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. 2006 Nov;18(11):2904-18.
doi: 10.1105/tpc.106.047274. Epub 2006 Nov 17.

Arabidopsis chromatin-associated HMGA and HMGB use different nuclear targeting signals and display highly dynamic localization within the nucleus

Dorte Launholt  1 , Thomas Merkle, Andreas Houben, Alexander Schulz, Klaus D Grasser
Affiliations

Arabidopsis chromatin-associated HMGA and HMGB use different nuclear targeting signals and display highly dynamic localization within the nucleus

Dorte Launholt et al. Plant Cell. 2006 Nov.

Abstract

In plants, the chromatin-associated high mobility group (HMG) proteins occur in two subfamilies termed HMGA and HMGB. The HMGA proteins are characterized by the presence of four AT-hook DNA binding motifs, and the HMGB proteins contain an HMG box DNA binding domain. As architectural factors, the HMG proteins appear to be involved in the regulation of transcription and other DNA-dependent processes. We have examined the subcellular localization of Arabidopsis thaliana HMGA, HMGB1, and HMGB5, revealing that they localize to the cell nucleus. They display a speckled distribution pattern throughout the chromatin of interphase nuclei, whereas none of the proteins associate with condensed mitotic chromosomes. HMGA is targeted to the nucleus by a monopartite nuclear localization signal, while efficient nuclear accumulation of HMGB1/5 requires large portions of the basic N-terminal part of the proteins. The acidic C-terminal domain interferes with nucleolar targeting of HMGB1. Fluorescence recovery after photobleaching experiments revealed that HMGA and HMGB proteins are extremely dynamic in the nucleus, indicating that they bind chromatin only transiently before moving on to the next site, thereby continuously scanning the genome for targets. By contrast, the majority of histone H2B is basically immobile within the nucleus, while linker histone H1.2 is relatively mobile.

