Research Article 3459 Dynamic interaction of HMGA1a proteins with

Monika Harrer1, Hardi Lührs2, Michael Bustin3, Ulrich Scheer1 and Robert Hock1,* 1Department of Cell and Developmental Biology, and 2Division of Gastroenterology, Department of Medicine, University of Würzburg, Würzburg, 97080, Germany 3Protein Section, LMC, DBS, NCI, NIH, Bethesda, MD 20892, USA *Author for correspondence (e-mail: [email protected])

Accepted 13 February 2004 Journal of Cell Science 117, 3459-3471 Published by The Company of Biologists 2004 doi:10.1242/jcs.01160

Summary High-mobility-group proteins A1 (HMGA1; previously proteins. Furthermore, by inhibiting kinase or named HMGI/Y) function as architectural chromatin- deacetylase activities, and with the help of fusion proteins binding proteins and are involved in the transcriptional lacking specific phosphorylation sites, we analyzed the regulation of several genes. We have used cells expressing effect of reversible modifications of HMGA1a on chromatin proteins fused to green fluorescent protein (GFP) and binding. Collectively our data show that the kinetic fluorescence recovery after photobleaching (FRAP) to properties of HMGA1a proteins are governed by the analyze the distribution and dynamics of HMGA1a in number of functional AT-hooks and are regulated by vivo. HMGA1-GFP proteins localize preferentially to specific phosphorylation patterns. The higher residence heterochromatin and remain bound to chromosomes time in heterochromatin and chromosomes, compared during mitosis. FRAP experiments showed that they with euchromatic regions, correlates with an increased are highly mobile components of euchromatin, phosphorylation level of HMGA1a. The regulated dynamic heterochromatin and of mitotic chromosomes, although properties of HMGA1a fusion proteins indicate that with different resident times. For a more-detailed HMGA1 proteins are mechanistically involved in local and investigation on the interaction of HMGA1a with global changes in chromatin structure. chromatin, the contribution of the AT-hook DNA-binding motifs was analyzed using point-mutated HMGA1a-GFP Key words: Chromatin, HMGA proteins, Dynamics, Phosphorylation

