A controlled and efficient coordination of gene expression is the key for normal development of an organism. In mammals, a subset of autosomal genes is expressed monoallelically depending on the sex of the transmitting parent, a phenomenon known as genomic imprinting. The imprinted state of the H19 and Igf2 genes is controlled by a short stretch of sequences upstream of H19 known as the imprinting control region (ICR). This region is differentially methylated and is responsible for the repression of the maternally inherited Igf2 allele. It harbors hypersensitive sites on the unmethylated maternal allele and functions as an insulator that binds a chromatin insulator protein CTCF. Hence the H19 ICR, which plays an important role in maintaining the imprinting status of H19 and Igf2, was shown to lose the insulator property upon CpG methylation. Another ICR in the Kcnq1 locus regulates long-range repression of p57Kip2 and Kcnq1 on the paternal allele, and is located on the neighboring subdomain of the imprinted gene cluster containing H19 and Igf2, on the distal end of mouse chromosome 7. Similarly to the H19 ICR, the Kcnq1 ICR appears to possess a unidirectional and methylation-sensitive chromatin insulator property in two different somatic cell types. Hence, methylation dependent insulator activity emerges as a common feature of imprinting control regions. The protein CTCF is required for the interpretation and propagation of the differentially methylated status of the H19 ICR. Work in this thesis shows that this feature applies genomewide. The mapping of CTCF target sites demonstrated not only a strong link between CTCF, formation of insulator complexes and maintaining methylation-free domains, but also a network of target sites that are involved in pivotal functions. The pattern of CTCF in vivo occupancy varies in a lineage-specific manner, although a small group of target sites show constitutive binding. In conclusion, the work of this thesis shows that epigenetic marks play an important role in regulating the insulator property. The studies also confirm the importance of CTCF in maintaining methylation-free domains and its role in insulator function. Our study unravels a new range of target sites for CTCF involved in divergent functions and their developmental control.

<p>A controlled and efficient coordination of gene expression is the key for normal development of an organism. In mammals, a subset of autosomal genes is expressed monoallelically depending on the sex of the transmitting parent, a phenomenon known as genomic imprinting.</p><p>The imprinted state of the <i>H19</i> and <i>Igf2</i> genes is controlled by a short stretch of sequences upstream of <i>H19</i> known as the imprinting control region (ICR). This region is differentially methylated and is responsible for the repression of the maternally inherited <i>Igf2</i> allele. It harbors hypersensitive sites on the unmethylated maternal allele and functions as an insulator that binds a chromatin insulator protein CTCF. Hence the <i>H19</i> ICR, which plays an important role in maintaining the imprinting status of <i>H19</i> and <i>Igf2</i>, was shown to lose the insulator property upon CpG methylation.</p><p>Another ICR in the <i>Kcnq1</i> locus regulates long-range repression of <i>p57Kip2</i> and <i>Kcnq1</i> on the paternal allele, and is located on the neighboring subdomain of the imprinted gene cluster containing <i>H19</i> and <i>Igf2</i>, on the distal end of mouse chromosome 7. Similarly to the <i>H19</i> ICR, the <i>Kcnq1</i> ICR appears to possess a unidirectional and methylation-sensitive chromatin insulator property in two different somatic cell types. Hence, methylation dependent insulator activity emerges as a common feature of imprinting control regions.</p><p>The protein CTCF is required for the interpretation and propagation of the differentially methylated status of the <i>H19</i> ICR. Work in this thesis shows that this feature applies genomewide. The mapping of CTCF target sites demonstrated not only a strong link between CTCF, formation of insulator complexes and maintaining methylation-free domains, but also a network of target sites that are involved in pivotal functions. The pattern of CTCF <i>in vivo</i> occupancy varies in a lineage-specific manner, although a small group of target sites show constitutive binding. </p><p>In conclusion, the work of this thesis shows that epigenetic marks play an important role in regulating the insulator property. The studies also confirm the importance of CTCF in maintaining methylation-free domains and its role in insulator function. Our study unravels a new range of target sites for CTCF involved in divergent functions and their developmental control.</p>

Chromatin insulators play a role in gene transcription regulation by defining chromatinboundaries. Genome-wide studies in Drosophila have shown that a large proportion of insulator sites are found in intergenic DNA sequences, supporting a role for these elements as boundaries. However, approximately 40% of insulator sites are also found in intragenic sequences, where they can potentially perform as yet unidentified functions. Here we show that multiple Su(Hw) insulator sites map within the 110 kb sequence of the muscleblind gene (mbl), which also forms a highly condensed chromatin structure in polytene chromosomes. Chromosome Conformation Capture assays indicate that Su(Hw) insulators mediate the organization of higher-order chromatin structures at the mbl locus, resulting in a barrier for the progression of RNA polymeraseII (PolII ), and producing a repressive effect on basal and active transcription. The interference of intragenic insulators in PolII progression suggests a role for insulators in the elongation process. Supporting this interpretation, we found that mutations in su(Hw) and mod(mdg4) also result in changes in the relative abundance of the mblD isoform, by promoting early transcription termination. These results provide experimental evidence for a new role ofintragenic Su(Hw) insulators in higher-order chromatin organization, repression of transcription, and RNA processing.

ABSTRACT Chromatin insulators are required for proper temporal and spatial expression of genes in metazoans. Insulators are thought to play an important role in the regulation of gene expression through the formation of higher-order chromatin structures. One of the best characterized insulators is the Drosophila gypsy insulator, which is located in the gypsy retrovirus. Several proteins are required for gypsy insulator function, including Su(Hw), Mod(mdg4), and CP190. In addition to the gypsy insulator, these proteins are located throughout the genome at sites which are thought to correspond to endogenous insulators. Analysis of the distribution of insulator proteins across a region of chromosome 2R in Drosophila polytene chromosomes shows that Su(Hw) is found in three structures differentially associated with insulator proteins: bands, interbands and domains of coexpressed genes. Bands are formed by condensation of chromatin within genes containing one or more Su(Hw) binding sites, while Su(Hw) sites in interbands appear to form structures normally associated with open chromatin. Bands characterized by the lack of CP190 and BEAF-32 insulator proteins are formed by clusters of coexpressed genes, and these bands correlate with the distribution of specific chromatin marks. Conservation of the band interband pattern, as well as the distribution of insulator proteins in nurse cells, suggests that this organization may represent the basic organization of interphasic chromosomes. We also show that, in addition to the gypsy insulator, sequence analysis predicts the presence of Su(Hw) binding sites within a number of transposable elements. Su(Hw) binds to predicted sites within gtwin and jockey, which possesses enhancer-blocking activity. Su(Hw) affects the tissue-specific expression of transposable elements, although this effect is unrelated to the presence of Su(Hw) binding sites within the element or control of the elements via the piRNA pathway. Additionally, the effect of Su(Hw) on transposable element expression often differs from that of Mod(mdg4). Taken together, these results suggest that insulator proteins associate specifically with, and may help to define, various levels of chromatin organization on polytene chromosomes. Also, gypsy insulator proteins may influence the expression of transposable elements in a way that does not depend on Su(Hw) binding sites within the elements themselves.