Modulation of chromatin position and gene expression by HDAC4 interaction with nucleoporins - PubMed (original) (raw)

Modulation of chromatin position and gene expression by HDAC4 interaction with nucleoporins

Izhak Kehat et al. J Cell Biol. 2011.

Abstract

Class IIa histone deacetylases (HDACs) can modulate chromatin architecture and transcriptional activity, thereby participating in the regulation of cellular responses such as cardiomyocyte hypertrophy. However, the target genes of class IIa HDACs that control inducible cardiac growth and the broader mechanisms whereby these deacetylases modulate locus-specific gene expression within chromatin remain a mystery. Here, we used genome-wide promoter occupancy analysis, expression profiling, and primary cell validation to identify direct class IIa HDAC4 targets in cardiomyocytes. Simultaneously, we identified nucleoporin155 (Nup155) as an HDAC4-interacting protein. Mechanistically, we show that HDAC4 modulated the association of identified target genes with nucleoporins through interaction with Nup155. Moreover, a truncated mutant of Nup155 that cannot bind HDAC4 suppressed HDAC4-induced gene expression patterns and chromatin-nucleoporin association, suggesting that Nup155-mediated localization was required for HDAC4's effect on gene expression. We thus propose a novel mechanism of action for HDAC4, suggesting it can function to dynamically regulate gene expression through changes in chromatin-nucleoporin association.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Validation of the expression and occupancy screens. (A) Validation of the transcription array profiling using normalized quantitative RT-PCR in NRVM at baseline or with HDAC4 (HD4) adenoviral-mediated overexpression showing HDAC4 repressed and activated transcripts. Gapdh was used as a control (P < 0.05 for all transcripts, n = 4). (B) Validation of the DamID screen with ChIP assays in NRVM overexpressing HDAC4-flag (adenoviral-mediated overexpression) followed by flag IP and PCR for the promoters of the indicated genes. Input represents 5% of chromatin before IP. (C) Western blot from control (β-galactosidase containing adenovirus) or adeno-HDAC4-flag infected NRVMs showing a decrease in several Ca2+ handling proteins and increased Fosl1 product Fra. Molecular weights in kD are shown. (D) Recordings of total cytoplasmic Ca2+ fluxing using the fluorescent Ca2+ probe Indo-1 in NRVMs paced at 0.5 Hz. Expression of HDAC4 resulted in an increase in baseline Ca2+ and a decrease in relaxation time (tau). (E) Quantitative analysis of the Ca2+ transient amplitude, tau, and baseline diastolic Ca2+ levels (*, P < 0.01; n = 18).

Figure 2.

Figure 2.

HDAC4 partners with Nup155 and associates with the NUPs. (A) Yeast two-hybrid growth assay with baits containing either the N terminus (aa 3–281 or 3–666) or C terminus (aa 632–1084) of HDAC4 (HD4) cotransfected with the library plasmid, which identified the Nup155 C terminus. Inducible promoters on library and bait plasmids allows differential activation of expression (−/+), showing growth and therefore interaction between Nup155 and both N-terminal constructs of HDAC4, but not with the C-terminal construct. (B) Western blots for the indicated proteins from mammalian cells transfected with the indicated constructs. For the bottom two blots, HA-Nup155 was immunoprecipitated (IP) followed by Western blotting for His-HDAC4 fragments or HA-Nup155. The asterisks show the fragments that interact. Input represents 10% of protein before IP. (C) Coomassie gel showing interaction between the C terminus of Nup155 as a GST fusion protein and the N terminus of HDAC4 as an MBP fusion protein. The asterisk shows the pull-down of MBP-HDAC4 with GST-Nup155 from the GST binding column. Input protein is shown on the left. (D) Western blots from NRVMs after endogenous HDAC4 immunoprecipitation (IP) or control IgG to investigate Nup155 and nucleoporin (mAb414) interaction. (E) Immunofluorescent staining of NRVMs for endogenous HDAC4 (green, arrowheads), the NUPs (mAb414, red), and DNA (ToPro3, blue) showing partial colocalization of HDAC4 with NUPs.