Introduction HMGA1† proteins preferentially bind to the minor groove The functional activity of the genome is controlled by its of AT-rich B-DNA with three AT-hook binding motifs (Reeves packaging into chromatin. Recent in vivo imaging approaches and Nissen, 1990). As shown by immunocytological revealed the dynamic behavior of structural chromatin proteins, approaches, HMGA1a/b proteins preferentially localize to the such as HMGN proteins and histone H1 (Lever et al., 2000; heterochromatin mass (Amirand et al., 1998; Martelli et al., Misteli et al., 2000; Phair and Misteli, 2000) or HP1 (Cheutin 1998). They bind to DNA elements termed scaffold attachment et al., 2003). Their kinetic properties were summarized in a regions (Zhao et al., 1993) and have been proposed to function ‘stop and go’ model, in which the molecules bind transiently as competitors of H1-mediated general repression of to chromatin, and diffuse through the nucleoplasm until they in vitro (Käs et al., 1993; Zhao et al., 1993). find another binding site (Misteli, 2001; Misteli et al., 2000). HMGA1 also has the ability to bind to DNA packaged in The residence times for histone H1 were found to be 3-4 nucleosomes and this ability is modulated by posttranslational minutes, compared with seconds in the case of HMGN modifications (Banks et al., 2000; Reeves, 2001; Reeves et al., proteins. Upon chromatin hyperacetylation, the residence times 2000). Furthermore, HMGA1 proteins are involved in were reduced indicating their dependence on the specific regulating the expression of specific genes, reviewed elsewhere properties of the chromatin as well, as the functional status (Reeves and Beckerbauer, 2001). The most accepted model of of the proteins itself (Misteli, 2001; Misteli et al., 2000). how they function in gene regulation is through either the The striking mobility of chromatin proteins has been facilitation, or inhibition, of the formation of ‘enhanceosomes’ mechanistically implicated in local and global reorganizations (Thanos and Maniatis, 1995). of chromatin, that is, the rapid association and dissociation of HMGA gene expression is maximal during embryonic chromatin proteins provide free binding sites for the same or development (Chiappetta et al., 1996), drops off in most adult other chromatin components, which could alter the chromatin tissues and is low, or undetectable, in fully differentiated or non- status (Misteli, 2001). Indeed, it was shown that HMGN dividing adult cells (Bustin and Reeves, 1996; Lundberg et al., proteins decrease the residence time of H1 on chromatin 1989). Overexpression of HMGA proteins correlates with and, therefore, may counteract the inhibitory effects of the †Formerly HMGI; the nomenclature of the HMG protein superfamily has been recently H1-induced higher-order chromatin structure (Catez et al., revised [see Bustin (Bustin, 2001) and http:/www.informatics.jax.org/mgihome/nomen/ 2002). genefamilies/hmgfamily.shtml]. 3460 Journal of Cell Science 117 (16) neoplastic transformation and tumor progression in many 3′. Combinations of mutations were made sequentially. Derived malign tumors (Tallini and Dal Cin, 1999). In most benign clones were verified by sequencing. mesenchymal neoplasias, chromosomal translocations lead to fusion proteins with multiple AT-hooks (Hess and Kossev, 2002). Transfection and cell treatments To gain insight into the in vivo distribution, function and dynamics of HMGA1a, we expressed GFP fusion proteins in HepG2 cells were transiently transfected using effectene (Quiagen) and 400 ng of plasmid DNA as specified by the manufacturer. HepG2 cells. First, the distribution and chromatin binding Transfected cells were grown on coverslips over night at 37°C and of the chimeric proteins was investigated by 5% CO2 to 50% confluence. Transfection rate was at least 50%, but immunolocalization and extraction experiments. Second, we routinely more than about 60%. Expression of HMGA1a-GFP did not used fluorescence recovery after photobleaching (FRAP) to interfere with cell growth over multiple cell cycles. For chromatin examine the dynamic behavior of the fusion proteins over acetylation, cells were incubated with 500 ng/ml Trichostatin A (TSA, euchromatin, heterochromatin and on mitotic chromosomes. Calbiochem) for 2 hours. Roscovitine (25 µM, Calbiochem) was These experiments revealed that HMGA1a proteins belong to incubated for 4 hours. A cell-permeable-specific inhibitor for protein the highly mobile components of interphase and mitotic kinase C, myristoyl-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH µ chromatin, although with different kinetic properties. Third, (ICN Biomedicals), was used at 10 g/ml for 4 hours. we used a set of point-mutated fusion proteins and FRAP to analyze the functional role of the AT-hook-binding motifs. We Live cell imaging and FRAP analyses identified the first two of the three AT hooks as main players For live-cell imaging, cells were grown on coverslips that were mediating DNA/chromatin binding. Fourth, the contribution mounted on a slide chamber with culture medium. Cells were analyzed of posttranslational modifications on HMGA1a dynamics in within the following 20-30 minutes at room temperature with a Leica vivo was analyzed by FRAP after inhibition of kinase and TCS-SP. FRAP experiments at 37°C, 5% CO2 showed now differences histone deacetylase activities. These experiments showed that in recovery kinetics compared with those obtained at room temperature the activity of p34/cdc2 kinase and protein kinase C as well as (not shown). Cells have been analyzed using the 488 nm laser line for histone deacetylases regulate the nuclear dynamic equilibrium GFP of an Ar/Kr laser (488 nm: 18 mW nominal output, pinhole setting × of HMGA1a. Fifth, we analyzed the contribution of specific at 1; 568 nm: 17 mW nominal output, pinhole setting at 1, 40 neofluar phosphorylation sites on the dynamic mobility of HMGA1a objective, N.A. 1.25). For bleaching, one single scan was acquired, followed by a single bleach pulse of 1 second using a spot of in vivo. The threonines in front of each AT hook were mutated approximately 1 µm in radius without imaging. For imaging, laser into alanines to yield single-, double- and triple-point-mutated power was attenuated to 4% of the bleach intensity. Single-section fusion proteins. FRAP experiments were performed to images were then collected at 1 second intervals (20 images), 2 seconds compare the mobility of the chimeric proteins in intervals (10 images), 5 seconds intervals (10 images) and in 10 heterochromatin and on chromosomes. Interestingly, these seconds intervals (20 images). To reduce chromosome movements in data suggested that, compared with euchromatin, the increased mitotic cells, FRAP experiments were performed in the presence of 10 residence time in heterochromatin and on mitotic µM taxol. Average fluorescence loss during imaging was 2-3%. FRAP chromosomes correlates with an increased phosphorylation. recovery curves were generated according to Phair and Misteli (Phair and Misteli, 2000) from background subtracted pictures. The quantitative values represent averages of at least 10 cells from three independent experiments. As standards we used GFP, H3-GFP and Materials and Methods fixed cells. Some recoveries were observed that were greater than Cloning of fusion proteins and site directed mutagenesis 100% because of the contribution of electronic noise in the detector The coding sequence of human HMGA1a was PCR amplified out of system. Student’s t-test was used to determine the statistical 2 µl reverse-transcribed HepG2 RNA using the primers 5′ significance of the results. ATGAGTGAGTCGAGCTCGAAGTCCAGC 3′ and 5′ ACTGCTCC- TCCTCCGAGGACTCC 3′ (Interactiva). This and all other PCR products were subcloned by TOPOTMTA cloning according the Fluorescence microscopy and native chromosome spreads manufacturers instructions (Invitrogen). The HMGA1 cDNA then was For DNA counterstaining with Hoechst 33258, transfected cells were EcoRI inserted into or pEGFPN1 (Clontech). briefly permeabilized with 0.1% Triton for 1 minute and then fixed in Site-directed mutagenesis of human HMGA1a was performed 2% formaldehyde in PBS for 10 minutes at room temperature. Cells essentially as described in the QuickChange® Site-Directed then were permeabilized with 100 µl 0,1% Triton X-100 in PBS for Mutagenesis Kit protocol (Stratagene). For mutation of hHMGA1a 10 minutes at room temperature and Hoechst 33258 was added at a phosphorylation sites, alanine codon substitutions were introduced at final concentration of 500 ng/ml. Cells were washed and mounted in the nucleotides coding for the amino acid threonine at positions 21 Mowiol as described (Hock et al., 1998). Hoechst-stained cells have (T21A), 53 (T53A) and 78 (T78A) of the hHMGA1a protein sequence been investigated with a confocal laser scanning microscope using the as reported elsewhere (Siino et al., 1995). 364 nm laser line of a Kr/Ar laser (Zeiss 510) or on a Zeiss Axiophot AT-Hook point mutations were introduced by glycine substitution equipped with a Pixera digital system. Immunfluorescence procedure of the arginine codon following the central glycine codon of the on formaldehyde fixed cells was essentially as previously described conserved AT-hook motif PRGRP at positions 28 (R28G), 60 (R60G) (Hock et al., 1998). Primary antibodies used for immunfluorescence and 86 (R86G). The following primers (Thermo Hybaid) were used experiments were anti-HMGA1 (gift from Ray Reeves), anti-sc35 in the PCR reactions, respectively: R28G 5′ CGGGGCCGGGG- (dilution 1:2000; Sigma) and anti-histone H1 (dilution 1:20; ICN). CGGGCCGCGCAAGCAG 3′; R28G reverse 5′ CTGCTTGCGC- Spreading of native chromosomes was essentially as described GGCCCGCCCCGGCCCCG 3′; R60G 5′ CCTAAGAGACCTC- (Christensen et al., 2002). GGGGCGGACCAAAGGGAAGC 3′; R60G reverse 5′ GCTT- CCCTTTGGTCCGCCCCGAGGTCTCTTAGG 3′; R86G 5′ GGAA- GGAAACCAAGGGGCGGACCCAAAAAACTGGAG 3′; R86G DNA-staining, run on transcription and salt extraction in situ reverse 5′ CTCCAGTTTTTTGGGTCCGCCCCTTGGTTTCCTTCC For in situ DNA-staining with Hoechst, transfected cells were Mobility of HMG proteins 3461

Fig. 1. (A) Distribution of HMGA1a-GFP fusion proteins in transfected cells. (a) Distribution of HMGA1a-GFP in living cells as revealed by fluorescence microscopy. Corresponding phase contrast image is shown in a′. (b) Optical section of a living cell expressing HMGA1a-GFP and overlay of the fluorescence picture with the corresponding dichromatic picture (b′). The distribution of the fusion proteins (c) is comparable with the distribution of endogenous HMGA1 in non-transfected cells (c′) and overlaps with endogenous HMGA1 in transfected cells (c′′). (B) Distribution of HMGA1a-GFP overlaps with Hoechst DNA staining in situ (a,a′) and in formaldehyde-fixed cells (b,b′). Bars correspond to 10 µm.

