CTCF regulates positioning of the human cystic fibrosis gene in association with a histone deacetylase

The nuclear positioning of mammalian genes often correlates with their functional state. For instance, the human cystic fibrosis transmembrane conductance regulator (CFTR) gene associates with the nuclear periphery in its inactive state, but occupies interior positions when active. Treatment with the histone deacetylase inhibitor trichostatin a (TSA) changes the radial positioning of the CFTR gene in HeLa S3 cells. The gene relocates from the nuclear periphery to the nuclear interior. In Calu-3 cells the gene is located in the nuclear interior. To identify potential regulatory elements for the positioning of CFTR, the histone H3 and H4 acetylation patterns of untreated and TSA-treated HeLa S3 and untreated Calu-3 cells were determined by ChIP–chip. Here is a detailed description of the datasets associated with the study by Muck et al. published in the Journal of Cellular Biochemistry in 2012

Pax7 is required for establishment of the xanthophore lineage in zebrafish embryos

The pigment pattern of many animal species is a result of the arrangement of different types of pigment-producing chromatophores. The zebrafish has three different types of chromatophores: black melanophores, yellow xanthophores, and shimmering iridophores arranged in a characteristic pattern of golden and blue horizontal stripes. In the zebrafish embryo, chromatophores derive from the neural crest cells. Using pax7a and pax7b zebrafish mutants, we identified a previously unknown requirement for Pax7 in xanthophore lineage formation. The absence of Pax7 results in a severe reduction of xanthophore precursor cells and a complete depletion of differentiated xanthophores in embryos as well as in adult zebrafish. In contrast, the melanophore lineage is increased in pax7a/pax7b double-mutant embryos and larvae, whereas juvenile and adult pax7a/pax7b double-mutant zebrafish display a severe decrease in melanophores and a pigment pattern disorganization indicative of a xanthophore-deficient phenotype. In summary, we propose a novel role for Pax7 in the early specification of chromatophore precursor cells

Differential regulation of myosin heavy chains defines new muscle domains in zebrafish

Numerous muscle lineages are formed during myogenesis within both slow-and fast-specific cell groups. In this study, we show that six fast muscle-specific myosin heavy chain genes have unique expression patterns in the zebrafish embryo. The expression of tail-specific myosin heavy chain (fmyhc2.1) requires wnt signaling and is essential for fast muscle organization within the tail. Retinoic acid treatment results in reduced wnt signaling, which leads to loss of the fmyhc2.1 domain. Retinoic acid treatment also results in a shift of muscle identity within two trunk domains defined by expression of fmyhc1.2 and fmyhc1.3 in favor of the anteriormost myosin isoform, fmyhc1.2. In summary, we identify new muscle domains along the anteroposterior axis in the zebrafish that are defined by individual nonoverlapping, differentially regulated expression of myosin heavy chain isoforms

Differential regulation of myosin heavy chains defines new muscle domains in zebrafish

Numerous muscle lineages are formed during myogenesis within both slow-and fast-specific cell groups. In this study, we show that six fast muscle-specific myosin heavy chain genes have unique expression patterns in the zebrafish embryo. The expression of tail-specific myosin heavy chain (fmyhc2.1) requires wnt signaling and is essential for fast muscle organization within the tail. Retinoic acid treatment results in reduced wnt signaling, which leads to loss of the fmyhc2.1 domain. Retinoic acid treatment also results in a shift of muscle identity within two trunk domains defined by expression of fmyhc1.2 and fmyhc1.3 in favor of the anteriormost myosin isoform, fmyhc1.2. In summary, we identify new muscle domains along the anteroposterior axis in the zebrafish that are defined by individual nonoverlapping, differentially regulated expression of myosin heavy chain isoforms

DNA compaction induced by a cationic polymer or surfactant impact gene expression and DNA degradation

