Robert Hock

Dynamic binding of histone H1 to chromatin in living cells

The linker histone H1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure. Histone H1 is generally viewed as a repressor of transcription as it prevents the access of transcription factors and chromatin remodelling complexes to DNA. Determining the binding properties of histone H1 to chromatin in vivo is central to understanding how it exerts these functions. We have used photobleaching techniques to measure the dynamic binding of histone H1–GFP to unperturbed chromatin in living cells. Here we show that almost the entire population of H1–GFP is bound to chromatin at any one time; however, H1–GFP is exchanged continuously between chromatin regions. The residence time of H1–GFP on chromatin between exchange events is several minutes in both euchromatin and heterochromatin. In addition to the mobile fraction, we detected a kinetically distinct, less mobile fraction. After hyperacetylation of core histones, the residence time of H1–GFP is reduced, suggesting a higher rate of exchange upon chromatin remodelling. These results support a model in which linker histones bind dynamically to chromatin in a stop-and-go mode.

Structure and function of the nucleolus.

The activity of the ribosomal RNA genes generates a distinct subnuclear structure, the nucleolus, which is the site of ribosome biogenesis. The signals that target proteins and snoRNAs (small nucleolar RNAs) to the nucleolus, the nuclear import of ribosomal proteins, the export of the completed ribosomal subunits and the molecular organization of the nucleolus have been the subject of intense research during the past year. Evidence is accumulating that nucleoli functionally interact with coiled bodies and are also involved in the maturation of non-ribosomal RNA species.

Dynamics of human DNA topoisomerases IIα and IIβ in living cells

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (α and β) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIα and IIβ behaved similarly in interphase but differently in mitosis, where only topo IIα was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIα was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.

A bisexually reproducing all-triploid vertebrate

Green toads are common in the Palaearctic region, where they have differentiated into several taxa. The toads exist with variable amounts of ploidy, similar to other anuran species or reptiles. In vertebrate biology, the very rare occurrence of triploidy is coupled with infertility or unisexuality, or requires the coexistence of individuals of different ploidy in a reproductive community. The reproduction of naturally occurring triploids has been reported to occur only through parthenogenesis, gynogenesis or hybridogenesis. The bisexual reproduction of pure triploids has been considered to be impossible because of the problem of equally distributing three chromosome sets in meiosis. Here we report geographically isolated populations of green toads (Bufo viridis complex) that are all-triploid and reproduce bisexually.

Dynamic interaction of HMGA1a proteins with chromatin

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.

Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins

In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo. This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G1 and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells.

Localization of glucocorticoid hormone receptors in mitochondria of human cells

Glucocorticoid hormones regulate the transcription of nuclear genes by way of their cognate receptors. In addition, these hormones also modulate mitochondrial gene transcription by mechanisms which are as yet poorly understood. Using immunofluorescence labeling and confocal laser scanning microscopy we show that the glucocorticoid receptor of HeLa and Hep-2 cells is specifically enriched at the sites of the mitochondria which were visualized by labeling with the vital dye CMX and antibodies against cytochrome oxidase subunit I. Immunogold electron microscopy demonstrated that the receptor was located within the inner space of the mitochondria. Immunoblotting experiments also revealed the presence of glucocorticoid receptor in mitochondria isolated from HeLa and Hep-2 cells. Finally, living HeLa cells expressing green fluorescent-glucocorticoid receptor fusion protein revealed a distinct mitochondrial GFP fluorescence. Our results support the concept of a receptor-mediated direct action of steroid hormones on mitochondrial gene transcription.

Sodium arsenite modulates histone acetylation, histone deacetylase activity and HMGN protein dynamics in human cells

Extensive epidemiological data indicate that inorganic arsenic is associated with several types of human cancer. Nevertheless, the underlying mechanisms are poorly understood. Among its mode of action are the alterations on DNA methylation, which provoke aberrant gene expression. However, beyond DNA methylation, little is known about arsenic’s effects on chromatin. In this study, we investigated the effects of sodium arsenite (NaAsO2) on global histone modifications and nucleosome-associated proteins. Our findings revealed that NaAsO2 exposure significantly increases global histone acetylation. This effect was related to the inhibition of histone deacetylase (HDAC) activity because NaAsO2 was able to inhibit HDACs comparable to the well-known HDAC inhibitor trichostatin A (TSA). Furthermore, analyses of the dynamic properties of the nucleosome-associated high mobility group N proteins demonstrate that NaAsO2 elevates their mobility. Thus, our data suggest that NaAsO2 induces chromatin opening by histone hyperacetylation due to HDAC inhibition and increase of the mobility of nucleosome-associated proteins. As the chromatin compaction is crucial for the regulation of gene expression as well as for genome stability, we propose that chromatin opening by NaAsO2 may play a significant role to impart its genotoxic effects.

Dynamic relocation of chromosomal protein HMG‐17 in the nucleus is dependent on transcriptional activity

Chromosomal proteins HMG‐14/‐17 are nucleosomal binding proteins, which alter the structure of the chromatin fiber and enhance transcription, but only from chromatin templates. Here we show that in tissue culture cells, HMG‐17 protein colocalizes with sites of active transcription. Incubation of permeabilized cells with a peptide corresponding to the nucleosomal binding domains of HMG‐14/‐17 specifically arrested polymerase II‐dependent transcription. In these cells the peptide displaces HMG‐17 from chromatin and reduces the cellular content of the protein. These results suggest that the presence of HMG‐14/‐17 in chromatin is required for efficient polymerase II transcription. In non‐permeabilized, actively transcribing cells, the protein is dispersed in a punctate pattern, throughout the nucleus. Upon transcriptional inhibition by α‐amanitin or actinomycin D, the protein gradually redistributes until it localizes fully to interchromatin granule clusters, together with the splicing factor SC35. The results suggest that the association of HMG‐17 with chromatin is dynamic rather than static, and that in the absence of transcription, HMG‐17 is released from chromatin and accumulates in interchromatin granule clusters. Thus, the intranuclear distribution of chromosomal proteins which act as architectural elements of chromatin structure may be dynamic and functionally related to the transcriptional activity of the cell.

Binding and interplay of HMG proteins on chromatin: lessons from live cell imaging.

Members of the superfamily of high mobility group (HMG) proteins are considered as architectural elements of chromatin. It is now clear that they belong to a network of dynamic chromatin proteins that constantly move around the chromatin fiber thereby dynamically modulating DNA-dependent processes. In this review we discuss how HMGs fused to fluorescent proteins and live cell imaging advanced our understanding in HMG dynamics and function. By presenting the regulation of the dynamic properties of each HMG family in comparison to one another we wish to highlight common themes among the three families, as well as stimulate new ideas from one HMG family in relation to the others and more generally in the dynamic world of chromatin.