cold acetone. The precipitated proteins were washed twice with 70% acetone, air dried and resuspended in SDS sample buffer. SDS-PAGE and transfer onto nitrocellulose was performed as described previously (Hock et al., 1998). Dilution of primary antibodies for immunoblots were 0.2 µg/ml for anti-GFP (Roche), 0.5 µg/ml for anti- HMGA1a (Santa Cruz), 0.3 µg/ml anti-pep2 (Hock et permeabilized with 0.05% Triton X-100 in PBS containing 10 µg/ml al., 1998). The monoclonal antibody directed against actin (Gonsior Hoechst. Cells were incubated for 2-5 minutes and monitored et al., 1999) was used at a dilution of 1:1000. Blocking and incubation immediately. Longer exposure to Hoechst led to displacement of was performed in 5% non-fat dry milk/TBS for 1 hour at room HMGA1a-GFP. temperature except for the HMGA1 antibody, where the blocking was In situ run-on transcription was performed as described previously in TBS plus 0.1% Tween-20. Washing and detection was performed (Hock et al., 1998). After BrUTP incorporation, cells were fixed and as described. permeabilized as described above. Incorporated BrUTP was detected using anti-BrdUTP (Roche, Mannheim, diluted 1:25). Optical sections were recorded with a Leica TCS-SP using the ×40 neofluar oil- Results immersion objective (N.A. 1.25) with a pinhole setting at 0.8 and HMGA1a-GFP proteins are enriched in heterochromatin AOTF for 488 nm at 35%, and AOTF for 568 nm at 50%. Fluorescent in vivo signals of GFP and Texas-red were recorded simultaneously. During interphase, HMGA1-GFP proteins were distributed Dichromatic pictures were recorded in parallel. No crosstalk was throughout the cell nucleus excluding the nucleoli and were observed. For in situ salt extraction, cells grown on coverslips were washed enriched in multiple nuclear domains or foci (Fig. 1A,a,b). The fusion proteins colocalized with their endogenous counterparts twice in PBS and incubated in 3 ml cytoskeleton buffer [CSK; 100 ′′ mM NaCl, 300 mM sucrose, 10 mM Pipes, pH 6.8, 3 mM MgCl2, in transfected cells and in an identical pattern (Fig. 1A,c-c ). 0.5% Triton X-100, 1 mM PMSF, Leupetin, Pepstatin, Bestatin and Furthermore, the distribution of the fusion proteins was RNasin (MBI)] for 10 minutes at 4°C. CSK was removed by pipetting comparable with that of endogenous proteins in non- and cells were fixed in 2% formaldehyde, or incubated further for salt transfected cells (Fig. 1A,c-c′′). The HMGA1a-GFP extraction in 3 ml CSK with 350 mM NaCl for 5 minutes at 4°C or containing foci overlapped with high local concentrations of incubated with DNase I in digestion buffer [like CSK but with 50 mM DNA as shown by in situ Hoechst staining of unfixed and NaCl and 20 U/ml RNasin (MBI) and 200 U/ml DNase I (Roche)] for gently permeabilized HepG2 cells (Fig. 1B,a-a′) as well as in 30 minutes at room temperature. Cells then were fixed and processed fixed HepG2 cells (Fig. 1B,b-b′). This indicated that for immunfluorescence analyses as described above. HMGA1a-GFP preferentially localizes to heterochromatin. Essentially the same results were obtained with frog, mouse Electron microscopy and other human cell lines (data not shown). Our finding that Localization of HMGA1a-GFP on ultrastructural level was perfomed HMGA1a-GFP is enriched in heterochromatin is compatible essentially as described previously (Hock et al., 1998) using with previous immunocytological data on the localization of monoclonal antibodies directed to GFP (Roche) at 3 µg/ml and endogenous HMGA1 proteins (Amirand et al., 1998; Martelli secondary antibodies coupled to 12 nm gold particles (Dianova, et al., 1998). diluted 1:10). The preferential association of HMGA1a-GFP with heterochromatin was further supported by the overlapping ′′ Western Blot analyses and salt extraction of proteins distribution of HMGA1a-GFP and histone H1 (Fig. 2A,a-a ). Furthermore, in HMGA1a-GFP-expressing cells, transcription To test salt extraction properties of HMGA1a-GFP fusion proteins, HepG2 cells were transfected with human HMGA1a-GFP or GFP as sites were labeled by in situ run-on transcription using BrUTP a control, were washed twice with PBS and then incubated with PBS incorporation. Notably, the majority of HMGA1a-containing containing 0.5% Triton X-100 or PBS containing 350 mM salt and foci did not overlap with the BrUTP incorporation sites, further 0.5% Triton X-100 for 10 minutes at room temperature. Supernatants indicating that HMGA1a-GFP is preferentially associated with were collected and solubilized. Proteins were precipitated with ice transcriptionally inactive chromatin (i.e. heterochromatin). In 3462 Journal of Cell Science 117 (16)

Fig. 2. (A) Co-localization of HMGA1a-GFP and histone H1. HMGA1a-GFP pattern is shown in (a), histone H1 distribution in (a′) and the merged picture in (a′′). (b-b′′) Localization of HMGA1a- GFP compared with that of nascent transcripts. Nascent transcripts were BrUTP-labeled by in situ run-on transcription. After fixation, incorporated BrUTP was visualized by immunofluorescence (b′). (c-c′′) Co-localization of HMGN2-GFP with nascent transcripts (c′). Merged picture is shown in c′′. Most right panels are magnifications of the boxed areas in a′′, b′′ and c′′, respectively. Bars correspond to 10 µm. (B) Electron microscopy of untransfected (a,a′) or HMGA1a-GFP expressing cells (b,b′). Higher magnifications of the boxed areas in (a) and (b) are shown in (a′) and (b′), respectively. HMGA1a-GFP was localized with secondary antibodies coupled to 12 nm gold particles (b′, arrows). Note that HMGA1a expression does not alter bulk chromatin structure on ultrastructurel level (compare a and b) and that HMGA1a-GFP proteins are concentrated in domains (arrows, b′). Bars represent 5 µm in (a,b) and 200 nm in (a′,b′).