Figure 3.

Figure 3.

HDAC4 binds and regulates the association of specific chromatin loci with NUPs. (A) ChIP assay in NRVM with the NUP (mAb414) antibody showing association of loci with NUPs with or without HDAC4, with or without TSA treatment. Input represents amplification of 5% of chromatin before IP. (B) Immunofluorescent staining of NRVMs for tagged full-length Nup155 or truncated Nup155 (Nup155ΔC) showing similar localization (arrowheads). Troponin (Tropo.) staining verified myocyte identity. (C) Immunofluorescent staining of NRVM for endogenous HDAC4 (HD4, green), the NUPs (mAb414, red), and DNA (ToPro3, blue) at control or after Nup155ΔC expression. The arrowheads show localization of HDAC4 at baseline, which is lost with Nup155ΔC expression, yet the NUPs maintain their peripheral location (red). (D) ChIP assay in NRVMs with mAb414 antibody during control (βgal, −), HDAC4, or HDAC4 + Nup155ΔC overexpression. (E) ChIP assay as in D, except NRVMs were infected with an adenovirus containing activated CaMKII, which reversed HDAC4 actions.

Figure 4.

Figure 4.

HDAC4 regulates spatial chromatin organization. (A) Confocal FISH of selected loci (green dots, arrows) with nuclear Topro3 counterstain (blue) in NRVMs showing predominant peripheral nuclear localization of the Nppb, Acta1, Cacna1c during control conditions, a shift to a more central nuclear position under HDAC4 (HD4) overexpression, and reversal of the HDAC4 effect by expression of Nup155ΔC. In contrast, the Pln locus demonstrates a reverse pattern. (B) Quantitative summary of the FISH data showing the percentage of peripheral nuclear FISH signals (white bar) versus central nuclear signals (black bar). *, P < 0.001; significance of HDAC4-expressing vs. control and HDAC4+ Nup155ΔC, n = 250.

Figure 5.

Figure 5.

Non-HDAC4 binding Nup155ΔC reverses HDAC4 gene expression patterns and alters the growth response of NRVMs. (A) Quantitative qRT-PCR in NRVMs (normalized to Gapdh) shows that combined expression of Nup155ΔC reverses the HDAC4 expression pattern for the indicated transcripts. (B) Immunofluorescent staining of NRVM for sarcomeric α-actinin (red) with Adβgal (control), AdHDAC4 (HD4), or AdHDAC4 + AdNup155ΔC overexpression. The data show that Nup155ΔC expression increased sarcomeric organization associated with hypertrophy, which is quantified in C (*, P < 0.001; n = 50). (D) HDAC4 overexpression also reduced myocyte size in culture, which was restored by expression of Nup155ΔC (*, P < 0.01).

Similar articles

Cited by

References

    1. Ago T., Liu T., Zhai P., Chen W., Li H., Molkentin J.D., Vatner S.F., Sadoshima J. 2008. A redox-dependent pathway for regulating class II HDACs and cardiac hypertrophy. Cell. 133:978–993 10.1016/j.cell.2008.04.041 - DOI - PubMed
    1. Akhtar A., Gasser S.M. 2007. The nuclear envelope and transcriptional control. Nat. Rev. Genet. 8:507–517 10.1038/nrg2122 - DOI - PubMed
    1. Aronheim A. 2004. Ras signaling pathway for analysis of protein-protein interactions in yeast and mammalian cells. Methods Mol. Biol. 250:251–262 - PubMed
    1. Backs J., Song K., Bezprozvannaya S., Chang S., Olson E.N. 2006. CaM kinase II selectively signals to histone deacetylase 4 during cardiomyocyte hypertrophy. J. Clin. Invest. 116:1853–1864 10.1172/JCI27438 - DOI - PMC - PubMed
    1. Bailey T.L., Elkan C. 1994. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2:28–36 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources