti-HA antibodies. Proteins were resolved by electrophoresis and probed with anti-HDAC1 or anti-HLCS antibodies. Empty vectors in various permutations and non-transfected cells were used as negative controls. (B) As for (A), but tags were swapped. (C) Limited proteolysis assays. Recombinant (r) HLCS and HDAC1 were pre-incubated before trypsin digestion (middle and right-hand panels). Negative controls were generated by omission of HLCS or HDAC1 (left-hand panel). Gels depict representative examples from three biological repeats.
Figure 6
Figure 6. Split luciferase complementation assays for detection of HLCS and HDAC1 interaction in HEK-293 cells
Fusion constructs of TP53 and MDM2 were used as positive controls and TP53 and CDK3 were used as negative controls. Values are means ± S.D. of independent experiments (n = 3). **P < 0.01 compared with the background control for self-association and negative control.
Figure 7
Figure 7. The roles of HLCS domains in mediating interactions with N-CoR and HDAC1
Myc-tagged HLCS and HA-tagged N-CoR C-terminus (A) or HDAC1 (B) were overexpressed in HEK-293 cells. Cell lysates were precipitated with an anti-Myc antibody and probed with an anti-HA antibody. Non-transfected cells were used as negative controls. Gels depict representative examples from three biological repeats.
Figure 8
Figure 8. N-CoR and HDAC1 are targets for biotinylation of HLCS
(A) Recombinant N-terminus, central domain and C-terminus in N-CoR were incubated with recombinant (r) HLCS, biotin and cofactors for biotinylation for up to 2 h. N-CoR-bound biotin was detected with an anti-biotin antibody. Negative controls were created by omitting N-CoR fragments or HLCS. Equal loading was confirmed by Coomassie Blue stain. (B) Similarly, biotinylation of recombinant HDAC1 by HLCS was conducted. Gels depict representative examples from three biological repeats.
Figure 9
Figure 9. Transcriptional repression of repeats by HLCS overexpression
(A) Transfection with the plasmid p3XFLAG-Myc-CMV-26-HLCS produced a stable overexpression of HLCS in HEK-293 cells, judged by qPCR (n = 3; *P < 0.05 compared with the untransfected controls) and Western blot analysis. GAPDH and histone H3 were used as loading controls. (B) The enrichment of H3K9ac marks in loci coding for LTR15, LTR22, Chr1alpha, Chr4alpha and GAPDH in HLCS-overexpressing HEK-293 cells and controls (n = 3). ****P < 0.0001 compared with the FLAG/Myc–HLCS controls. The insert depicts a magnified version of the Chr1alpha and Chr4alpha data. (C) The abundance of transcripts coding for LTRs, Chr1alpha and Chr4alpha in HLCS-overexpressing HEK-293 cells and controls. Values are means ± S.D. of independent experiments (n = 3). ****P < 0.0001 compared with the FLAG/Myc–HLCS controls.
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References

    1. Suzuki Y, Aoki Y, Ishida Y, Chiba Y, Iwamatsu A, Kishino T, Niikawa N, Matsubara Y, Narisawa K. Isolation and characterization of mutations in the human holocarboxylase synthetase cDNA. Nat. Genet. 1994;8:122–128. - PubMed
    1. Zempleni J, Wijeratne SSK, Kuroishi T. Biotin. In: Erdman JW Jr, Macdonald I, Zeisel SH, editors. Present Knowledge in Nutrition. Washington: International Life Sciences Institute; 2012. pp. 587–609.
    1. Camporeale G, Giordano E, Rendina R, Zempleni J, Eissenberg JC. Drosophila holocarboxylase synthetase is a chromosomal protein required for normal histone biotinylation, gene transcription patterns, lifespan and heat tolerance. J. Nutr. 2006;136:2735–2742. - PMC - PubMed
    1. Singh D, Pannier AK, Zempleni J. Identification of holocarboxylase synthetase chromatin binding sites using the DamID technology. Anal. Biochem. 2011;413:55–59. - PMC - PubMed
    1. Bao B, Rodriguez-Melendez R, Wijeratne SS, Zempleni J. Biotin regulates the expression of holocarboxylase synthetase in the miR-539 pathway in HEK-293 cells. J. Nutr. 2010;140:1546–1551. - PMC - PubMed

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