During mitosis, HMGA1a-GFP proteins were associated with the chromosomes of live cells without indications of banding patterns or axial concentrations (Fig. 4A,a,b). When we prepared native chromosome control experiments, when we examined the distribution of spreads from HMGA1a-GFP-expressing cells (see Christensen HMGN proteins, which are known to localize preferentially to et al., 2002), the homogenous distribution of HMGA1a-GFP transcriptionally active chromatin (Hock et al., 1998), there was lost over time and resulted in a more-banded pattern after was a clear overlap between the sites of HMGN2-GFP proteins an incubation time of 1-2 hours in 75 mM KCl (Fig. 4B). In and that of BrUTP-labeled nascent transcripts (Fig. 2A,c-c′′). addition, after prolonged incubation of the chromosomes, Interestingly, electron microscopic (EM) immunogold HMGA1a-GFP disappeared from the peripheral regions and localizations of HMGA1a-GFP revealed an accumulation of became enriched in the more-axial regions of the chromosomal gold particles at electron-dense chromatin structures, most arms (Fig. 4B,b′′). Thus, HMGA1a proteins are not stably likely representing heterochromatin foci (Fig. 2B,b′). The EM- bound to mitotic chromosomes but slowly dissociate, or studies also showed that HMGA1a-GFP expression did not redistribute, upon exposure of isolated chromosomes to an alter bulk chromatin structure at the ultrastructural level (Fig. isotonic buffer. 2B). The extractability of the fusion proteins was investigated in situ and by western blot experiments using permeabilized HMGA1a proteins are dynamically associated with transfected cells. In both assays, HMGA1a-GFP proteins were chromatin throughout the cell cycle salt extractable with 350 mM salt (Fig. 3A,e-h and 3B,a), To investigate HMGA1a dynamics in living cells, we used which is characteristic for members of all HMG protein fluorescence recovery after photobleaching (FRAP). Upon families. Endogenous HMGA1a (as well as HMGN) proteins bleaching of a small nuclear area of approximately 1 µm in shared the same solubility properties as the fusion proteins, diameter over euchromatin or heterochromatin, recovery of indicating proper DNA binding of HMGA1a-GFP (Fig. fluorescence was measured (Fig. 5, Table 1). For these 3B,b,c). In addition, digestion of DNA by DNase I resulted in experiments, heterochromatin was defined on the basis of a total release of HMGA1a-GFP, indicating that the fusion morphological criteria as areas strongly labeled with proteins were not associated with non-chromatin structures HMGA1a-GFP; euchromatin was defined morphologically as (Fig. 3A,j). As a control for the in situ salt extraction and areas weakly labeled with HMGA1a-GFP. DNase I digestion experiments, we localized sc35, a non- In euchromatin, complete fluorescence recovery of the snRNP splicing factor (Spector et al., 1991). Both treatments bleached spot occurred in approximately 23 seconds. After less did not solubilize sc35, which is in striking contrast to the than 1 second, 50% of the prebleach fluorescence and after behavior of HMGA1a-GFP (Fig. 3A,g,h,l,m). Significantly, 4.2 seconds already 80% of the prebleach fluorescence was when cells were treated with a buffer of low ionic strength regained. (CSK buffer), sc35 localized outside of regions with high In heterochromatin, fluorescence recovery was complete HMGA1a concentrations (Fig. 3A,c,d). after approximately 37 seconds with 50% and 80% recovery Mobility of HMG proteins 3463

Fig. 3. (A) In situ extraction experiments of HMGA1a-GFP. (a) Permeabilized cells expressing HMGA1a-GFP incubated in low salt extraction buffer retained nuclear fluorescence. Incubation in buffer containing 350 mM salt resulted in a loss of nuclear fluorescence (e) as well as after treatment with DNase I (j). Localization of the non-snRNP splicing factor sc35 was used as a control for the extraction experiments (c,g,l). Note that the distribution of sc35 and HMGA1a-GFP does not overlap (d). DNA was counterstained with Hoechst 33258 (b-k). Overlays are shown in (d,h,m). Cells were monitored using a Zeiss Axiophot equipped with a Pixera Digital Imaging System. Bars represent 10µm. (B) Extractability of HMGA1a-GFP as analyzed in western blot experiments. Cells transfected with HMGA1a-GFP were extracted with PBS (140 mM salt) or PBS containing 350mM salt, respectively. Solubilized proteins were precipitated and submitted to western blot analyses. Stripped blots were reprobed with antibodies directed to HMGA1 proteins, HMGN proteins or actin. HMGA1a-GFP was detected with an antibody directed against GFP. Note that comparable with endogenous HMGA1 (b) and HMGN (c) proteins, HMGA1a-GFP (a) are extracted in 350 mM salt. Control cells expressing GFP were treated like described above. Solubility of GFP is independent of salt treatment (e). Detection of cellular actin was used to show equal loading of extracted proteins (d,f).

Fig. 4. (A) In vivo localization of HMGA1a- GFP on chromosomes (a,b). Overlay of HMGA1a-GFP with the corresponding dichromatic picture is shown in (a′) and comparison with Hoechst staining in (b′). Bars represent 10 µm. (B) Native chromosome spreads. Cells were swollen in 75 mM KCl and spotted on glass slides and incubated in isotonic buffer. Chromosomes were monitored after 30 minutes (a-a′′) and 1.5 hours (b-b′′) with a confocal microscope. The HMGA1a-GFP patterns are shown in (a,b). Counterstaining with propidium-iodide is shown in (a′) and (b′). Note, that after prolonged incubation the homogenous chromosomal distribution is lost. Bar represents 1 µm. of the prebleach intensity after 3.3 seconds and 12 seconds, recoveries to 80% of the prebleach intensity were compared. respectively (Table 1). Thus, the mobility of HMGA1a-GFP in Recovery to 80% of bleached heterochromatin located directly heterochromatin is significantly slower (P<0.004) than in near the nucleolus was significantly faster (7.3 seconds) euchromatin, indicating more-tightly bound HMGA1a-GFP in as compared with heterochromatin located within the heterochromatin. Irrespective of these differences, the FRAP nucleoplasm (19.5 seconds; P<0.0001; Fig. 5C). Notably, data show that HMGA1a proteins belong to the highly mobile kinetic differences as a function of intranuclear localization components of chromatin. Remarkably, the recovery kinetics were found exclusively in the case of HMGA1a proteins and over heterochromatin were quite variable depending on where were undetectable when the dynamic properties of HMGN- or the bleach spot was set. This was especially striking when the HMGB-GFP were probed. In these cases, fluorescence 3464 Journal of Cell Science 117 (16)

Fig. 5. (A) Fluorescence recovery after photobleaching (FRAP) of cells expressing HMGA1a-GFP. Cells were bleached for 1 second in an area of approximately 1 µm (circle) and the recovery of fluorescence was measured in interphase (arrows, upper panels) or mitotic cells (arrows, lower panels). Pictures of selected time points of fluorescence recovery are shown as indicated. Bars represent 10 µm. (B-D) Quantitative analyses of FRAP experiments in euchromatin (B; compared with heterochromatin, hc), in heterochromatin (C; compared with heterochromatin near nucleolus and heterochromatin in nucleoplasm) and in chromosomes (D; compared with heterochromatin). Note the difference in the kinetic properties after bleaching heterochromatin near the nucleolus or within the cytoplasm (C) and the reduced mobility of HMGA1a-GFP bound to chromosomes (D). (E) Comparison of recovery kinetics with members of the HMGB- and the HMGN-families.

