Transgenic mosquitoes are beneficial for the design and implementation of various pathogen control programs. However, low and variable expression of transgenes caused by position effects is a hindrance to the characterization and effective use of transgenes in mosquito species. The use of insulator sequences to flank transgenes may have the ability to overcome position effects caused by the genomic environment surrounding the insertion site. CTCF is a multifunctional protein, conserved from humans to Drosophila. Its role as an enhancer blocker in the Drosophila bithorax complex and its proximal binding to other insulator proteins on Drosophila chromosomes makes it a good candidate for identifying insulator sequences throughout the mosquito genome that may be used to improve mosquito transgenesis. Its multi-functionality as a transcription factor and genome organizer also makes CTCF worthy of investigation for an improved understanding of the regulation of the mosquito genome. This study uses chromatin immunoprecipitation with an An. gambiae CTCF antibody followed by Illumina deep sequencing (ChIP-Seq) to identify regions of CTCF binding throughout the An. gambiae genome. A subset of the CTCF binding site peaks was validated using ChIP-PCR. Another subset of this data set, including the ChIP-PCR validated peaks, was input into the motif finding tool, AlignACE, in order to identify a CTCF binding site consensus. Four motifs were identified, none of which were found in more than 11.9% of the ChIP-Seq data set. These results lead us to conclude that An. gambiae CTCF binds to a wider variety of sequences compared to Drosophila CTCF. This work also includes a comparison of the expression profiles of the dipteran insulator proteins, Su(Hw) and CP190, with that of CTCF across multiple life stages in Ae. aegypti. The results of this study suggest the possibility of genomic colocalization, as has been recently discovered in Drosophila. The identification of CTCF binding site peaks throughout the An. gambiae genome provides a large data set of potential insulator sequences that may be used to improve mosquito transgenesis, and provide a new model for the study of CTCF function in a species with medical significance.

Polytäne Riesenchromosomen von Drosophila melanogaster bieten ein ideales Modellsystem für die Untersuchung der Mechanismen zur strukturellen Bildung chromosomaler Domänen. Über Manipulation und Rekonstruktion des chromosomalen Bandenmusters polytäner Chromosomen können artifiziell kondensierte als auch dekondensierte Domänen etabliert werden. Diese Eigenschaft habe ich in meiner Arbeit genutzt für die Etablierung eines experimentellen Systems zur Analyse der strukturellen Vorraussetzungen zur Bildung offener Chromatindomänen. Dafür wurde eine transgene kondensierte Chromatindomäne ektopisch innerhalb offenen Chromatins generiert. Diese kondensierte „Modellbande“ bietet einen definierten genetischen Hintergrund für die gezielte Insertion von DNA-Sequenzen unter Ausschluss variabler Positionseffekte und ermöglicht dadurch eine vergleichende Analyse dieser Sequenzen auf ihr Vermögen offenes Chromatin zu bilden. Zu diesem Zweck wurde über Sequenz-spezifische Rekombination die 61C7/8 Interbandensequenz gezielt in die „Modellbande“ eingefügt. Die zytogenetische Untersuchung dieser Insertion zeigt, dass infolge der Insertion offenes Chromatin gebildet wird, was in einer Aufsplittung der kondensierten „Modellbande“ resultiert. Molekulare Analysen weisen darauf hin, dass auch die epigenetischen Charakteristika wie z.B. die Rekrutierung typischer Proteine oder transkriptionelle Eigenschaften zur endogenen Domäne immitiert werden. Über Deletionsanalysen konnte von mir die essentielle DNA-Sequenz zur Bildung offenen Chromatins auf ein ~490bp großes Fragment im proximalen Bereich der 61C7/8 Sequenz kartiert werden. Dieses Fragment überlappt mit Bindungsstellen spezifischer Proteine, welche dafür bekannt sind eine Rolle in der chromosomalen Domänenbildung zu spielen wie z.B. das Chromatin-Protein Chriz, die Histon-Kinase Jil1 oder das Insulator-Protein CP190. Desweiteren überlappt es mit einer Promoterregion, welche zwischen den Genen Rev1 und Med30 lokalisiert ist. / Polytene chromosomes of Drosophila melanogaster provides an ideal model-system for the analysis of the mechanisms needed for chromosomal domain formation. Condensed as well as decondensed chromosomal domains can be formed by manipulating and reconstructing the polytene banding pattern. This possibility i ha-ve used for the establishment of an experimental system to study the structural requirements for open chromatin formation. Therefor i have generated a condensed chromatin domain at ectopic positions. This condensed „model“ domain provides a defined genetic context for the targeted insertion of sequences of interest, excluding any variable position effects. This allows comarative analysis of different sequences in order to identify the structural requirements for open chromatin formation. For this purpose the 61C7/8 interband sequence was targetly integrated into the condensed „model“ domain by site-specific recombination. Thereby i could show that the 61C7/8 interband sequence maintains the capacity to form open chromatin cognizable by the splitting of the condensed „model“ domain. Furthermore the newly formed open chromatin domain also keeps epigenetic characteristica like transcriptional activity or the recruitment of typical proteins. By deletion analysis, i have mapped the essential region needed for open chromatin formation to a ~490bp fragment located in the proximal part of the 61C7/8 interband sequence. This fragment overlaps binding sites for characteristic proteins known to be involved in chromosomal domain formation like the chromatin protein Chriz, the histone kinase Jil1 or the insulator protein CP190. Furthermore the fragment overlaps a promoter region that locates between the Rev1 and Med30 genes.