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

The CCCTC-Binding Factor (CTCF) of Drosophila Contributes to the Regulation of the Ribosomal DNA and Nucleolar Stability

In the repeat array of ribosomal DNA (rDNA), only about half of the genes are actively transcribed while the others are silenced. In arthropods, transposable elements interrupt a subset of genes, often inactivating transcription of those genes. Little is known about the establishment or separation of juxtaposed active and inactive chromatin domains, or preferential inactivation of transposable element interrupted genes, despite identity in promoter sequences. CTCF is a sequence-specific DNA binding protein which is thought to act as a transcriptional repressor, block enhancer-promoter communication, and delimit juxtaposed domains of active and inactive chromatin; one or more of these activities might contribute to the regulation of this repeated gene cluster. In support of this hypothesis, we show that the Drosophila nucleolus contains CTCF, which is bound to transposable element sequences within the rDNA. Reduction in CTCF gene activity results in nucleolar fragmentation and reduced rDNA silencing, as does disruption of poly-ADP-ribosylation thought to be necessary for CTCF nucleolar localization. Our data establish a role for CTCF as a component necessary for proper control of transposable element-laden rDNA transcription and nucleolar stability

Novel CTCF binding at a site in exon1A of BCL6 is associated with active histone marks and a transcriptionally active locus

BCL6 is a zinc-finger transcriptional repressor, which is highly expressed in germinal centre B-cells and is essential for germinal centre formation and T-dependent antibody responses. Constitutive BCL6 expression is sufficient to produce lymphomas in mice. Deregulated expression of BCL6 due to chromosomal rearrangements, mutations of a negative autoregulatory site in the BCL6 promoter region and aberrant post-translational modifications have been detected in a number of human lymphomas. Tight lineage and temporal regulation of BCL6 is, therefore, required for normal immunity, and abnormal regulation occurs in lymphomas. CCCTC-binding factor (CTCF) is a multi-functional chromatin regulator, which has recently been shown to bind in a methylation-sensitive manner to sites within the BCL6 first intron. We demonstrate a novel CTCF-binding site in BCL6 exon1A within a potential CpG island, which is unmethylated both in cell lines and in primary lymphoma samples. CTCF binding, which was found in BCL6-expressing cell lines, correlated with the presence of histone variant H2A.Z and active histone marks, suggesting that CTCF induces chromatin modification at a transcriptionally active BCL6 locus. CTCF binding to exon1A was required to maintain BCL6 expression in germinal centre cells by avoiding BCL6-negative autoregulation. Silencing of CTCF in BCL6-expressing cells reduced BCL6 mRNA and protein expression, which is sufficient to induce B-cell terminal differentiation toward plasma cells. Moreover, lack of CTCF binding to exon1A shifts the BCL6 local chromatin from an active to a repressive state. This work demonstrates that, in contexts in which BCL6 is expressed, CTCF binding to BCL6 exon1A associates with epigenetic modifications indicative of transcriptionally open chromatin

Widespread Expression of BORIS/CTCFL in Normal and Cancer Cells

BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a “cancer-testis” antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function

A Cell Cycle Role for the Epigenetic Factor CTCF-L/BORIS

CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, downregulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis

The Chromatin Remodelling Complex B-WICH Changes the Chromatin Structure and Recruits Histone Acetyl-Transferases to Active rRNA Genes

The chromatin remodelling complex B-WICH, which comprises the William syndrome transcription factor (WSTF), SNF2h, and nuclear myosin 1 (NM1), is involved in regulating rDNA transcription, and SiRNA silencing of WSTF leads to a reduced level of 45S pre-rRNA. The mechanism behind the action of B-WICH is unclear. Here, we show that the B-WICH complex affects the chromatin structure and that silencing of the WSTF protein results in a compaction of the chromatin structure over a 200 basepair region at the rRNA promoter. WSTF knock down does not show an effect on the binding of the rRNA-specific enhancer and chromatin protein UBF, which contributes to the chromatin structure at active genes. Instead, WSTF knock down results in a reduced level of acetylated H3-Ac, in particular H3K9-Ac, at the promoter and along the gene. The association of the histone acetyl-transferases PCAF, p300 and GCN5 with the promoter is reduced in WSTF knock down cells, whereas the association of the histone acetyl-transferase MOF is retained. A low level of H3-Ac was also found in growing cells, but here histone acetyl-transferases were present at the rDNA promoter. We propose that the B-WICH complex remodels the chromatin structure at actively transcribed rRNA genes, and this allows for the association of specific histone acetyl-transferases

