Cell Biology

XCI in preimplantation mouse and human embryos: first there is remodelling... (Article)

Berg, I.M. van den — 2011-06-07T00:00:00Z

Female eutherians silence one of their X chromosomes to accomplish an equal dose of X-linked gene expression compared with males. The mouse is the most widely used animal model in XCI research and has proven to be of great significance for understanding the complex mechanism of X-linked dosage compensation. Although the basic principles of XCI are similar in mouse and humans, differences exist in the timing of XCI initiation, the genetic elements involved in XCI regulation and the form of XCI in specific tissues. Therefore, the mouse has its limitations as a model to understand early human XCI and analysis of human tissues is required. In this review, we describe these differences with respect to initiation of XCI in human and mouse preimplantation embryos, the extra-embryonic tissues and the in vitro model of the epiblast: the embryonic stem cells.

The EMT regulator Zeb2/Sip1 is essential for murine embryonic hematopoietic stem/progenitor cell differentiation and mobilization (Article)

Goossens, S. — 2011-06-01T00:00:00Z

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box - binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-tomesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult.

Xist regulation and function eXplored (Article)

Pontier, D.B. — 2011-05-31T00:00:00Z

X chromosome inactivation (XCI) is a process in mammals that ensures equal transcript levels between males and females by genetic inactivation of one of the two X chromosomes in females. Central to XCI is the long non-coding RNA Xist, which is highly and specifically expressed from the inactive X chromosome. Xist covers the X chromosome in cis and triggers genetic silencing, but its working mechanism remains elusive. Here, we review current knowledge about Xist regulation, structure, function and conservation and speculate on possible mechanisms by which its action is restricted in cis. We also discuss dosage compensation mechanisms other than XCI and how knowledge from invertebrate species may help to provide a better understanding of the mechanisms of mammalian XCI.

Long-term follow-up and treatment in nine boys with X-linked creatine transporter defect (Article)

van de Kamp, J.M. — 2011-05-10T00:00:00Z

The creatine transporter (CRTR) defect is a recently discovered cause of X-linked intellectual disability for which treatment options have been explored. Creatine monotherapy has not proved effective, and the effect of treatment with L-arginine is still controversial. Nine boys between 8 months and 10 years old with molecularly confirmed CRTR defect were followed with repeated1H-MRS and neuropsychological assessments during 4-6 years of combination treatment with creatine monohydrate, L-arginine, and glycine. Treatment did not lead to a significant increase in cerebral creatine content as observed with H1-MRS. After an initial improvement in locomotor and personal-social IQ subscales, no lasting clinical improvement was recorded. Additionally, we noticed an age-related decline in IQ subscales in boys affected with the CRTR defect.

Defective deacetylation of histone 4 K12 in human oocytes is associated with advanced maternal age and chromosome misalignment (Article)

Berg, I.M. van den — 2011-05-01T00:00:00Z

Background: Chromosome segregation errors during human oocyte meiosis are associated with low fertility in humans and the incidence of these errors increases with advancing maternal age. Studies of mitosis and meiosis suggest that defective remodeling of chromatin plays a causative role in aneuploidy. We analyzed the histone deacetylation pattern during the final stages of human oocyte maturation to investigate whether defective epigenetic regulation of chromatin remodeling in human oocytes is related to maternal age and leads to segregation errors.MethodsHuman surplus oocytes of different meiotic maturation stages [germinal vesicle (GV), metaphase (M)I and MII] were collected from standard IVF/ICSI treatments. Oocytes were analyzed for acetylation of different lysines of histone 4 (H4K5, H4K8, H4K12 and H4K16) and for α-tubulin. ResultsHuman GV oocytes had an intense staining of the chromatin for all four histone 4 lysine acetylations. MI and MII stage oocytes showed either normal deacetylation or various amounts of defective histone deacetylation. Residual H4K12 acetylation was more frequently found in oocytes obtained from older women, with a significant correlation between defective deacetylation and maternal age (r 0.185, P 0.007). Eighty-eight percent of the oocytes with residual acetylation had misaligned chromosomes, whereas only 33 of the oocytes that showed correct deacetylated chromatin had misaligned chromosomes (P < 0.001). Conclusions We conclude that defective deacetylation during human female meiosis increases with maternal age and is correlated with misaligned chromosomes. As chromosome misalignment predisposes to segregation errors, our data imply that defective regulation of histone deacetylation could be an important factor in age-related aneuploidy.

Medulloblastoma comprises four distinct molecular variants (Article)

Northcott, P.A. — 2011-05-01T00:00:00Z

Purpose Recent genomic approaches have suggested the existence of multiple distinct subtypes of medulloblastoma. We studied a large cohort of medulloblastomas to determine how many subgroups of the disease exist, how they differ, and the extent of overlap between subgroups. Methods We determined gene expression profiles and DNA copy number aberrations for 103 primary medulloblastomas. Bioinformatic tools were used for class discovery of medulloblastoma subgroups based on the most informative genes in the data set. Immunohistochemistry for subgroup-specific signature genes was used to determine subgroup affiliation for 294 nonoverlapping medulloblastomas on two independent tissue microarrays. Results Multiple unsupervised analyses of transcriptional profiles identified the following four distinct, nonoverlapping molecular variants: WNT, SHH, group C, and group D. Supervised analysis of these four subgroups revealed significant subgroup-specific demographics, histology, metastatic status, and DNA copy number aberrations. Immunohistochemistry for DKK1 (WNT), SFRP1 (SHH), NPR3 (group C), and KCNA1 (group D) could reliably and uniquely classify formalin-fixed medulloblastomas in approximately 98% of patients. Group C patients (NPR3-positive tumors) exhibited a significantly diminished progression-free and overall survival irrespective of their metastatic status. Conclusion Our integrative genomics approach to a large cohort of medulloblastomas has identified four disparate subgroups with distinct demographics, clinical presentation, transcriptional profiles, genetic abnormalities, and clinical outcome. Medulloblastomas can be reliably assigned to subgroups through immunohistochemistry, thereby making medulloblastoma subclassification widely available. Future research on medulloblastoma and the development of clinical trials should take into consideration these four distinct types of medulloblastoma.

CFTR-deficiency renders mice highly susceptible to cutaneous symptoms during mite infestation (Article)

Hashimoto, Y. — 2011-04-01T00:00:00Z

Pruritus, also known as itch, is a sensation that causes a desire to scratch. Prolonged scratching exacerbates skin lesions in several skin diseases such as atopic dermatitis. Here, we identify the cystic fibrosis transmembrane conductance regulator (CFTR/Cftr), an integral membrane protein that mediates transepithelial chloride transport, as a determinant factor in mice for the susceptibility to several cutaneous symptoms during mite infestation. Mice that endogenously express dysfunctional Cftr (CftrΔF508/ΔF508) show significant increase of scratching behavior and skin fibrosis after mite exposure. These phenotypes were due to the increased expression of nerve growth factor (NGF) that augments the sensitization of peripheral nerve fibers. Moreover, protein gene product 9.5 (PGP9.5)-positive neurites were abundant in the epidermis of mite-infested CftrΔF508/ΔF508mice. Furthermore, mite-infested Cftr+/+mice orally administered with a chloride channel inhibitor glibenclamide had higher scratching count and increased level of NGF than vehicle-treated mice. Consistently, mite extract-exposed primary and transformed human keratinocytes, treated with CFTR inhibitor, had significantly higher level of NGF mRNA compared with vehicle-treated, mite extract-exposed cells. These results reveal that CFTR in keratinocytes plays a critical role for the regulation of peripheral nerve function and pruritus sensation, and suggest that CftrΔF508/ΔF508mice may serve as a novel mouse model that represents NGF-dependent generation of pruritus.

