Introduction The functional activity of the genome is controlled by its packaging into chromatin. Recent in vivo imaging approaches revealed the dynamic behavior of structural chromatin proteins, such as HMGN proteins and histone H1 (Lever et al., 2000; Misteli et al., 2000; Phair and Misteli, 2000) or HP1 (Cheutin et al., 2003). Their kinetic properties were summarized in a ‘stop and go’ model, in which the molecules bind transiently to chromatin, and diffuse through the nucleoplasm until they find another binding site (Misteli, 2001; Misteli et al., 2000). The residence times for histone H1 were found to be 3-4 minutes, compared with seconds in the case of HMGN proteins. Upon chromatin hyperacetylation, the residence times were reduced indicating their dependence on the specific properties of the chromatin as well, as the functional status of the proteins itself (Misteli, 2001; Misteli et al., 2000). The striking mobility of chromatin proteins has been mechanistically implicated in local and global reorganizations of chromatin, that is, the rapid association and dissociation of chromatin proteins provide free binding sites for the same or other chromatin components, which could alter the chromatin status (Misteli, 2001). Indeed, it was shown that HMGN proteins decrease the residence time of H1 on chromatin and, therefore, may counteract the inhibitory effects of the H1-induced higher-order chromatin structure (Catez et al., 2002). HMGA1 † proteins preferentially bind to the minor groove of AT-rich B-DNA with three AT-hook binding motifs (Reeves and Nissen, 1990). As shown by immunocytological approaches, HMGA1a/b proteins preferentially localize to the heterochromatin mass (Amirand et al., 1998; Martelli et al., 1998). They bind to DNA elements termed scaffold attachment regions (Zhao et al., 1993) and have been proposed to function as competitors of H1-mediated general repression of transcription in vitro (Käs et al., 1993; Zhao et al., 1993). HMGA1 also has the ability to bind to DNA packaged in nucleosomes and this ability is modulated by posttranslational modifications (Banks et al., 2000; Reeves, 2001; Reeves et al., 2000). Furthermore, HMGA1 proteins are involved in regulating the expression of specific genes, reviewed elsewhere (Reeves and Beckerbauer, 2001). The most accepted model of how they function in gene regulation is through either the facilitation, or inhibition, of the formation of ‘enhanceosomes’ (Thanos and Maniatis, 1995). HMGA gene expression is maximal during embryonic development (Chiappetta et al., 1996), drops off in most adult tissues and is low, or undetectable, in fully differentiated or non- dividing adult cells (Bustin and Reeves, 1996; Lundberg et al., 1989). Overexpression of HMGA proteins correlates with 3459 High-mobility-group proteins A1 (HMGA1; previously named HMGI/Y) function as architectural chromatin- binding proteins and are involved in the transcriptional regulation of several genes. We have used cells expressing proteins fused to green fluorescent protein (GFP) and fluorescence recovery after photobleaching (FRAP) to analyze the distribution and dynamics of HMGA1a in vivo. HMGA1-GFP proteins localize preferentially to heterochromatin and remain bound to chromosomes during mitosis. FRAP experiments showed that they are highly mobile components of euchromatin, heterochromatin and of mitotic chromosomes, although with different resident times. For a more-detailed investigation on the interaction of HMGA1a with chromatin, the contribution of the AT-hook DNA-binding motifs was analyzed using point-mutated HMGA1a-GFP proteins. Furthermore, by inhibiting kinase or histone deacetylase activities, and with the help of fusion proteins lacking specific phosphorylation sites, we analyzed the effect of reversible modifications of HMGA1a on chromatin binding. Collectively our data show that the kinetic properties of HMGA1a proteins are governed by the number of functional AT-hooks and are regulated by specific phosphorylation patterns. The higher residence time in heterochromatin and chromosomes, compared with euchromatic regions, correlates with an increased phosphorylation level of HMGA1a. The regulated dynamic properties of HMGA1a fusion proteins indicate that HMGA1 proteins are mechanistically involved in local and global changes in chromatin structure. Key words: Chromatin, HMGA proteins, Dynamics, Phosphorylation Summary Dynamic interaction of HMGA1a proteins with chromatin Monika Harrer 1 , Hardi Lührs 2 , Michael Bustin 3 , Ulrich Scheer 1 and Robert Hock 1, * 1 Department of Cell and Developmental Biology, and 2 Division of Gastroenterology, Department of Medicine, University of Würzburg, Würzburg, 97080, Germany 3 Protein Section, LMC, DBS, NCI, NIH, Bethesda, MD 20892, USA *Author for correspondence (e-mail: rhock@biozentrum.uni-wuerzburg.de) Accepted 13 February 2004 Journal of Cell Science 117, 3459-3471 Published by The Company of Biologists 2004 doi:10.1242/jcs.01160 Research Article † Formerly HMGI; the nomenclature of the HMG protein superfamily has been recently revised [see Bustin (Bustin, 2001) and http:/www.informatics.jax.org/mgihome/nomen/ genefamilies/hmgfamily.shtml]. JCS ePress online publication date 22 June 2004