The CTCF protein is involved in several important aspects of gene regulation including transcriptional activation, transcriptional repression and insulator ability. It is also involved in the regulation of epigenetic processes including X-chromosome inactivation and the maintenance of genomic imprinting. CTCF has been shown to bind to approximately 15 000 sites in the mammalian genome and has been implicated in nuclear organization. The CTCF protein mediates long-range chromatin interactions and is believed to form DNA loops. It also acts to block the communication of an enhancer with a promoter by acting as an insulator. Despite its importance in gene regulation, the molecular mechanisms that govern CTCF’s ability to perform its myriad functions remain enigmatic. In this thesis, I add insight into our understanding of the mechanisms behind CTCF’s function. I show that CTCF is post-translationally modified by the Small Ubiquitin-like Modifier proteins and that this post-translational modification contributes to its repressive ability at the c-myc P2 promoter. I also show that CTCF is localized to the sub-nuclear compartment called the Polycomb bodies. The Polycomb protein Pc2 acts as an E3 ligase to enhance the SUMOylation of CTCF by SUMOs 2 and 3. These findings help to explain CTCF’s ability to act as a transcriptional repressor. I also report biochemical evidence to support the role for CTCF in forming an unusual DNA structure, possibly a loop. I hypothesize that a single CTCF binding site is able to form DNA loops. These findings suggest mechanisms by which CTCF is able to organize the mammalian genome and to function as an insulator protein. In addition to these findings I have also purified CTCF interacting proteins through the use of the tandem affinity purification technique. The interacting proteins contain many chromatin and DNA binding proteins further suggesting a role for CTCF in chromatin organization. The results in this thesis enhance our knowledge of the molecular mechanisms of CTCF function and provide a basis for the improved understanding of CTCF mediated gene expression.

The CTCF protein is involved in several important aspects of gene regulation including transcriptional activation, transcriptional repression and insulator ability. It is also involved in the regulation of epigenetic processes including X-chromosome inactivation and the maintenance of genomic imprinting. CTCF has been shown to bind to approximately 15 000 sites in the mammalian genome and has been implicated in nuclear organization. The CTCF protein mediates long-range chromatin interactions and is believed to form DNA loops. It also acts to block the communication of an enhancer with a promoter by acting as an insulator. Despite its importance in gene regulation, the molecular mechanisms that govern CTCF’s ability to perform its myriad functions remain enigmatic. In this thesis, I add insight into our understanding of the mechanisms behind CTCF’s function. I show that CTCF is post-translationally modified by the Small Ubiquitin-like Modifier proteins and that this post-translational modification contributes to its repressive ability at the c-myc P2 promoter. I also show that CTCF is localized to the sub-nuclear compartment called the Polycomb bodies. The Polycomb protein Pc2 acts as an E3 ligase to enhance the SUMOylation of CTCF by SUMOs 2 and 3. These findings help to explain CTCF’s ability to act as a transcriptional repressor. I also report biochemical evidence to support the role for CTCF in forming an unusual DNA structure, possibly a loop. I hypothesize that a single CTCF binding site is able to form DNA loops. These findings suggest mechanisms by which CTCF is able to organize the mammalian genome and to function as an insulator protein. In addition to these findings I have also purified CTCF interacting proteins through the use of the tandem affinity purification technique. The interacting proteins contain many chromatin and DNA binding proteins further suggesting a role for CTCF in chromatin organization. The results in this thesis enhance our knowledge of the molecular mechanisms of CTCF function and provide a basis for the improved understanding of CTCF mediated gene expression.

Erlangen-Nürnberg, Univ., Diss., 2009.

Papillomaviruses (PV) are epithelium specific DNA viruses that can cause health problems ranging from harmless warts to invasive cancer. Papillomavirus induced tumours most often arise in the cervix where human papillomavirus (HPV) infections were shown to cause 99.7 % of all malignancies. This study aims to map binding sites of the multifunctional host protein CCCTC binding factor (CTCF) to the papillomavirus genome, validate them and determine the function of CTCF in the papillomavirus life cycle. Computer predictions of CTCF binding sites in the sequence of 8 different PV revealed a CTCF binding pattern including a conserved high-affinity binding site around nucleotide 3000 in high risk HPV and around nucleotide 5400 in low risk HPV. This binding pattern was experimentally confirmed using electrophoretic mobility shift assays (EMSA). The binding site around nucleotide 3000 in HPV18 was mutated and human foreskin keratinocytes (HFK) were transfected with mutant and wild type HPV18 to analyse the effect of the mutation on viral gene expression and life cycle. Western blotting of methylcellulose differentiated HFK revealed earlier expression of E2 and decreased expression of E1^E4 in the mutant compared to the wild type. Immunostaining of organotypic raft cultures grown from the mutant maintaining cells showed a significant increase in proliferating cells compared to the HFK maintaining the wild type. This was accompanied by pseudo-differentiation of keratinocytes since the cells of the granular layer of the raft expressed the terminal differentiation marker loricrin but maintained the morphology of undifferentiated cells. Thus CTCF was shown to have a major impact on the HPV life cycle and it may play a role in HPV induced carcinogenesis. Furthermore a function of CTCF in long term maintenance of the viral episome was revealed as cells maintaining the CTCF mutant were shown to lose episomes more quickly compared to wild type maintaining cells.