Table 1. Recovery kinetics of wild type and mutant HMGA1a-GFP fusion proteins in interphase cells t50 t80 Complete recovery Experiment Time (s)±s.d. t-test Time (s)±s.d. t-test Time (s)±s.d. t-test HMGA1a (euchromatin) <1 N.A. 4.2±1.3 P<0.0001 23.0±5.0 P<0.004 HMGA1a (heterochromatin) 3.3±2.0 12.0±2.7 37.0±12.3 HMGA1a (heterochromatin/nucleoplasm) 5.6±1.6 P<0.005 19.5±5.6 P<0.0001 45.5±11.3 P<0.08 HMGA1a (heterochromatin/near nucleolus) 2.2±0.9 P<0.05 7.3±2.7 P<0.0001 31.3±9.4 P=0.1

HMGA1a+PKC inhibitor 1.6±0.7 P<0.05 6.1±2.9 P<0.0001 22.8±6.4 P<0.005 HMGA1a+roscovitine 2.0±0.4 P=0.1 5.5±1.7 P<0.0001 18.7±5.5 P<0.001 HMGA1a+TSA 1.9±0.3 P<0.05 6.4±1.8 P<0.0001 24.7±6.4 P<0.005

T21A 2.2±0.7 P=0.2 8.5±2.8 P<0.01 27.8±7.7 P<0.06 T53A 2.8±0.8 P=0.5 12.9±2.7 P=0.5 37.5±3.5 P=1.0 T78A 2.1±0.3 P<0.08 9.2±3.4 P<0.06 30.0±4.5 P=0.1 T21,53A <1 N.A. 3.5±0.7 P<0.0001 17.2±2.9 P<0.001 T21,78A 1.3±0.4 P<0.006 5.5±2.1 P<0.0001 27.5±4.1 P<0.04 T53,78A 2.1±0.8 P=0.1 8.5±3.9 P=0.03 36.6±9.4 P=0.9 T3xA 2.8±1.0 P=0.5 10.0±3.1 P=0.14 36.2±4.7 P=0.85

R28G 1.1±0.2 P=0.003 4.8±1.4 P<0.0001 19.3±2.1 P=0.0003 R60G 1.2±0.4 P=0.005 4.2±1.5 P<0.0001 18.5±2.2 P=0.0002 R86G 2±0.8 P=0.07 7.6±2.2 P=0.0008 38.1±4.6 P=0.8 R28,60G <1 N.A. 2.7±1.0 P<0.0001 12.4±4.4 P<0.0001 R28,86G <1 N.A. 2,4±0.6 P<0.0001 17.3±2.1 P<0.0001 R60,86G <1 N.A. 2,2±0.6 P<0.0001 13.4±1.8 P<0.0001 R3xG <1 N.A. 2.0±0.3 P<0.0001 7.9±1.6 P<0.0001

In all cases nucleoplasmic heterochromatin or specific regions as notified in the table were bleached. The time required to reach 50% (t50) and 80% (t80) of the prebleach fluorescence intensity or to reach complete recovery was determined from each curve. In all cases, unless indicated otherwise, nucleoplasmic heterochromatin was bleached. The values given are means from recovery curves of at least 10 cells. The standard deviation (±s.d.) is given for each value. Values were compared with the average recovery values obtained after bleaching heterochromatin. The statistical significance was determined by Student’s t-test. Mobility of HMG proteins 3465

Table 2. Recovery times for wild type and mutant HMGA1a-GFP fusion proteins after bleaching mitotic chromosomes t50 t80 Complete recovery Experiment Time (s)±s.d. t-test Time (s)±s.d. t-test Time (s)±s.d. t-test HMGA1a (chromosomes) 9.1±4.0 23.0±10.8 52.6±10.1

T21A 1.3±0.5 P<0.0001 3.8±1.0 P<0.0001 11.8±2.0 P<0.0001 T53A 2.2±0.6 P<0.0001 7.5±3.0 P=0.0004 18.0±6.5 P<0.0001 T78A 2.3±0.4 P<0.0001 5.7±1.1 P<0.0001 16.9±8.2 P<0.0001 T21,53A <1 n.a. 4.0±1.4 P<0.0001 13.1±1.6 P<0.0001 T21,78A 1.2±0.3 P<0.0001 4.9±1.3 P<0.0001 11.8±2.4 P<0.0001 T53,78A 2.1±0.3 P<0.0001 6.7±1,1 P=0.0002 16.4±1.7 P<0.0001 T3xA 3.5±1.2 P=0.0005 9.3±3.6 P=0.001 23.9±8.2 P<0.0001

Recovery times required to achieve 50% (t50) and 80% (t80) of the prebleach fluorescence intensity and the time required for complete recovery are given for wild type and mutant HMGA1a-GFP fusion proteins after bleaching mitotic chromosomes. The values given are means from recovery curves of at least 10 cells. Recovery kinetics of mutant proteins were compared with those of the wild-type fusion protein. Standard deviations are given. Statistical significance was determined by Student’s t-test.