Directed adenovirus evolution using engineered mutator viral polymerases (Article)

Uil, T.G. — 2011-03-01T00:00:00Z

Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad's intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, 'accelerated-evolution' approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad's intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.

Genetic alterations in Glioma (Article)

Bralten, L.B.C. — 2011-03-01T00:00:00Z

Gliomas are the most common type of primary brain tumor and have a dismal prognosis. Understanding the genetic alterations that drive glioma formation and progression may help improve patient prognosis by identification of novel treatment targets. Recently, two major studies have performed in-depth mutation analysis of glioblastomas (the most common and aggressive subtype of glioma). This systematic approach revealed three major pathways that are affected in glioblastomas: The receptor tyrosine kinase signaling pathway, the TP53 pathway and the pRB pathway. Apart from frequent mutations in the IDH1/2 gene, much less is known about the causal genetic changes of grade II and III (anaplastic) gliomas. Exceptions include TP53 mutations and fusion genes involving the BRAF gene in astrocytic and pilocytic glioma subtypes, respectively. In this review, we provide an update on all common events involved in the initiation and/or progression across the different subtypes of glioma and provide future directions for research into the genetic changes.

RNF12 activates Xist and is essential for X chromosome inactivation (Article)

Barakat, T.S. — 2011-02-15T00:00:00Z

In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12-/- knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.

RNF12 activates Xist and is essential for X chromosome inactivation (Article)

Barakat, T.S. — 2011-02-15T00:00:00Z

In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12-/-knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach (Article)

Giardine, B. — 2011-02-01T00:00:00Z

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.

Endothelial nitric oxide synthase overexpression restores the efficiency of bone marrow mononuclear cell-based therapy (Article)

Mees, B.M.E — 2011-01-01T00:00:00Z

Bone marrow-derived mononuclear cells (BMMNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. However, cardiovascular risk factors, including diabetes mellitus and hypercholesterolemia, lead to the abrogation of BMMNCs proangiogenic potential. NO has been shown to be critical for the proangiogenic function of BMMNCs, and increased endothelial NO synthase (eNOS) activity promotes vessel growth in ischemic conditions. We therefore hypothesized that eNOS overexpression could restore both the impaired neovascularization response and decreased proangiogenic function of BMMNCs in clinically relevant models of diabetes and hypercholesterolemia. Transgenic eNOS overexpression in diabetic, atherosclerotic, and wild-type mice induced a 1.5- to 2.3-fold increase in postischemic neovascularization compared with control. eNOS overexpression in diabetic or atherosclerotic BMMNCs restored their reduced proangiogenic potential in ischemic hind limb. This effect was associated with an increase in BMMNC ability to differentiate into cells with endothelial phenotype in vitro and in vivo and an increase in BMMNCs paracrine function, including vascular endothelial growth factor A release and NO-dependent vasodilation. Moreover, although wild-type BMMNCs treatment resulted in significant progression of atherosclerotic plaque in ischemic mice, eNOS transgenic atherosclerotic BMMNCs treatment even had antiatherogenic effects. Cell-based eNOS gene therapy has both proangiogenic and antiatherogenic effects and should be further investigated for the development of efficient therapeutic neovascularization designed to treat ischemic cardiovascular disease.

Nicotinamide adenine dinucleotide (NAD)-regulated DNA methylation alters CCCTC-binding factor (CTCF)/cohesin binding and transcription at the BDNF locus (Article)

