Touched by CTCF: Analysis of a multi-functional zinc finger protein

Multicellular organisms contain a complete set of genes in nearly all of their cells. However most cells are very different to each other and are able to form organs with distinct functions. The identity and survival of the cell is regulated by the activity of specific genes in time and space. Specific sets of genes encoding proteins become activated, whereas others are repressed. CTCF is a protein that mediates distinct processes of gene regulation, including transcription and the structural organization of the genome. The aim of this thesis is to investigate the different functions of CTCF by a combined analysis of CTCF-interacting proteins and by deletion of CTCF in vivo and in vitro. Using these approaches we aimed to improve our understanding of the molecular mechanism underlying its functions. Chapter 1 gives an overview of the information required to understand the foundations of studies presented and discussed in this thesis. It gives an introduction to gene regulation and how this process is influenced by chromatin modifications, nuclear organization and compartmentation. A specific nuclear compartment, the nucleolus, and its involvement in ribosomal RNA synthesis, are highlighted. Furthermore the characteristics of CTCF and its homolog CTCFL are described in detail

New Tobacco and Tobacco-Related Products: Early Detection of Product Development, Marketing Strategies, and Consumer Interest.

A wide variety of new tobacco and tobacco-related products have emerged on the market in recent years

CTCF regulates the local epigenetic state of ribosomal DNA repeats

Background: CCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription. CTCF may act by binding tightly to DNA and recruiting other proteins to mediate its various functions in the nucleus. To further explore the role of this essential factor, we used a mass spectrometry-based approach to screen for novel CTCF-interacting partners. Results. Using biotinylated CTCF as bait, we identified upstream binding factor (UBF) and multiple other components of the RNA polymerase I complex as potential CTCF-interacting partners. Interestingly, CTCFL, the testis-specific paralog of CTCF, also binds UBF. The interaction between CTCF(L) and UBF is direct, and requires the zinc finger domain of CTCF(L) and the high mobility group (HMG)-box 1 and dimerization domain of UBF. Because UBF is involved in RNA polymerase I-mediated ribosomal (r)RNA transcription, we analyzed CTCF binding to the rDNA repeat. We found that CTCF bound to a site upstream of the rDNA spacer promoter and preferred non-methylated over methylated rDNA. DNA binding by CTCF in turn stimulated binding of UBF. Absence of CTCF in cultured cells resulted in decreased association of UBF with rDNA and in nucleolar fusion. Furthermore, lack of CTCF led to reduced binding of RNA polymerase I and variant histone H2A.Z near the rDNA spacer promoter, a loss of specific histone modifications, and diminished transcription of non-coding RNA from the spacer promoter. Conclusions. UBF is the first common interaction partner of CTCF and CTCFL, suggesting a role for these proteins in chromatin organization of the rDNA repeats. We propose that CTCF affects RNA polymerase I-mediated events globally by controlling nucleolar number, and locally by regulating chromatin at the rDNA spacer promoter, similar to RNA polymerase II promoters. CTCF may load UBF onto rDNA, thereby forming part of a network that maintains rDNA genes poised for transcription

The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear. RESULTS: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF. CONCLUSIONS: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes

Sensory analysis of characterising flavours : Evaluating tobacco product odours using an expert panel

Objectives: Tobacco flavours are an important regulatory concept in several jurisdictions, for example in the USA, Canada and Europe. The European Tobacco Products Directive 2014/40/EU prohibits cigarettes and roll-your-own tobacco having a characterising flavour. This directive defines characterising flavour as 'a clearly noticeable smell or taste other than one of tobacco [⋯]'. To distinguish between products with and without a characterising flavour, we trained an expert panel to identify characterising flavours by smelling. Methods: An expert panel (n=18) evaluated the smell of 20 tobacco products using self-defined odour attributes, following Quantitative Descriptive Analysis. The panel was trained during 14 attribute training, consensus training and performance monitoring sessions. Products were assessed during six test sessions. Principal component analysis, hierarchical clustering (four and six clusters) and Hotelling's T-tests (95% and 99% CIs) were used to determine differences and similarities between tobacco products based on odour attributes. Results: The final attribute list contained 13 odour descriptors. Panel performance was sufficient after 14 training sessions. Products marketed as unflavoured that formed a cluster were considered reference products. A four-cluster method distinguished cherry-flavoured, vanilla-flavoured and menthol-flavoured products from reference products. Six clusters subdivided reference products into tobacco leaves, roll-your-own and commercial products. Conclusions: An expert panel was successfully trained to assess characterising odours in cigarettes and roll-your-own tobacco. This method could be applied to other product types such as e-cigarettes. Regulatory decisions on the choice of reference products and significance level are needed which directly influences the products being assessed as having a characterising odour.</p

Critical role for the transcription regulator CCCTC-binding factor in the control of Th2 cytokine expression

Differentiation of naive CD4+ cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine locus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctcf gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-γ production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein 1 (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATA3- and SATB1-dependent regulation of Th2 cytokine gene expression

Critical Role for the Transcription Regulator CCCTC-Binding Factor in the Control of Th2 Cytokine Expression

Differentiation of naive CD4(+) cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine focus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctef gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-gamma production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein I (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATAX and SATB1-dependent regulation of Th2 cytokine gene expression. The Journal of Immunology, 2009, 182: 999-1010