Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Jan;18(1):133-45.
doi: 10.1105/tpc.105.036855. Epub 2005 Dec 16.

Different domains control the localization and mobility of LIKE HETEROCHROMATIN PROTEIN1 in Arabidopsis nuclei

Assaf Zemach  1 , Yan Li, Hagit Ben-Meir, Moran Oliva, Assaf Mosquna, Vladimir Kiss, Yigal Avivi, Nir Ohad, Gideon Grafi
Affiliations
Free PMC article

Different domains control the localization and mobility of LIKE HETEROCHROMATIN PROTEIN1 in Arabidopsis nuclei

Assaf Zemach et al. Plant Cell. 2006 Jan.
Free PMC article

Abstract

Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type-dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localization signal was identified within the hinge region. Fluorescence recovery after photobleaching showed that Sl LHP1 is a highly mobile protein whose localization and retention are controlled by distinct domains; retention at the nucleolus and chromocenters is conferred by the CSD. Our results imply that LHP1 recruitment to chromatin is mediated, at least in part, through interaction with methyl K9 and that LHP1 controls different nuclear processes via transient binding to its nuclear sites.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Sl LHP1 Binds in Vitro K9-Methylated Histone H3 in a CD-Dependent Manner. (A) In vitro binding of Sl LHP1 to histone H3 prepared from tobacco leaves. GST pull-down assays were performed with the indicated GST fusion proteins. GST-CD contains the CD and GST-CSD contains the CSD. Bound proteins were resolved by 18% SDS-PAGE and immunoblotted with anti-H3 (lanes 1 to 5). Input indicates 20% of the input histones. (B) Sl LHP1 protein binds histone H3 that was methylated at Lys-9 by SUV39H1 HMTase. The acid-soluble fraction from tobacco leaves was subjected to methylation by GST-SUV39H1 (H320R). Methylated histones were collected and used in GST pull-down assays with GST alone or GST-Sl LHP1 (lanes 5 to 8). Input indicates 35% of the input methylated H3.
Figure 2.
Figure 2.
Subnuclear Localization of LHP1-GFP Proteins in Arabidopsis Cells. (A) Sequential confocal optical sections (slices of 0.4 μm; panels 1 to 8) showing the subnuclear localization of Sl LHP1 fused to GFP. Section 9 is an extended view of slices 1 to 8. Note the uneven distribution of the GFP signal within the nucleolus and the exclusion of Sl LHP1 from the center of the chromocenters (arrows in panels 3 and 4). ChC, chromocenters; nuc, nucleolus. Bars = 5 μm. (B) Transient expression of At LHP1-GFP in Arabidopsis protoplasts showing different types of subnuclear localization. Note that At LHP1-GFP is absent from the nucleolus. Bars = 5 μm.
Figure 3.
Figure 3.
Localization of Sl LHP1-GFP and At LHP1-GFP at Chromocenters and Their Punctuated Distribution Require the Aromatic Cage Residues That Recognize Methyl K9. (A) Arabidopsis protoplasts transiently expressing the indicated LHP1-GFP proteins were stained with DAPI and inspected with a confocal microscope equipped with a laser diode system for DAPI detection. ChC, chromocenters; nuc, nucleolus. Bar = 2 μm. (B) Amino acid sequences of the CDs of Sl LHP1 and At LHP1. The aromatic cage residues that recognize methyl K9 are highlighted. Asterisks indicate Trp (W) residues converted to Gly (G). (C) Arabidopsis protoplasts transiently expressing the mutated forms of LHP1 proteins, At LHP1(W132G)-GFP and Sl LHP1(W114G/W117G)-GFP, were stained with DAPI and inspected with a confocal microscope. Note that the mutated form of At LHP1 is evenly dispersed within the nucleus, whereas that of Sl LHP1 is localized to the nucleolus. DAPI is pseudocolored red. Bar = 2 μm.
Figure 4.
Figure 4.