recovery resulted in kinetics shown in Fig. 5E and were In mitotic cells, mutations of either AT hook I (R28G), II independent from the location of the bleached region. (R60G) or III (R86G) induce no (R28G), or only a slight, To apply FRAP experiments on mitotic chromosomes, increase of the cytoplasmic non-chromosome-bound fraction positional movements of the metaphase plate had to be of the GFP fusion proteins (Fig. 6A, lower panel) compared prevented by taxol treatment. Upon bleaching a small region with wild-type constructs (Fig. 6A, wt). The mitotic of metaphase chromosomes, the fluorescence signal recovered chromosomes still fluoresced, indicating that two functional completely within approximately 53 seconds (Fig. 5D; AT-hook domains are sufficient to mediate HMGA1a binding Table 2), which is significantly slower than the average to chromosomes. Expression of fusion proteins with two recovery time of 37 seconds over interphase heterochromatin mutated hook domains clearly compromised DNA binding as (P<0.005; Table 1). This observation shows that the HMGA1a shown by the decrease of chromosomal fluorescence and proteins of mitotic chromosomes are permanently exchanged increase in cytoplasmic fluorescence (Fig. 6A). When all three but that their residence time on mitotic chromosomes is higher hook domains were mutated (R3×G), the fusion proteins were compared with interphase heterochromatin. distributed evenly without any preferential chromosomal binding (Fig. 6A, lower panel, R3×G). To quantify the interaction of the various AT-hook mutants AT-hook motifs I and II of HMGA1a are main mediators with DNA, we performed FRAP experiments. Here, the bleach of DNA binding in vivo spots were set over areas strongly labeled with mutant HMGA proteins contain three DNA-binding domains termed HMGA1a-fusion proteins located within the nucleoplasm and AT hooks. In mammals, the AT-hook motifs share the the recovery kinetics were compared with those obtained after consensus sequence P-R-G-R-P flanked by other positively bleaching similar regions labeled by the wild-type fusion charged residues (Reeves and Beckerbauer, 2001). To protein. Mutation of either AT hook I (R28G) or II (R60G) investigate how the individual AT-hook motifs I, II and III significantly increased the mobility of the fusion proteins contribute to DNA binding in vivo, we have replaced the compared with those obtained for the wild-type protein (Fig. second arginine of the P-R-G-R-P consensus with glycine. 6B, Table 1). Interestingly, both single-domain mutants The arginines were chosen because the side chains of the showed a comparable increase in the kinetics of fluorescence arginines are responsible for the contact of the AT hook with recovery (Fig. 6B, Table 1), indicating an equal contribution DNA (Huth et al., 1997). GFP-fusions of the point-mutated of AT hook I and II for DNA binding. By contrast, the mobility proteins were transiently expressed in human cells and their of AT hook III mutants (R86G) was only moderately increased DNA-binding properties studied by FRAP. The fusion (Fig. 6B, Table 1). Thus, this domain contributes to the proteins examined were point mutated in the AT hook I interaction of HMGA1a with DNA to a lesser degree than AT (R28G), AT hook II (R60G) or AT hook III (R86G), double- hook I or II. point-mutated in AT hook I+II (R28,60G), I+III (R28,86G), The double domain mutants (AT hook I + II, I + III and II II+III (R60,86G) or triple-point-mutated in AT hook I+II+III + III) caused an increased mobility as compared with the (R3×G). single domain mutants (Fig. 6B, Table 1). When we mutated Compared with wild-type HMGA1a, point-mutations of all three AT-hook domains, the interaction of the mutant either AT hook I, II or III caused a more diffuse and less protein (R3×G) with DNA was even more impaired as punctuate distribution in interphase cell nuclei (Fig. 6A, upper evidenced by its relatively high mobility and full fluorescence row, images R28G, R60G and R86G). However, in all three recovery within 8 seconds compared with 12 seconds of the cases the mutated fusion proteins still accumulated in double mutant (Fig. 6B, Table 1). Taken together, our results heterochromatin blocks. By contrast, mutation of two (Fig. 6A, demonstrate that the AT-hook motif governs the HMGA images R28,60G, R28,86G and R60,86G) or all three AT-hook distribution in vivo and that all three AT-hook motifs motifs (R3×G) caused an almost homogenous distribution of contribute to the interaction of HMGA1a proteins with DNA, the fusion proteins throughout the entire nucleus. although to different extents. 3466 Journal of Cell Science 117 (16)

Fig. 6. In vivo distribution (A) and dynamics (B) of fusion proteins with point mutations in the AT hook DNA- binding motifs. The mutants contained a substitution of the second arginine by glycine in the consensus AT hook peptide PRGRP. GFP-fusion proteins analyzed contained a single point mutation within AT hook I (R28G), II (R60G), III (R86G), two point mutations in AT hooks I and II (R28,60G), I and III (R28,60G), II and III (R60,86G) or three point mutations in AT hook I, II and III (R3×G). (A) Distribution of mutated fusion proteins in interphase (upper panels) or during mitosis (lower panels). Pictures are optical sections made with a confocal laser scanning microscope. Note the increased homogeneous distribution in interphase and the reduced chromosomal localization during mitosis compared with the wild-type fusion protein (wt). (B) Recovery kinetics of the point-mutated proteins introduced in (A) as revealed by FRAP.

Inhibition of protein kinase C, p34-kinase or histone cell permeable PKC-specific inhibitor and p34 activity by deacetylases increases HMGA1a mobility in roscovitine. Inhibition of both protein kinases resulted in a heterochromatin more diffuse distribution of HMGA1a-GFP (Fig. 7A,a,b). HMGA1 proteins are among the most extensively modified FRAP analyses of heterochromatin after drug treatments nuclear proteins [e.g. phosphorylations, acetylations and showed that inhibition of PKC led to a significantly increased methylations; reviewed in (Banks et al., 2000; Reeves and mobility of HMGA1a-GFP compared with untreated cells (Fig. Beckerbauer, 2001)]. Protein kinase C (PKC) and p34-kinase 7B, Table 1). After inhibition of PKC, full recovery of a have both been implicated in the phosphorylation of HMGA1 bleached region already occurred in 17-25 seconds. A similar proteins in vivo (Banks et al., 2000; Schwanbeck and increased mobility was measured after p34 inhibition by Wisniewski, 1997; Xiao et al., 2000). To test whether inhibition roscovitine (Fig. 7B, Table 1). Here, the fluorescence recovery of phosphorylation affects the distribution and dynamics of the bleached region occurred after 14-23 seconds. These of HMGA1a-GFP proteins, we blocked PKC activity by a experiments demonstrate that reduced phosphorylation by Mobility of HMG proteins 3467

Fig. 7. In vivo distribution (A) and dynamics (B) of HMGA1a-GFP proteins after inhibition of p34 by roscovitine (a), after inhibition of protein kinase C (PKC) by a specific cell permeable inhibitory peptide (b) and after inhibition of histone deacetylases by Trichostatin A [TSA, shown in (c)]. Note the altered distribution of HMGA1a-GFP after the different drug treatments. Bar represents 10 µm. (B) Recovery kinetics of HMGA1a-GFP after bleaching of drug treated cells. Treatment of cells with Roscovitin, PKC inhibitor or TSA increased the HMGA1a-GFP mobility compared with untreated cells.

The mobility of the point-mutated proteins in heterochromatin (located within the nucleoplasm) was investigated. FRAP analyses of the single mutants T21A and T78A revealed a slightly, but significantly, increased mobility (Fig. 9, Table 1). By contrast, the kinetic behavior of the T53A point mutant was identical to that of wild-type HMGA1a-GFP (Fig. 9, Table 1). These results indicate that both phosphorylation sites T21 and T78, but not T53, are involved in regulating binding to PKC and/or p34 weakens the interaction of HMGA1a with heterochromatin. When we analyzed the double-point mutants DNA/chromatin in vivo. (T21,53A; T21,78A; T53,78A) by FRAP, the T21,53A and the To test whether acetylation has an impact on HMGA1a-GFP T21,78A mutants showed a significant increase in mobility dynamics, histone deacetylases (HDACs) were inhibited by compared with the wild-type protein (Fig. 9, Table 1). This trichostatin A (TSA). This treatment also resulted in a less- indicates that T21 acts cooperatively with other sites in confined distribution of HMGA1a-GFP (Fig. 7A,c). In FRAP- regulating heterochromatin binding. By contrast, the recovery experiments of TSA-treated cells, fluorescence recovery of a kinetics of the double-point-mutant T53,78A was comparable bleached region was complete in 20-28 seconds compared with with that of the single point mutant T21A or T78A (Fig. 9, approximately 37 seconds in untreated cells (Fig. 7B, Table 1). Table 1), respectively. Furthermore, additional mutation of T53 These results indicate that chromatin acetylation increases reduced the cooperative effect of the T21,78A double mutant HMGA1a dynamics in vivo. in the triple mutant T3×A. Significantly, in cells expressing Thus, after kinase or HDAC inhibition, the kinetic T3xA, fluorescence recovery was slower than that of T21,78A differences of HMGA1a-GFP found in heterochromatin were and identical to that of the wild-type protein (Fig. 9, Table 1). lost. This suggests that the protein kinases and histone These results indicate that phosphorylation of T53 does not deacetylases are involved in targeting HMGA1a to directly contribute to HMGA1a heterochromatin binding in heterochromatin. interphase cells but weakens the effect of phosphorylations at T21 and T78. However, they also suggest that different combinations of phosphorylation patterns act cooperatively Point mutations of phosphorylation sites increase (in the case of T21,53A and T21,78A) in stabilizing the HMGA1a mobility in heterochromatin and on heterochromatin binding but also non-cooperatively (in T3×A) chromosomes in weakening heterochromatin binding. To rule out non-specific or indirect effects by drug-induced In mitotic cells, all GFP-fusion proteins mutated at kinase inhibition, we used HMGA1a-GFP fusion proteins that phosphorylation sites were preferentially located to were point mutated at the phosphorylation sites T21, T53 and chromosomes, but were also found in the cytoplasm, indicating T78 by substituting the threonine by alanine residues (Siino et a reduced binding to chromosomes (Fig. 8). FRAP-analyses of al., 1995). At T53 and T78, HMGA1a proteins are known to chromosomes showed that all point-mutated fusion proteins be heavily phosphorylated, especially during mitosis (Nissen have significantly altered dynamics during mitosis (Fig. 9, et al., 1991). Mutants T21A, T53A and T78A bore a point Table 2). Compared with the wild-type HMGA1a-GFP, mutation in one phosphorylation site, T21,78A, T21,53A fluorescence recovery of a bleached chromosome occurred in and T53,78A were double-point mutants and all three a quarter of the time taken in T21A (Table 2). For T53A and phosphorylation sites were mutated in the triple mutant T3×A T78A it occurred in a third of the time (Table 2). Comparable (T21,53,78A). Mobility and distribution of these point-mutants with the kinetic properties found in interphase were analyzed by FRAP during interphase and mitosis. heterochromatin, the double-mutated T21,53A and T21,78A The cellular distribution of the mutant HMGA1a-GFP fusion proteins displayed cooperative effects resulting in a proteins is depicted in Fig. 8. Compared with the wild-type faster recovery of bleached chromosomes compared with the HMGA1a-GFP the single, double or triple mutants showed a single mutants. Likewise, the mobility of the T53,78A mutant less-prominent accumulation in heterochromatin, and during or the triple mutated T3×A was reduced compared with the mitosis the mutated fusion proteins were enriched in the T21,78A double mutant (Table 2). Thus, as in interphase cells, cytoplasm. this indicates the same cooperative and non-cooperative 3468 Journal of Cell Science 117 (16)

Fig. 8. In vivo distribution of HMGA1a-GFP fusion proteins mutated in threonine phosphorylation sites. Threonines at position 21, 53 or 78 were substituted by alanine to yield the single point mutated proteins T21A, T53A and T78A, the double point mutated proteins T21,53A, T21,78A and T53,78A and the triple point mutated T3×A (with alanine substitutions at positions 21, 53 and 78). Note, that the distribution of the fusion proteins is less distinct in heterochromatin and shows only a slightly increased cytoplasmic fluorescence during mitosis as compared with the wild type (wt) fusion protein. Bar represents 10 µm.