Low level and variable transgene expression plague efforts to produce and characterize transgenic lines in many species. When transformation efficiency is high, productive transgenic lines can be generated with reasonable effort. However, most efforts to date in mosquitoes have resulted in suboptimal levels of transformation. This, coupled with the large space and intensive labor requirements of mosquito colony maintenance makes the optimization of transformation in mosquitoes a research priority. This study proposes two strategies for improving transgene expression and transformation efficiency. The first is to explore exogenous promoter/enhancer combinations to direct expression of either the transgene itself, or the transposase required for insertion of the transgene into the genome. An extension of this strategy is to investigate the use of a powerful viral transactivating protein and its cognate enhancer to further increase expression of these targets. The second strategy involves the identification of an endogenous boundary element for use in insulating transgenes and their associated regulatory elements. This would mitigate the inappropriate expression or silencing of many transgenes inserted into “unfavorable” genomic environments as a consequence of an inability to specifically target the integration of transposons currently used in mosquito transgenesis. The IE1 transactivating protein and its cognate enhancer from a baculovirus were shown to significantly increase expression of a reporter gene from three different promoters in cultured mosquito cells. Other heterologous enhancer/promoter combinations resulted in minimal increases or insignificant changes in expression. Orthologues of the vertebrate insulator-binding factor, CTCF, were cloned and characterized in two mosquito species, Aedes aegypti and Anopheles gambiae. The expression profile of mosquito CTCF is consistent with its role as a putative insulatorbinding protein. Preliminary binding site studies reveal a C/G-rich binding site consistent with that known in vertebrates and indicate that CTCF may bind widespread sites within mosquito genomes.

The renin angiotensin system (RAS) is critical for the regulation of blood pressure, electrolyte/fluid, and metabolic homeostasis. Regulation of the RAS is important in the development and treatment of hypertension. As part of the rate-limiting step in a cascade of events ending in the production of angiotensin II, renin is a major regulator of the RAS. Its expression is localized to the juxtaglomerular (JG) cells of the JG apparatus where it is exquisitely located to respond to various physiological cues. Understanding the regulation of renin expression and development of the juxtaglomerular cells is critical. Two regulatory elements, the enhancer and proximal promoter, have been found to be important in controlling cell- and tissue- specific baseline expression of the renin gene. Within the enhancer is a hormone response element (HRE) which confers a high level of activity to the enhancer. Nuclear receptors that bind this element have been found to bind the HRE and regulate renin promoter transcriptional activity. We have previously characterized the role of the orphan nuclear receptor Nr2f6 as a negative regulator of renin expression that mediates its effects through the HRE. However, gel shift assays indicate that there are other transcription factors binding this element. We have identified other orphan nuclear receptors that regulate renin expression. The first, Nr2f2 acts as a negative regulator of renin promoter activity but does not appear to affect baseline expression of the endogenous renin gene. The other, Nr4a1, is a positive regulator of renin expression, but it does not appear to mediate its effects through the HRE. The transcriptional regulation of gene expression is controlled by regulatory elements separated by large distances from promoters. We and others have found that short transgenes of the human renin (hREN) locus are not sufficient to protect them from positional effects that can be exerted upon them by neighboring regulatory elements. We discovered a random truncation in a large genomic construct of the hREN gene that resulted in ubiquitous expression of renin not seen with the intact form. By locating the genomic insertion site of that transgene in the Zbtb20 gene, we found that the hREN promoter had come under control of that gene's regulatory elements. The gene downstream of renin however maintained its tissue-specific expression. We found that CCCTC-binding factor (CTCF) bound to chromatin in and around the renin locus. The presence of CTCF suggests that insulator elements are present in the renin locus, and their loss likely explains the results above. Finally, we assessed the role of microRNAs in the development of renin expressing cells in the mouse kidneys by cell-specific deletion of the processing enzyme Dicer. This resulted in reduction of renin expression and a decrease in the number of renin expressing cells in the kidney. Mice were hypotensive and had several kidney abnormalities including a hypertrophied vasculature and striped fibrosis. These results indicate that Dicer and the miRNAs it processes are critical for the development and maintenance of renin expressing cells that contribute to normal kidney development.