Subnuclear Localization of Sl LHP1-GFP in Transgenic Arabidopsis lhp1 Plants. (A) Leaf tissue inspected with a confocal microscope showing distinctive subnuclear localization of Sl LHP1-GFP in the indicated cell types. ChC, chromocenter; nuc, nucleolus. Bars = 5 μm. (B) Immunolabeling/FISH analysis of nuclei from transgenic lhp1 expressing Sl LHP1-GFP showing that this protein is associated with the centromeric 180-bp repeats (CEN180). Arrows indicate the nucleolus.
Figure 5.
Figure 5.
Localization of Sl LHP1 at Chromocenters Is Not Affected in Mutants Displaying Reduced H3-K9 Methylation. (A) Immunofluorescence assays showing reduced H3-K9 methylation in kyp-2 and ddm1-2 mutants compared with wild-type plants. Bar = 5 μm. (B) Localization of Sl LHP1-GFP in kyp-2, suvh2, ddm1-2, and met1-1 is indistinguishable from that of wild-type plants. ChC, chromocenters; nuc, nucleolus.
Figure 6.
Figure 6.
Subnuclear Localization of Various Sl LHP1 Domains. (A) Scheme of Sl LHP1 and its derivatives fused to GFP. FL, full-length; NLS, nuclear localization signal. (B) The indicated constructs were transiently expressed in Arabidopsis protoplasts and inspected with a confocal microscope. Note that neither the CSD (202 to 399) nor the CD (90 to 145) has site-specific subnuclear targeting properties and that the nucleolar targeting signal is located between amino acids 141 and 171. Bar = 5 μm.
Figure 7.
Figure 7.
FRAP Analysis Showing That Nucleolar Retention Is Conferred by the CSD. (A) Distribution of Sl LHP1 truncated proteins within the nucleolus. Note that the unique, uneven distribution pattern within the nucleolus is retained by Sl LHP1(141–399), whereas other Sl LHP1 truncated proteins show diffused patterns. Bar = 2 μm. (B) Selected images were taken at various times after a band across the nucleolus was photobleached (bar at top), showing fluorescence recovery within the nucleolus of the indicated Sl LHP1-GFP proteins. Bar = 2 μm. (C) Kinetics of fluorescence recovery of the various Sl LHP1-GFP proteins. The average (Av.) 50% recovery time for each protein and the sd are given.
Figure 8.
Figure 8.
The CSD Stabilizes the Association of Sl LHP1 with Chromocenters. (A) Selected images were taken at various times after a chromocenter (arrows) was photobleached, showing the fluorescence recovery of the full-length (FL) and truncated (1 to 306) Sl LHP1-GFP proteins. Bars = 2 μm. (B) Kinetics of fluorescence recovery of the Sl LHP1-GFP proteins. The average 50% recovery time for each protein and the sd are given in brackets.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aasland, R., and Stewart, A.F. (1995). The chromo shadow domain, a second chromo domain in heterochromatin-binding protein 1, HP1. Nucleic Acids Res. 23 3168–3174. - PMC - PubMed
    1. Andersen, J.S., Lyon, C.E., Fox, A.H., Leung, A.K., Lam, Y.W., Steen, H., Mann, M., and Lamond, A.I. (2002). Directed proteomic analysis of the human nucleolus. Curr. Biol. 12 1–11. - PubMed
    1. Avivi, Y., Morad, V., Ben-Meir, H., Zhao, J., Kashkush, K., Tzfira, T., Citovsky, V., and Grafi, G. (2004). Reorganization of specific chromosomal domains and activation of silent genes in plant cells acquiring pluripotentiality. Dev. Dyn. 230 12–22. - PubMed
    1. Bannister, A.J., Zegerman, P., Partridge, J.F., Miska, E.A., Thomas, J.O., Allshire, R.C., and Kouzarides, T. (2001). Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410 120–124. - PubMed
    1. Brasher, S.V., Smith, B.O., Fogh, R.H., Nietlispach, D., Thiru, A., Nielsen, P.R., Broadhurst, R.W., Ball, L.J., Murzina, N.V., and Laue, E.D. (2000). The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer. EMBO J. 19 1587–1597. - PMC - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources