Efficient and correct responses to double stranded breaks (DSB) in chromosomal DNA are critical for maintaining genomic stability and preventing chromosomal alterations leading to cancer1. The generation of DSB is associated with structural changes in chromatin and the activation of the protein kinase ataxia-telangiectasia mutated (ATM), a key regulator of the signaling network of the cellular response to DSB 2,3. The interrelationship between DSB-induced changes in chromatin architecture and the activation of ATM is unclear 3. Here we show that the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin both prior to and after DSB formation thereby optimizing its activation. Loss of HMGN1, or ablation of its ability to bind to chromatin, reduces the levels of IR-induced ATM autophosphorylation and the activation of several ATM targets. IR treatments lead to a global increase in the acetylation of Lys14 of histone H3 (H3K14) in an HMGN1 dependent manner and treatment of cells with a histone deacetylase inhibitor bypasses the HMGN1 requirement for efficient ATM activation. Thus, by regulating the levels of histone modifications, HMGN1 affects ATM activation. Our studies identify a new mediator of ATM activation and demonstrate a direct link between the steady-state intranuclear organization of ATM and the kinetics of its activation following DNA damage.

SummaryCell migration is a fundamental process that is necessary for the development and survival of multicellular organisms. Here, we show that cell migration is contingent on global condensation of the chromatin fiber. Induction of directed cell migration by the scratchwound assay leads to decreased DNaseI sensitivity, alterations in the chromatin binding of architectural proteins and elevated levels of H4K20me1, H3K27me3 and methylated DNA. All these global changes are indicative of increased chromatin condensation in response to induction of directed cell migration. Conversely, chromatin decondensation inhibited the rate of cell migration, in a transcription-independent manner. We suggest that global chromatin condensation facilitates nuclear movement and reshaping, which are important for cell migration. Our results support a role for the chromatin fiber that is distinct from its known functions in genetic processes.

In previous reports we showed that the long 5 untranslated region (5 UTR) of c-sis, the gene encoding the B chain of platelet-derived growth factor, has translational modulating activity due to its differentiationactivated internal ribosomal entry site (D-IRES). Here we show that the 5 UTR contains three regions with a computer-predicted Y-shaped structure upstream of an AUG codon, each of which can confer some degree of internal translation by itself. In nondifferentiated cells, the entire 5 UTR is required for maximal basal IRES activity. The elements required for the differentiation-sensing ability (i.e., D-IRES) were mapped to a 630-nucleotide fragment within the central portion of the 5 UTR. Even though the region responsible for IRES activation is smaller, the full-length 5 UTR is capable of mediating the maximal translation efficiency in differentiated cells, since only the entire 5 UTR is able to confer the maximal basal IRES activity. Interestingly, a 43-kDa protein, identified as hnRNP C, binds in a differentiation-induced manner to the differentiationsensing region. Using UV cross-linking experiments, we show that while hnRNP C is mainly a nuclear protein, its binding activity to the D-IRES is mostly nuclear in nondifferentiated cells, whereas in differentiated cells such binding activity is associated with the ribosomal fraction. Since the c-sis 5 UTR is a translational modulator in response to cellular changes, it seems that the large number of cross-talking structural entities and the interactions with regulated trans-acting factors are important for the strength of modulation in response to cellular changes. These characteristics may constitute the major difference between strong IRESs, such as those seen in some viruses, and IRESs that serve as translational modulators in response to developmental signals, such as that of c-sis.

The long uORF-burdened 5'UTRs of many genes encoding regulatory proteins involved in cell growth and differentiation contain internal ribosomal entry site (IRES) elements. In a previous study we showed that utilization of the weak IRES of platelet-derived growth factor (PDGF2) is activated during megakaryocytic differentiation. The establishment of permissive conditions for IRES-mediated translation during differentiation has been confirmed by our demonstration of the enhanced activity of vascular endothelial growth factor, c-Myc and encephalomyocarditis virus IRES elements under these conditions, although their mRNAs are not naturally expressed in differentiated K562 cells. In contrast with the enhancement of IRES-mediated protein synthesis during differentiation, global protein synthesis is reduced, as judged by polysomal profiles and radiolabelled amino acid incorporation rate. The reduction in protein synthesis rate correlates with increased phosphorylation of the translation initiation factor eIF2 alpha. Furthermore, IRES use is decreased by over-expression of the dominant-negative form of the eIF2 alpha kinase, PKR, the vaccinia virus K3L gene, or the eIF2 alpha-S51A variant which result in decreased eIF2 alpha phosphorylation. These data demonstrate a connection between eIF2 alpha phosphorylation and activation of cellular IRES elements. It suggests that phosphorylation of eIF2 alpha, known to be important for cap-dependent translational control, serves to fine-tune the translation efficiency of different mRNA subsets during the course of differentiation and has the potential to regulate expression of IRES-containing mRNAs under a range of physiological circumstances.