TAD-Grenzen sind genomische Regionen mit Isolatorpotenzial, die zwischen benachbarten Chromatindomänen liegen und deren Unterbrechung zu einer pathologischen Genexpression führen kann. Die meisten TAD-Grenzen werden durch das CTCF gebunden, ein Architekturprotein, das Chromatinschleifen bevorzugt zwischen distalen Paaren von CTCF-Bindungsstellen (CBS) mit einer konvergenten Motivausrichtung bildet. An TAD-Grenzen sind die CBS häufig geclustert, wobei die Motive eine divergente Ausrichtung aufweisen und Chromatinschleifen in Richtung der stromaufwärts und stromabwärts gelegenen Regionen projizieren. Wie die CTCF-Besetzung die Isolierung an TAD-Grenzen moduliert, ist immer noch nicht ganz klar. Hier habe ich die regulatorische Logik von CTCF-geclusterten TAD-Grenzen untersucht, indem ich genomweite Analysen und in vivo-Mausexperimente an der Epha4-Pax3-TAD-Grenze kombiniert habe. Analysen einzelner Deletionen zeigten einen deutlichen hierarchischen Beitrag von CBS zur Grenzfunktion. Im Gegensatz dazu zeigten kombinierte CBS-Deletionen ein gewisses Maß an funktioneller Redundanz und Kooperativität zwischen den Stellen. Diese Analysen zeigten auch, dass die abweichende Konfiguration der CBS, die immer wieder an TAD-Grenzen zu finden ist, für eine robuste Isolierung nicht unbedingt erforderlich ist. Genomweite Analysen haben gezeigt, dass es eine Untergruppe von CBS gibt, die unabhängig von der konvergenten Ausrichtung Chromatinschleifen bilden, wofür ich einen Mechanismus der "Schleifeninterferenz" vorschlage. Weitere Vergleiche ergaben, dass das Niveau der Genexpression von den Abständen zwischen Enhancer und Promoter im linearen Genom abhängen könnte. Durch die Quantifizierung der Isolierung der Grenzen, der Pax3-Fehlexpression und der Schwere der Gliedmaßenfehlbildungen konnte ich schließlich zeigen, dass die TAD-Grenzen die Genexpression und den Phänotyp quantitativ beeinflussen. / TAD boundaries are genomic regions with insulator potential located between adjacent chromatin domains, which disruption can cause pathological gene expression. Most TAD boundaries are bound by the CTCF, an architectural protein that forms chromatin loops preferentially between distal pairs of CTCF binding sites (CBSs) with a convergent motif orientation. At TAD boundaries, CBSs are frequently clustered, with motifs displaying a divergent orientation and projecting chromatin loops towards up and downstream regions. How CTCF occupancy modulates insulation at TAD boundaries still remains elusive. Here, I dissected the regulatory logic of CTCF-clustered TAD boundaries by combining genome-wide analysis and in vivo mouse experiments at the Epha4-Pax3 TAD boundary. Analyses of individual deletions revealed a distinct hierarchical contribution of CBS to boundary function. In contrast, combined CBSs deletions revealed a certain degree of functional redundancy and cooperativity between sites. These analyses also demonstrated that the divergent configuration of CBSs, recurrently found at TAD boundaries, is not strictly required for robust insulation. Genome-wide analysis highlighted the existence of a subset of CBSs that establish chromatin loops independently of the convergent orientation bias, for which I propose a mechanism of “loop interference”. This mechanism suggests that CBS forming a robust convergent loop can simultaneously form a non-convergent loop, by stalling Cohesin complexes extruded from both sides. Further comparisons revealed that gene expression levels might depend on enhancer-promoter distances in the linear genome. Finally, by quantifying boundary insulation, Pax3 misexpression and the severity of limb malformation, I demonstrate that TAD boundaries are quantitative modulators of gene expression and phenotypes. Overall, I highlight that TAD boundary composition and strength constitute a fundamental regulatory layer in developmental processes and disease.

The expansion of repetitive sequences leads to more than 40 neurological, neurodegenerative and neuromuscular diseases. These diseases are frequently characterized by ongoing DNA repeat instability upon transmission, worsening of disease severity and decreasing age of onset with each successive generation. The mechanism of repeat instability and contribution of repeat instability to disease pathogenesis are unknown. My thesis examines the contribution of cis-elements – sequences around and within repeats as well as surrounding epigenetic environments – to repeat instability, and discusses their possible contribution to repeat diseases. Here I identify the first cis-element regulating repeat instability, a DNA binding site for a trans factor protein, CTCF. Loss of CTCF binding at the spinocerebellar ataxia type 7 disease locus induces somatic and germline instability in an age- and tissue-specific manner in mice. CTCF protects against instability in an epigenetic manner, and may function at other disease loci also possessing CTCF binding sites near the repeat. Given that CTCF flanks many repeat loci and is often situated between a replication origin and disease-associated repeat, I assess the role of CTCF on replication and instability at the myotonic dystrophy repeat locus. Templates with CTCF binding sites reduce overall replication efficiency in primate cells that may be partly due to replication fork stalling. Mutating CTCF binding sites can alter the stability of the repeat depending on the distance from the origin of replication to the repeat. Finally I examine chromatinization of (ATTCT)n repeats from the spinocerebellar ataxia type 10 locus. These repeats induce very strong nucleosome formation, and at physiological conditions form even more strongly on (ATTCT)n repeats with interruptions that are also found in some patients. These data contribute to the understanding of repeat instability in the causation of many diseases, and suggest that the presence of cis-elements at repeat-associated disease loci alter the behaviour of repeats.

