Tobias A. Knoch

Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells

Significance For the 2m DNA to fit into the tiny cell nucleus, it is wrapped around nucleosomes and folded into loops clustering together in domains. Genome function depends on this 3D-organization, especially on-going dynamic processes like transcription. Techniques studying the network of DNA contacts genome-wide have recently revealed this 3D architecture, but the protein factors behind this are not understood. We study two proteins that are known to help form DNA loops: cohesin and CTCC-binding factor (CTCF). Respective depletion and analysis of DNA contacts genome-wide show that CTCF is required to separate neighboring folding domains and keep cohesin in place, whereas cohesin is important for shaping the domains. Consistently, we observe different changes of gene expression. Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences between domains are enriched for binding sites of CTCC-binding factor (CTCF) and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on higher-order chromatin organization, as well as the transcriptome. We observed a general loss of local chromatin interactions upon disruption of cohesin, but the topological domains remain intact. However, we found that depletion of CTCF not only reduced intradomain interactions but also increased interdomain interactions. Furthermore, distinct groups of genes become misregulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute differentially to chromatin organization and gene regulation.

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 μm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends.

Microtubule (MT) plus end–tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.

ERYTHROPOIETIN AND G-CSF RECEPTORS IN HUMAN TUMOR CELLS: EXPRESSION AND ASPECTS REGARDING FUNCTIONALITY

Aims and Background Recombinant human erythropoietin (Epo) and granulocyte-colony-stimulating factor (G-CSF) are used to stimulate hematopoiesis in patients with malignant diseases. These cytokines transduce their biological signal via the Epo receptor (EpoR) and G-CSF receptor (G-CSF-R) into the cell. We therefore investigated in human tumor cell lines the expression of these receptors in tumor cells as well as their response to Epo and G-CSF. Methods and Study Design The expression of EpoR and G-CSF-R mRNA was analyzed with reverse transcription-polymerase chain reaction (RT-PCR). EpoR protein expression was further monitored with Western blot and immunocytochemistry analysis. The cellular response to various concentrations of Epo was evaluated using 3[H]-thymidine uptake, Northern blot of c-fos expression and tyrosine kinase activity assay. The proliferation after G-CSF incubation was analyzed with the MTS assay. Results In this study EpoR mRNA and protein were detected in various human tumor cell lines. Treatment with Epo did not influence the proliferation rate of examined EpoR-positive tumor cell lines. Epo did not stimulate the tyrosine kinase activity nor did it affect the c-fos mRNA in these cell lines. G-CSF-R mRNA was only detected in two myeloid cell lines. Treatment with G-CSF did not increase the proliferation of these cells. Conclusions These results demonstrate that Epo and G-CSF did not modulate the growth rate of examined receptor-positive tumor cell lines; the presence of the Epo receptor seems not essential for cell growth of these tumor cells in cell culture.

Spatio-temporal permanence and plasticity of foraging trails in young and mature leaf-cutting ant colonies (Atta spp.)

The distribution and formation of foraging trails have largely been neglected as factors explaining harvesting patterns of leaf-cutting ants. We applied fractal analysis, circular, and conventional statistics to published and newly recorded trail maps of seven Atta colonies focusing on three aspects: permanence, spatio-temporal plasticity and colony life stage. In the long term, trail patterns of young and mature Atta colonies revealed that foraging activities were focused on distinct, static sectors that made up only parts of their potentially available foraging range. Within these foraging sectors, trails were typically ephemeral and highly variable in space and time. These ephemeral trails were concentrated around permanent trunk trails in mature and around nest entrances in young colonies. Besides these similarities, the comparison of trail systems between the two life stages indicated that young colonies exploited fewer leaf sources, used smaller and less-complex systems of foraging trails, preferred different life forms as host plants, and switched hosts more often compared with mature colonies. Based on these analyses, we propose a general hypothesis which describes the foraging pattern in Atta as a result of initial foraging experiences, spatio-temporal distribution of suitable host plants, energetic constraints, and other factors such as seasonality and interspecific predation.

GRIMP: A web- and grid-based tool for high-speed analysis of large-scale genome-wide association using imputed data

Summary: The current fast growth of genome-wide association studies (GWAS) combined with now common computationally expensive imputation requires the online access of large user groups to high-performance computing resources capable of analyzing rapidly and efficiently millions of genetic markers for ten thousands of individuals. Here, we present a web-based interface—called GRIMP—to run publicly available genetic software for extremely large GWAS on scalable super-computing grid infrastructures. This is of major importance for the enlargement of GWAS with the availability of whole-genome sequence data from the 1000 Genomes Project and for future whole-population efforts. Contact: ta.knoch@taknoch.org; f.rivadeneira@erasmusmc.nl

Super-resolution imaging reveals three-dimensional folding dynamics of the β-globin locus upon gene activation

Summary The chromatin architecture is constantly changing because of cellular processes such as proliferation, differentiation and changes in the expression profile during gene activation or silencing. Unravelling the changes that occur in the chromatin structure during these processes has been a topic of interest for many years. It is known that gene activation of large gene loci is thought to occur by means of an active looping mechanism. It was also shown for the &bgr;-globin locus that the gene promoter interacts with an active chromatin hub by means of an active looping mechanism. This means that the locus changes in three-dimensional (3D) nuclear volume and chromatin shape. As a means of visualizing and measuring these dynamic changes in chromatin structure of the &bgr;-globin locus, we used a 3D DNA-FISH method in combination with 3D image acquisition to volume render fluorescent signals into 3D objects. These 3D chromatin structures were geometrically analysed, and results prior to and after gene activation were quantitatively compared. Confocal and super-resolution imaging revealed that the inactive locus occurs in several different conformations. These conformations change in shape and surface structure upon cell differentiation into a more folded and rounded structure that has a substantially smaller size and volume. These physical measurements represent the first non-biochemical evidence that, upon gene activation, an actively transcribing chromatin hub is formed by means of additional chromatin looping.

Super-resolution imaging reveals 3D folding dynamics of the β-globin locus upon gene activation

Summary The chromatin architecture is constantly changing because of cellular processes such as proliferation, differentiation and changes in the expression profile during gene activation or silencing. Unravelling the changes that occur in the chromatin structure during these processes has been a topic of interest for many years. It is known that gene activation of large gene loci is thought to occur by means of an active looping mechanism. It was also shown for the &bgr;-globin locus that the gene promoter interacts with an active chromatin hub by means of an active looping mechanism. This means that the locus changes in three-dimensional (3D) nuclear volume and chromatin shape. As a means of visualizing and measuring these dynamic changes in chromatin structure of the &bgr;-globin locus, we used a 3D DNA-FISH method in combination with 3D image acquisition to volume render fluorescent signals into 3D objects. These 3D chromatin structures were geometrically analysed, and results prior to and after gene activation were quantitatively compared. Confocal and super-resolution imaging revealed that the inactive locus occurs in several different conformations. These conformations change in shape and surface structure upon cell differentiation into a more folded and rounded structure that has a substantially smaller size and volume. These physical measurements represent the first non-biochemical evidence that, upon gene activation, an actively transcribing chromatin hub is formed by means of additional chromatin looping.

Cell cycle-dependent 3D distribution of telomeres and telomere repeat-binding factor 2 (TRF2) in HaCaT and HaCaT-myc cells

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.