1987; Strick and Laemmli, 1995; Zhao et al., 1993). Nevertheless, we found that HMGA1a-GFP fusion proteins associate dynamically with DNA/chromatin throughout the cell cycle. This implies that HMGA1a proteins do not represent an immobile constituent of either the nuclear matrix or of a chromosome scaffold. Comparable with this conclusion is the recently published finding that topoisomerase II, another member of the matrix or scaffold fraction, is highly dynamic (Christensen et al., 2002). However, as for topoisomerase II, the mobility of HMGA1a does not rule out a dynamic interaction with such structures. Conversely, the HMGA1a mobility provides us with scope to explain the multiple functions of these proteins in regulating transcription and as architectural elements of the chromatin. Despite their preferential influence of different phosphorylation patterns. However, by association with heterochromatin, the mobile HMGA1a contrast with interphase cells, our results show that all proteins can move to transcription sites and participate in the threonines next to each AT hook play a major role in regulating transcriptional regulation of several genes. In addition, the HMGA1a DNA/chromatin binding during mitosis. HMGA1a mobility would also ensure a dynamic interplay with other chromatin components, as discussed elsewhere (Käs et al., 1993; Zhao et al., 1993). For example, HMGA proteins Discussion were considered as anti-repressor molecules that could locally Recent studies using photobleaching techniques have shown displace inhibitory chromatin proteins, such as histone H1, at that many chromatin-associated proteins, such as histone H1 AT-rich SAR sequences. (Lever et al., 2000; Misteli et al., 2000), HMGN proteins Our investigations using point-mutated fusion proteins show (Catez et al., 2002; Phair and Misteli, 2000), HMGB proteins that distribution and dynamics are essentially governed by (Bianchi and Beltrame, 2000), topoisomerase II (Christensen functional AT-hook motifs. Actually, all three AT hooks et al., 2002) or heterochromatin protein 1 (Cheutin et al., 2003), contribute to DNA/chromatin-binding in vivo. AT hooks I and move rapidly through the nucleus of a living cell. Here we II are the main mediators of DNA binding, whereas AT hook present data on the dynamic properties of another class of III plays only a secondary, but nevertheless cooperative part. HMG proteins, the HMGA1a proteins. By transient expression For proper binding to chromatin in vivo, two functional AT of GFP-tagged wild type and mutant fusion proteins in human hooks are necessary and sufficient. This conclusion is in cells, we have studied the in vivo distribution and dynamic accordance with several prior in vitro investigations (Claus et interaction with DNA/chromatin by using FRAP experiments. al., 1994; Frank et al., 1998; Maher and Nathans, 1996; Yie et HMGA1a-GFP fusion proteins were distributed throughout al., 1997). the nuclear interior, excluding the nucleoli, with a distinct As the HMGA proteins are one of the most extensively enrichment in heterochromatin blocks. Double-localization modified nuclear proteins (Reeves, 2001), we also investigated experiments localizing histone H1 or nascent transcripts the contribution of modifications on the HMGA1a revealed that HMGA1a-GFP proteins were preferentially DNA/chromatin binding. Treatment of cells with drugs enriched in transcriptionally inactive, condensed chromatin. inhibiting kinases, specifically protein kinase C (PKC) and p34- Likewise, HMGA1a-GFP proteins were associated with kinase, or histone deacetylases (HDACs), increased the chromosomes during mitosis. This distribution corresponds to HMGA1a-GFP mobility in heterochromatin. Interestingly, the previous immunfluorescence studies (Amirand et al., 1998; different kinetic properties found in nucleoplasmic or Martelli et al., 1998) and earlier observations, describing that perinucleolar heterochromatin regions of untreated cells were HMGA1a proteins bind preferentially to A-tracts within lost after drug treatment. This observation indicates that heterochromatic regions (Radic et al., 1992; Reeves and Elton, phosphorylation and acetylation of HMGA1a modulates its Mobility of HMG proteins 3469 binding to heterochromatin. In fact, previous studies identified phosphorylation at T53 regulates heterochromatin binding in PKC as a possible kinase acting on HMGA proteins in vitro combination with T21 and T78. These FRAP data show that (Schwanbeck and Wisniewski, 1997) and in vivo (Banks et al., different combinations of phosphorylations result in different, 2000; Xiao et al., 2000), and the increased dynamics after but not necessarily cooperative, changes in HMGA1a inhibition of HDACs is consistent with the finding that dynamics. Consequently, different combinations of HMGA1a acetylation results in destabilization of the phosphorylations lead to different kinetic properties and fine- enhanceosome (Munshi et al., 1998). tune HMGA1a dynamics especially in heterochromatin. In Our data suggest that the consequence of HMGA1a sum, these data imply that, compared with euchromatin, the hypophosphorylation is a reduced affinity to heterochromatin. increased residence times found in heterochromatin and in Conversely, we can assume that hyperphosphorylation should mitotic chromosomes are caused by an increased increase HMGA1a binding to heterochromatin. This phosphorylation of HMGA1a proteins. However, whether assumption is supported by a decreased mobility of HMGA1a- HMGA1a hyperphosphorylation induces chromatin GFP over mitotic chromosomes, when HMGA1a proteins are condensation in vivo still has to be examined. Similar to known to be hyperphosphorylated (Nissen et al., 1991). HMGA1a, heterochromatin binding of histone H1 is also FRAP studies using point-mutated proteins, where one or affected by the phosphorylation status (Contreras et al., 2003; several mitosis-specific phosphorylation sites were replaced, Dou et al., 2002). However, in contrast with HMGA1a confirmed the regulatory role of HMGA1a phosphorylation on proteins, histone H1 in heterochromatin is in an its dynamic behavior. All point-mutated fusion proteins studied unphosphorylated state (Contreras et al., 2003). showed increased mobility during mitosis, which strongly Many previous investigations showed that phosphorylation indicates that reduced phosphorylation (mimicked here by the of HMGA proteins reduces DNA binding in vitro (reviewed in mutations T21A, T53A, T78A, or double and triple mutations Reeves and Beckerbauer, 2001). In vivo, DNA is packed into at these sites) reduces binding to DNA/chromatin. Notably, chromatin and HMGA proteins were also shown to interact this implies that besides the known mitosis-specific with nucleosomes (Reeves et al., 2000). Therefore, we can phosphorylation sites T53 and T78 (Nissen et al., 1991), the assume that phosphorylated HMGA1a proteins preferentially threonine at position 21 plays a crucial role for the regulation of HMGA1a binding to chromosomes. Thus, all threonines that are next to the AT-hook-binding motifs are involved in regulating the chromatin binding during mitosis and phosphorylation of these threonines increases binding of HMGA1a to chromosomes in vivo. FRAP experiments also showed that some of these threonines play a crucial role for heterochromatin binding during interphase. The threonines at the positions 21 and 78 appear to be most relevant to this phenomenon, which have already been shown to be phosphorylated in vitro and in vivo (Banks et al., 2000; Schwanbeck and Wisniewski, 1997; Xiao et al., 2000). Alone, mutation of the threonine at position 53 next to AT hook II does not play a significant role for heterochromatin binding. However, investigation of double- or triple-point-mutated proteins by FRAP indicates that

Fig. 9. Kinetics of recovery after bleaching nucleoplasmic heterochromatin (A) or mitotic chromosomes (B) in cells expressing the point-mutated fusion proteins shown in Fig. 8. The kinetics of the mutant fusion proteins (red curves) are compared with those of the wild-type HMGA1a-GFP (black curves). 3470 Journal of Cell Science 117 (16) interact with positively charged , while Dou, Y., Bowen, J., Liu, Y. and Gorovsky, M. A. (2002). Phosphorylation dephosphorylated HMGA1a proteins preferentially interact and an ATP-dependent process increase the dynamic exchange of H1 in with negatively charged DNA. This phosphorylation- chromatin. J. Cell Biol. 158, 1161-1170. Frank, O., Schwanbeck, R. and Wisniewski, J. R. (1998). Protein dependent movement between DNA binding and histone footprinting reveals specific binding modes of a high mobility group protein binding might contribute to activation and inactivation of I to DNAs of different conformation. J. Biol. 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