Chang, J. — 2010-12-14T00:00:00Z

Cellular metabolism alters patterns of gene expression through a variety of mechanisms, including alterations in histone modifications and transcription factor activity. Nicotinamide adenine dinucleotide (NAD)-dependent proteins such as poly(ADP ribose) polymerases (PARPs) and sirtuin deacetylases play important roles in this regulation, thus NAD provides a crucial link between metabolism and these cellular signaling processes. Here, we found that lowering NAD levels in mouse primary cortical neurons led to decreased activity-dependent BDNF expression. The altered BDNF transcription occurred independently of Sirt or Parp activities; instead, low NAD levels promoted increased DNA methylation of the activity-dependent BDNF promoter. Increased methylation at this promoter triggered the dissociation of the insulator protein CTCF as well as the accompanying cohesin from the BDNF locus. The loss of these proteins resulted in histone acetylation and methylation changes at this locus consistent with chromatin compaction and gene silencing. Because BDNF is critical for neuronal function, these results suggest that age- or nutrition-associated declines in NAD levels as well as deficits in cohesin function associated with disease modulate BDNF expression and could contribute to cognitive impairment.

Functional Analysis of CTCF During Mammalian Limb Development (Article)

Soshnikova, N. — 2010-12-14T00:00:00Z

CCCTC-binding factor (CTCF) is a nuclear zinc-finger protein that displays insulating activity in a variety of biological assays. For example, CTCF-binding sites have been suggested to isolate Hox gene clusters from neighboring transcriptional interference. We investigated this issue during limb development, where Hoxd genes must remain isolated from long-range effects to allow essential regulation within independent sub-groups. We used conditional Ctcf inactivation in incipient forelimbs and show that the overall pattern of Hoxd gene expression remains unchanged. Transcriptome analysis using tiling arrays covering chromosomes 2 and X confirmed the weak effect of CTCF depletion on global gene regulation. However, Ctcf deletion caused massive apoptosis, leading to a nearly complete loss of limb structure at a later stage. We conclude that, at least in this physiological context, rather than being an insulator, CTCF is required for cell survival via the direct transcriptional regulation of target genes critical for cellular homeostasis.

Microcalcifications in atherosclerotic lesion of apolipoprotein E-deficient mouse (Article)

Debernardi, N. — 2010-12-01T00:00:00Z

Evidence is accumulating that calcium-rich microdeposits in the vascular wall might play a crucial role in the onset and progression of atherosclerosis. Here we investigated an atherosclerotic lesion of the carotid artery in an established murine model, i.e. the apolipoprotein E-deficient (APOE-/-) mouse to identify (i) the presence of microcalcifications, if any, (ii) the elemental composition of microcalcifications with special reference to calcium/phosphorus mass ratio and (iii) co-localization of increased concentrations of iron and zinc with microcalcifications. Atherosclerosis was induced by a flow-divider placed around the carotid artery resulting in low and high shear-stress regions. Element composition was assessed with a proton microprobe. Microcalcifications, predominantly present in the thickened intima of the low shear-stress region, were surrounded by areas with normal calcium levels, indicating that calcium-precipitation is a local event. The diameter of intimal microcalcifications varied from 6 to 70 μm. Calcium/phosphorus ratios of microcalcifications varied from 0.3 to 4.8, mainly corresponding to the ratio of amorphous calcium-phosphate. Increased iron and zinc concentrations commonly co-localized with microcalcifications. Our findings indicate that the atherosclerotic process in the murine carotid artery is associated with locally accumulated calcium, iron and zinc. The calcium-rich deposits resemble amorphous calcium phosphate rather than pure hydroxyapatite. We propose that the APOE-/- mouse, in which atherosclerosis was evoked by a flow-divider, offers a useful model to investigate the pathophysiological significance of accumulation of elements such as calcium, iron and zinc.