Cannabis contaminants

Alleen digitaal verschenenIn nederwiet uit verschillende koffieshops zijn restanten van bestrijdingsmiddelen aangetroffen. De hoeveelheden zijn dermate laag dat het geen risico vormt voor de gezondheid van gebruikers. Dit geldt voor de consumptie van nederwiet in de vorm van thee en voor gerookte nederwiet. Er zijn geen giftige plantenschimmels (aflatoxines) aangetroffen. Dit blijkt uit verkennend onderzoek van het RIVM, waarvoor bij 25 koffieshops een monster is genomen en onderzocht op de aanwezigheid van vervuilende stoffen. De focus lag op de aanwezigheid van bestrijdingsmiddelen en aflatoxines. Het onderzoek is uitgevoerd in opdracht van het ministerie van VWS. Het is van belang dat er niet te grote hoeveelheden of verboden bestrijdingsmiddelen in nederwiet zitten, bijvoorbeeld om mensen met een kwetsbare gezondheid te beschermen. Mensen die om medische redenen wiet gebruiken nemen die namelijk niet altijd af bij de apotheek. Alleen de door apotheek verstrekte mediwiet is volgens kwaliteitsrichtlijnen geproduceerd. In 23 van de 25 monsters zijn één of meerdere bestrijdingsmiddelen aangetroffen. In 11 monsters was de hoeveelheid hoger dan de grens die in de kruidengeneesmiddelenrichtlijn wordt gesteld. Eén monster bevatte een verboden bestrijdingsmiddel, maar ook deze concentratie was dermate laag dat het geen risico voor de gezondheid vormt.Traces of pesticides were found in cannabis samples (nederwiet) from different coffee shops in the Netherlands. The levels are very low and of no consequence to the users' health. This applies to the samples used as a tea or smoked. Aflatoxines produced by fungi commonly found on plants were not found. In this exploratory study, RIVM analysed nederwiet samples from 25 coffee shops for the presence of contaminants, specifically aflatoxines and residues of pesticides. This study was commissioned by the Ministry of Health, Wellfare and Sports. It is important that cannabis does not contain significant levels of pesticides because it may harm the user, particularly when already in poor health. This is relevant because people that take cannabis for medical reasons do not always turn to a pharmacy. Only the cannabis available at pharmacies (mediwiet) is produced under quality conditions. In 23 of the 25 samples one or more pesticides were identified. In 11 samples the observed levels exceed the limits set for herbal medicines. One sample contained a banned pesticide but at a level that is of no consequence to the users' health.Ministerie van VW

Detection methods to assist enforcement of the smoking ban

Ondanks het rookverbod kunnen mensen last hebben van rook die 'weglekt' uit ruimten waar roken is toegestaan. Dat is de belangrijkste oorzaak van overlast in rookvrije ruimten. Het rookverbod is ingesteld om onvrijwillige blootstelling aan tabaksrook te voorkomen op de werkplek, in het openbaar vervoer en in publiek toegankelijke ruimten. Er bestaan draagbare en gebruiksvriendelijke meetapparaten die inspecteurs van de NVWA kunnen helpen te bepalen of het rookverbod is overtreden. Dit kan bijdragen aan een veel gevoeligere en objectievere manier van handhaven. Momenteel worden deze inspecties nog uitgevoerd door in ruimten te kijken en te ruiken (zintuigelijke waarneming). De mogelijkheid om meetapparaten voor dit doeleinde te gebruiken is door het RIVM onderzocht. Met de apparaten kan lucht worden bemonsterd, die daarna in het laboratorium geanalyseerd wordt. De concentratie tabaksrook wordt dan bepaald door de omgevingslucht te analyseren op de geselecteerde indicatorstoffen nicotine en 3-EP. Aanbevolen wordt welke apparaten het meest geschikt zijn voor tabaksrook metingen. Vervolgonderzoek moet uitwijzen of de apparaten voldoende gevoelig zijn om de concentratie tabaksrook te kunnen bepalen. Het onderzoek is uitgevoerd in opdracht van de Nederlandse Voedsel en Waren Autoriteit (NVWA), die erop toeziet dat de tabakswet wordt nageleefd. Om het rookverbod te handhaven en overlast zoveel mogelijk tegen te gaan wil de de NVWA tijdens inspecties beschikken over ondersteunende detectiemethoden.There are portable and easy to use measuring devices that can help NVWA surveillance teams to determine whether the smoking ban has been violated. The use of such devices can contribute to a much more sensitive and objective way of enforcement. Currently, surveillances are carried out by visiting premises and observing the presence of tobacco smoke visually and by its odor (sensory perception). The possibility to use devices for this purpose has been investigated by the RIVM. The tested devices sample ambient air, which subsequently will be analyzed in the laboratory. The tobacco smoke concentration is then determined by analyzing the ambient air for selected indicator substances as nicotine and 3- EP. Despite the smoking ban, people can be hindered by smoke ' leaking ' from areas where smoking is allowed. This is the main cause of nuisance in smokefree areas. The smoking ban is set to ensure prevention of involuntarily exposure to environmental tobacco smoke (ETS) at work, in public transport and in public spaces. In this study recommendations are made regarding the most suitable devices for ETS measurements. Future research will clarify whether the devices are sufficiently sensitive to determine the level of smoke leakage. This study was commissioned by the Dutch Food and Consumer Product Safety Authority (NVWA), which ensures the Tobacco Act is complied with. To enforce the smoking ban and prevent inconvenience to non-smokers as much as possible the NVWA wishes to include supporting detection methods during their surveillances.NVW