Numerous nuclear proteins bind to chromatin by targeting unique DNA sequences or specific histone modifications. In contrast, HMGN proteins recognize the generic structure of the 147-bp nucleosome core particle. HMGNs alter the structure and activity of chromatin by binding to nucleosomes; however, the determinants of the specific interaction of HMGNs with chromatin are not known. Here we use systematic mutagenesis, quantitative fluorescence recovery after photobleaching, fluorescence imaging, and mobility shift assays to identify the determinants important for the specific binding of these proteins to both the chromatin of living cells and to purified nucleosomes. We find that several regions of the protein affect the affinity of HMGNs to chromatin; however, the conserved sequence RRSARLSA, is the sole determinant of the specific interaction of HMGNs with nucleosomes. Within this sequence, each of the 4 amino acids in the R-S-RL motif are the only residues absolutely essential for anchoring HMGN protein to nucleosomes, both in vivo and in vitro. Our studies identify a new chromatin-binding module that specifically recognizes nucleosome cores independently of DNA sequence or histone tail modifications.

The presence of a conserved protein motif usually implies common functional features. Here, we focused on the LisH (LIS1 homology) domain, which is found in multiple proteins, and have focused on three involved in human genetic diseases; LIS1, Transducin beta-like 1X (TBL1) and Oral-facial-digital type 1 (OFD1). The recently solved structure of the LisH domain in the N-terminal region of LIS1 depicted it as a novel dimerization motif. Our findings indicated that the LisH domain of both LIS1 and TBL1 is essential for in vitro oligomerization. Furthermore, our study disclosed novel in vivo features of the LisH motif. Mutations in conserved LisH amino acids significantly reduced both the protein half-life of LIS1, TBL1, and OFD1, and dramatically affected specific intracellular localizations of these proteins. LIS1 mutated in the LisH domain induced its localization to the actin filaments. TBL1 mutated in the LisH domain was not imported into the nucleus. Mutations in OFD1 modified its localization to the Golgi apparatus and in some cases also to the nucleus. In summary, the LisH domain may participate in protein dimerization, affect protein half-life, and may influence specific cellular localizations. Our results allow the prediction that mutations within the LisH motif are likely to result in pathogenic consequences in genes associated with genetic diseases.

Michael Bustin, bustin@helix.nih.gov Directed cell migration is a property central to multiple basic biological processes. Here, we show that directed cell migration is associated with global changes in the chromatin fiber. Polarized posttranslational changes in histone H1 along with a transient decrease in H1 mobility were detected in cells facing the scratch in a wound healing assay. In parallel to the changes in H1, the levels of the heterochromatin marker histone H3 lysine 9 trimethylation were elevated. Interestingly, reduction of the chromatin-binding affinity of H1 altered the cell migration rates. Moreover, migration-associated changes in histone H1 were observed during nuclear motility in the simple multicellular organism Neurospora crassa. Our studies suggest that dynamic reorganization of the chromatin fiber is an early event in the cellular response to migration cues. Directed cell migration is a fundamental property of both simple and complex organisms, which is necessary for the proper execution of various biological processes including foraging, embryonic development, immunity, tissue repair and homeostasis. Improper cell migration is an underlying cause of numerous pathological conditions such as vascular diseases, chronic inflammatory diseases, cancer and cognitive disorders. Induction of directed cell migration results in cellular polarization, a process that involves dynamic changes in the actin cytoskeleton and in the adhesion molecules. In parallel, the microtubule-organizing center and the Golgi apparatus are reoriented (1,2) through nuclear movement (3). The nuclei of migrating cells display a wide range of structural changes. Developmentally related nuclear polarization has been noted in the single cell algae Chlamydomonas, where nuclear pore complexes localize to the posterior side of the nucleus and heterochromatin to the anterior side of the nucleus (4). Cancer cells change their nuclear shape and assume an elongated structure in capillaries (5). Migrating neurons change the structure of the nucleus during migration in a prototypic fashion (6,7). These types of morphological changes of the cell nucleus raise the possibility that cell migration is associated with the reorganization of the chromatin fiber. This possibility, which has obvious functional consequences, has not been investigated in detail yet.To monitor possible chromatin structural changes, we focused on histone H1, one of the most abundant and ubiquitous families of chromatin-binding proteins. H1 molecules are involved in diverse nuclear processes, and their intranuclear organization is affected by various cellular conditions and stimuli (8,9). We now show that following migration cues, the mobility of histone H1 is decreased in correlation with increased level of the heterochromatin marker histone H3 lysine 9 tri-methylation (H3K9me3). Altered properties of linker histone in migrating nuclei are evolutionary conserved and were also found in the fungus Neurospora crassa. Furthermore, alterations in the interaction of H1 wi...

Chromatin dynamics play a major role in regulating genetic processes. Now, accumulating data suggest that chromatin structure may also affect the mechanical properties of the nucleus and cell migration. Global chromatin organization seems to modulate the shape, the size and the stiffness of the nucleus. Directed-cell migration, which often requires nuclear reshaping to allow cellular passage through narrow openings, is dependent not only on changes in cytoskeletal elements, but also on the global chromatin condensation. Conceivably, during cell migration a physical link between the chromatin and the cytoskeleton facilitates coordinated structural changes in these two components. Thus, in addition to regulating genetic processes, we suggest that alterations in chromatin structure may facilitate cellular reorganizations necessary for efficient migration.