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First published online 22 June 2004
doi: 10.1242/jcs.01160

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Dynamic interaction of HMGA1a proteins with chromatin

¹ Department of Cell and Developmental Biology, University of Würzburg, Würzburg, 97080, Germany
² Division of Gastroenterology, Department of Medicine, University of Würzburg, Würzburg, 97080, Germany
³ Protein Section, LMC, DBS, NCI, NIH, Bethesda, MD 20892, USA

View larger version (60K): [in a new window]   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.

View larger version (103K): [in a new window]   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').

View larger version (42K): [in a new window]   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).

View larger version (39K): [in a new window]   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.

View larger version (54K): [in a new window]   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.

View larger version (40K): [in a new window]   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 (R3xG). (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.

View larger version (37K): [in a new window]   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.

View larger version (58K): [in a new window]   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 T3xA (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.

View larger version (37K): [in a new window]   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).