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Figures

Figure 1.
Figure 1.
HMGA and HMGB1 Display a Similar Speckled Distribution Pattern in the Chromatin of Interphase Nuclei, but They Are Not Associated with Metaphase Chromosomes. Immunolocalization of HMGA ([A] and [B]) and HMGB1 ([C] and [D]) in mitotic interphase chromatin and condensed metaphase/anaphase chromosomes (indicated by arrows) of Arabidopsis root cells. Note that HMGA and HMGB1 immunolabeling occurs only with interphase chromatin but not with condensed mitotic chromosomes. Double immunolabeling for anti-HMGB1 and histone H3 phosphorylated at Ser-28 [p(Ser28)H3] in mitotic cells ([C] and [D]). The nuclear DNA has been counterstained with DAPI, which is depicted in blue in the merged images, while the HMGA and HMGB1 immunofluorescence is in red and the immunofluorescence of p(Ser28)H3 in green. Insets show individual nuclei at higher magnification.
Figure 2.
Figure 2.
Domain Structure of HMGA, HMGB1, and HMGB5. Plant HMGA proteins consist of a C-terminal domain containing four AT-hook motifs and have an additional plant-specific N-terminal domain with similarity to the globular domain of linker histone H1. The GRP core motifs of the four AT hooks of Arabidopsis HMGA (22.0 kD) are highlighted in gray and the N-terminal H1-like domain in black. The Arg residues (Arg-98 and Arg-164) that are part of putative NLSs and that have been mutated here are indicated in bold. Within the amino acid sequences of HMGB1 (20.2 kD) and HMGB5 (14.2 kD), the central HMG box DNA binding domain has been highlighted in black. Note the different length of the basic N-terminal and acidic C-terminal domains of HMGB1 and HMGB5. NLSs predicted by the Psort algorithm (http://psort.nibb.ac.jp/form.html) are indicated by asterisks, and basic residues in the N-terminal region of HMGB1 and HMGB5 (that may contribute to the nuclear targeting) are underlined.
Figure 3.
Figure 3.
DNA Binding Properties of HMGA, HMGB1, and the Corresponding GFP Fusion Proteins. The DNA interaction of HMGA and HMGB proteins examined using EMSA. Increasing amounts (indicated in nM, final concentration) of HMGA and GFP-HMGA have been incubated with an A/T-rich 106-bp DNA fragment (L106) originating from a maize zein gene promoter (left panels). Increasing amounts of HMGB1 and HMGB1-GFP were incubated with a mixture of linear (L78) and circular (C78) 78-bp DNA fragment (right panels). Protein-DNA complexes display a reduced electrophoretic mobility relative to the unbound DNA fragments.
Figure 4.
Figure 4.
A Basic Monopartite NLS Close to the Third AT Hook Is Critical for Nuclear Localization of HMGA. (A) Schematic representation of the analyzed HMGA-GFP fusions. The H1-like domain is highlighted in black, while the AT hooks are highlighted in gray. The position of the GFP relative to HMGA is also indicated for the different constructs. Asterisks indicate the Arg residues (Arg-98 and Arg-164) that have been mutated within the putative NLSs. The predominant subcellular localization of the fusion proteins is indicated (N, nuclear accumulation; N+C, localization in nucleus and cytoplasm) based on inspecting 60 to 80 transformed protoplasts each. (B) to (P) CLSM images of tobacco BY-2 cell protoplasts transiently transformed with plasmids driving the expression of the GFP fusion constructs indicated in (A). In (D), (F), and (H), overlays of the GFP fluorescence (seen in [C], [E], and [G], respectively) and the transmission image of the same protoplast are shown. Various control construct have been used for the subcellular localization studies: GFP alone ([B]; localizing to the nucleus and cytosol), GFP-NLS-CHS ([C] and [D]; localizing to the nucleus), and GFP-H1.2 ([E] and [F]; localizing to the nucleus). The different HMGA-GFP fusion constructs are shown in (G) to (P). In (O) and (P), the nuclei seen in (G) and (I) are shown in higher magnification. Bars = 10 μm.
Figure 5.
Figure 5.
Different Regions of HMGB1 and HMGB5 Determine Their Subcellular Localization. (A) Schematic representation of the HMGB1-GFP and HMGB5-GFP fusions used for protoplast transformation. The central HMG box DNA binding domain is highlighted in gray. The position of the GFP relative to the HMGB protein is also indicated for the different constructs. The predominant subcellular localization of the fusion proteins is indicated (N, nuclear accumulation; N+C, localization in the nucleus and cytosol; N+n, nuclear accumulation including localization in the nucleolus; N≤C or N≥C, indicating the tendency of higher fluorescence in the cytosol or in the nucleus, respectively, compared with the other compartment) based on inspecting 60 to 80 transformed protoplasts each. (B) to (Q) CLSM images of tobacco BY-2 cell protoplasts transiently transformed with plasmids driving the expression of the GFP fusion constructs indicated in (A). In (C) and (K), overlays of the GFP fluorescence (seen in [B] and [J]) and the transmission image of the same protoplast are shown. Nuclei of protoplasts expressing GFP-HMGB1 ([H] and [I]) and GFP-HMGB5 (Q) are shown at higher magnification. Images shown in (O) and (P) are slightly overexposed to clearly show the additional cytosolic localization. Bars = 10 μm.
Figure 6.
Figure 6.
Nuclear Localization of HMGA, HMGB1, HMGB5, and Histone H2B in Stably Transformed Arabidopsis Seedlings. Detection of nuclear GFP and YFP fluorescence by CLSM of roots of 3-d-old transgenic seedlings, expressing the indicated GFP/YFP constructs. Bars = 40 μm.
Figure 7.
Figure 7.
Measurement of the Mobility of Linker Histone H1.2 in Tobacco Protoplast Nuclei by FRAP. (A) Images of the GFP fluorescence of the nucleus of a transiently transformed BY-2 cell protoplast expressing the GFP-H1.2 fusion protein. Images have been taken before and 2 and 90 s after bleaching a small area within the nucleus. The bleached area is indicated by a circle. (B) Quantitative analysis of FRAP experiments summarizing the results from ≥10 individual cells. The time course of the relative fluorescence intensity has been determined within the bleached area. The initial fluorescence intensity measured before bleaching is reduced by ∼80% upon bleaching by a short laser pulse before it recovers over time due to influx of unbleached molecules from the area surrounding the bleached spot. The time required for half-maximal recovery (t1/2) and the mobile fraction (Mf) of the protein can be determined from the recovery data.
Figure 8.
Figure 8.
HMGA and HMGB Proteins Are Highly Mobile in Plant Cell Nuclei. FRAP curves depicting the time course of fluorescence recovery after bleaching (starting at 0 s). Root cells of stably transformed Arabidopsis seedlings (indicated by s) and transiently transformed BY-2 cell protoplast (indicated by p) expressing HMGA, HMGB1, and HMGB5 proteins and control proteins (H1.2 and H2B) fused to GFP/YFP were analyzed. (A) to (C) The results obtained with HMGA, HMGB1, and HMGB5 fused to GFP are shown in comparison to the GFP-H1.2 fusion. (D) The mobility of the control proteins GFP, GFP-H1.2, and H2B-YFP was measured in parallel (note the different time scale in [D] compared with [A] to [C]). GFP that does not interact with chromatin served as an inert nonbinding reference protein (Sprague and McNally, 2005), displaying an extremely rapid fluorescence recovery.
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