The expansion of repetitive sequences leads to more than 40 neurological, neurodegenerative and neuromuscular diseases. These diseases are frequently characterized by ongoing DNA repeat instability upon transmission, worsening of disease severity and decreasing age of onset with each successive generation. The mechanism of repeat instability and contribution of repeat instability to disease pathogenesis are unknown. My thesis examines the contribution of cis-elements – sequences around and within repeats as well as surrounding epigenetic environments – to repeat instability, and discusses their possible contribution to repeat diseases. Here I identify the first cis-element regulating repeat instability, a DNA binding site for a trans factor protein, CTCF. Loss of CTCF binding at the spinocerebellar ataxia type 7 disease locus induces somatic and germline instability in an age- and tissue-specific manner in mice. CTCF protects against instability in an epigenetic manner, and may function at other disease loci also possessing CTCF binding sites near the repeat. Given that CTCF flanks many repeat loci and is often situated between a replication origin and disease-associated repeat, I assess the role of CTCF on replication and instability at the myotonic dystrophy repeat locus. Templates with CTCF binding sites reduce overall replication efficiency in primate cells that may be partly due to replication fork stalling. Mutating CTCF binding sites can alter the stability of the repeat depending on the distance from the origin of replication to the repeat. Finally I examine chromatinization of (ATTCT)n repeats from the spinocerebellar ataxia type 10 locus. These repeats induce very strong nucleosome formation, and at physiological conditions form even more strongly on (ATTCT)n repeats with interruptions that are also found in some patients. These data contribute to the understanding of repeat instability in the causation of many diseases, and suggest that the presence of cis-elements at repeat-associated disease loci alter the behaviour of repeats.

<p>Understanding the mechanisms whereby the sequence of the human genome is interpreted into diverse cellular phenotypes is a critical endeavor in modern biology. A major determinant of cellular phenotype is the spatial and temporal pattern gene expression, which is regulated in part by epigenomic properties such as histone post-translational modifications, DNA methylation, chromatin accessibility and the 3-dimensional architecture of the genome within the nucleus. These properties regulate the dynamic assembly of transcription factors and their co-regulatory proteins upon chromatin. To properly understand the interplay between the epigenomic framework of a cell and transcription factors, integrated analysis of transcription factor-DNA binding, chromatin status, and transcription is required. This work integrates information about chromatin accessibility, as measured by DNaseI hypersensitivity, transcription factor binding, as measured by chromatin immunoprecipitation, and transcription, as measured by microarray or transcriptome sequencing, to further understand the functional role of two important transcription factors, the androgen receptor (AR) and CTCF, in cancer cell line models. Data gathered from a prostate cancer cell line model demonstrate that the AR does not exclusively bind accessible chromatin upon ligand-activation, and induces significant changes in chromatin accessibility upon binding. Regions of quantitative change in chromatin accessibility contain motifs corresponding to potential collaborators for AR function, and are also significantly associated with AR-regulated transcriptional changes. Furthermore, base pair resolution of the DNaseI cleavage profile revealed three distinct patterns of AR-DNA interaction, suggesting multiple modes of AR interacting with the genome. A novel role for the nuclear receptor REV-ERB&#945; in AR-mediated transcription was explored within the same model system. Though preliminary, results thus far indicate that REV-ERB&#945; is required for AR-induced increases in target gene transcription in a manner that is likely dependent on HDAC3. Genetic knockdown of REV-ERB&#945; resulted in notable changes in chromatin accessibility around AR-target genes both before and after AR activation. The function of CTCF was interrogated using stable knockdown in a breast cancer cell line model. CTCF knockdown led to widespread changes in chromatin accessibility that were dependent on DNA sequence. Further analysis suggested that AP-1 and FOXA1 are involved in CTCF function. Together, the work presented in this dissertation offers novel insight into the behavior of two critical transcription factors in cancer cell lines, and describe a framework of analysis that can be extended and applied to any transcription factor within any desired cellular context.</p> / Dissertation