There is an increasing interest in achieving gene regulation in biotechnological and biomedical applications by using synthetic DNA-binding agents. Most studies have so far focused on synthetic sequence-specific DNA-binding agents. Such approaches are relatively complicated and cost intensive and their level of sophistication is not always required, in particular for biotechnological application. Our study is inspired by in vivo data that suggest that DNA compaction might contribute to gene regulation. This study exploits the potential of using synthetic DNA compacting agents that are not sequence-specific to achieve gene regulation for in vitro systems. The semi-synthetic in vitro system we use include common cationic DNA-compacting agents, poly(amido amine) (PAMAM) dendrimers and the surfactant hexadecyltrimethylammonium bromide (CTAB), which we apply to linearized plasmid DNA encoding for the luciferase reporter gene. We show that complexing the DNA with either of the cationic agents leads to gene expression inhibition in a manner that depends on the extent of compaction. This is demonstrated by using a coupled in vitro transcription-translation system. We show that compaction can also protect DNA against degradation in a dose-dependent manner. Furthermore, our study shows that these effects are reversible and DNA can be released from the complexes. Release of DNA leads to restoration of gene expression and makes the DNA susceptible to degradation by Dnase. A highly charged polyelectrolyte, heparin, is needed to release DNA from dendrimers, while DNA complexed with CTAB dissociates with the non-ionic surfactant C12E5. Our results demonstrate the relation between DNA compaction by non-specific DNA-binding agents and gene expression and gene regulation can be achieved in vitro systems in a reliable dose-dependent and reversible manner

Nuclei of chicken neurons in tissues and three-dimensional cell cultures are organized into distinct radial zones.

We used chicken retinospheroids (RS) to study the nuclear architecture of vertebrate cells in a three-dimensional (3D) cell culture system. The results showed that the different neuronal cell types of RS displayed an extreme form of radial nuclear organization. Chromatin was arranged into distinct radial zones which became already visible after DAPI staining. The distinct zones were enriched in different chromatin modifications and in different types of chromosomes. Active isoforms of RNA polymerase II were depleted in the outermost zone. Also chromocenters and nucleoli were radially aligned in the nuclear interior. The splicing factor SC35 was enriched at the central zone and did not show the typical speckled pattern of distribution. Evaluation of neuronal and non-neuronal chicken tissues showed that the highly ordered form of radial nuclear organization was also present in neuronal chicken tissues. Furthermore, the data revealed that the neuron-specific nuclear organization was remodeled when cells spread on a flat substrate. Monolayer cultures of a chicken cell line did not show this extreme form of radial organization. Rather, such monolayer cultures displayed features of nuclear organization which have been described before for many different types of monolayer cells. The finding that an extreme form radial nuclear organization, which has not been described before, is present in RS and tissues, but not in cells spread on a flat substrate, suggests that it would be important to complement studies on nuclear architecture performed with monolayer cells by studies on 3D cell culture systems and tissues

Luciferase gene expression and DNA accessibility as a function of <i>r</i>_charge using pre-casted RNA gels.

<p>(A) G4 dendrimers and (B) CTAB. The synthesized amounts of RNA are displayed and samples were not pretreated with Dnase I. References are displayed in B where lane 1 shows the sample consisting only of DNA and without any compacting agent or transcriptional activity. Lane 2 shows the control sample containing DNA and the <i>in vitro</i> transcription mixture in the absence of compacting agents. Gels were post-stained using GelStar.</p

DNA condensation using G4 dendrimers and CTAB surfactants.

<p>(A) The fluorescence intensity of GelStar bound to DNA, shown as a function of <i>r</i>_charge in solutions containing 10 mM NaBr for G4 dendrimers (▵) and CTAB (•). Data are normalized to the amounts produced in the samples only containing DNA (in the absence of dendrimer or surfactant) and the <i>I</i>_max value is linearly dependent on the amount of DNA that is available to bind GelStar. The DNA concentration is 2 μg mL⁻¹ and error bars are smaller or equal to the size of the markers. (B) The mean number of G4 per compacted DNA chain at varying <i>r</i>_charge, calculated as described in the text. Note that below charge neutralization the amount of bound G4 per DNA strand is constant, that is each complex contains the same number of G4. Once the neutralization point is reached, the solution only contains compacted DNA and the number of G4 per compacted DNA increases. The results from the electrophoreses study - DNA condensation by G4 dendrimers and CTAB surfactants - are shown in (C) (D), respectively. Lane 1 in both C and D displays free linearized plasmid DNA in the absence of any compacting agent (control, 4331 bp). Samples in lanes 2–11 contain increasing amounts of the compacting agent and the corresponding <i>r</i>_charge values are indicated. The DNA concentration was 25 μg mL⁻¹ and the gels were stained with Ethidium Bromide (EtBr).</p