Evaluating the relationship between spermatogenic silencing of the X chromosome and evolution of the Y chromosome in chimpanzee and human (Article)

Achame, E.M. — 2010-12-01T00:00:00Z

Chimpanzees and humans are genetically very similar, with the striking exception of their Y chromosomes, which have diverged tremendously. The male-specific region (MSY), representing the greater part of the Y chromosome, is inherited from father to son in a clonal fashion, with natural selection acting on the MSY as a unit. Positive selection might involve the performance of the MSY in spermatogenesis. Chimpanzees have a highly polygamous mating behavior, so that sperm competition is thought to provide a strong selective force acting on the Y chromosome in the chimpanzee lineage. In consequence of evolution of the heterologous sex chromosomes in mammals, meiotic sex chromosome inactivation (MSCI) results in a transcriptionally silenced XY body in male meiotic prophase, and subsequently also in postmeiotic repression of the sex chromosomes in haploid spermatids. This has evolved to a situation where MSCI has become a prerequisite for spermatogenesis. Here, by analysis of microarray testicular expression data representing a small number of male chimpanzees and men, we obtained information indicating that meiotic and postmeiotic X chromosome silencing might be more effective in chimpanzee than in human spermatogenesis. From this, we suggest that the remarkable reorganization of the chimpanzee Y chromosome, compared to the human Y chromosome, might have an impact on its meiotic interactions with the X chromosome and thereby on X chromosome silencing in spermatogenesis. Further studies will be required to address comparative functional aspects of MSCI in chimpanzee, human, and other placental mammals.

Fetal globin expression is regulated by Friend of Prmt1 (Article)

Dijk, T.B. van — 2010-11-18T00:00:00Z

Anestimated 6% to 7% of the earth's population carries a mutation affecting red blood cell function. The β-thalassemias and sickle cell disease are the most common monogenic disorders caused by these mutations. Increased levels of γ-globin ameliorate the severity of these diseases because fetal hemoglobin (HbF; α2γ2) can effectively replace adult hemoglobin (HbA; α2β2) and counteract polymerization of sickle hemoglobin (HbS;α2βS2). Therefore, understanding the molecular mechanism of globin switching is of biologic and clinical importance. Here, we show that the recently identified chromatin factor Friend of Prmt1 (FOP) is a critical modulator of γ-globin gene expression. Knockdown of FOP in adult erythroid progenitors strongly induces HbF. Importantly, γ-globin expression can be elevated in cells from β-thalassemic patients by reducing FOP levels. These observations identify FOP as a novel therapeutic target in β-hemoglobinopathies.

Approaching the internalization challenge of grid technologies into e-society by e-human "grid" ecology (Article)

Knoch, T.A. — 2010-11-15T00:00:00Z

The amount of information is growing exponentially with ever-new technologies emerging and is believed to be always at the limit. In contrast, huge resources are obviously available, which are underused in the IT sector, similar as e.g. in the renewable energy sector. This is especially for grid with its fast turnover rates very astonishing considering the barriers for further development put forward by the inability to satisfy the need for such resources. The phenomenon is a typical example of the Inverse Tragedy of the Commons, i.e. resources are underexploited in contrast to the unsustainable and destructing overexploitation in the Classic Tragedy of the Commons. An analysis of IT and the grid sector which attempts to share resources for better usage efficiency, reveals two challenges, which lead to the heart of the paradox: i) From a macro perspective all grid infrastructures involve not only mere technical solutions but also dominantly all of the autopoietic social sub-systems ranging from religion to policy. ii) On the micro level the individual players and their psychology and risk behaviour are of major importance for acting within the macro autopoietic framework. Consequently, the challenges of grid implementation are similar to those of other pressing global issues as e.g. climate protection. This is well described by extending the Human Ecology triangle to a rectangle: invironment-individual-society-environment. By applying this extension of this classical field of interdisciplinary basic and applied research to the grid sector, i.e. by further extension to an e-Human Grid Ecology rational, the Grid Inverse Tragedy of the Commons can be understood and approached regarding the internalization challenge into e-Society and e-Life, from which then guidelines for the day-to-day management can be derived. This is of general importance for many complex fields and thus with similar paradoxes and challenges.

CTCF regulates the local epigenetic state of ribosomal DNA repeats (Article)

Nobelen, S. van de — 2010-11-10T00:00:00Z

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.