Der SET-Bereich befindet sich unter den verschiedenen Proteinsequenzbereichen, die mit epigentischer Regulation hauptsächlich durch die Präsenz von Trihtorax (trxG) und Polycomb (PcG) Gruppen von Chromatinmodifikatoren in Zusammenhang gebracht werden. Nach der Entdeckung des ersten SET-Bereichs vor einigen Jahren, welcher die Histon-Lysin-Methyltransferase (Su(var)39) enthält, wurde den Proteinen mit SET-Bereich sehr viel Aufmerksamkeit geschenkt. Obwohl die Histon-Lysin-Methylierung schon länger als 30 Jahre bekannt ist, war ihre Funktion vor diesem überragenden Ergebnis größten Teils unbekannt. In meiner Arbeit beschreibe ich die kombinatorische und funktionale Charakterisierung von 3 Hefe Proteinkomplexen durch die Anwendung von proteomischer SEAM (Sequential Epitope Tagging and Mass Spectrometry). Zwei dieser Komplex enthalten einen SET-Bereich und die Dritte ist der Rad6 Komplex aus S. pombe (Sp_Rad6C). Der Set1 Komplex (Set1C) beinhaltet 8 Bausteine, methylisiert Lysin 4 in Histon H3 und ist die erste, entdeckte Histone H3 Lysin 4 Methyltransferase. Es beinhaltet ein Ash2 Homolog (Bre2), einen bekannten Baustein von trxG. Kürzlich wurden Rad6 beinhaltende Komplexe gezeigt, die in engem Bezug zu der H3-K4 und H3-K79 Methylation durch ubiquitinierung von Histon H2B und die Etablierung von trans-histonen Signalwegen stehen. Unsere Analysen von Sp-Rad6C führten zu mehreren interessanten Ansichten. Der Set3 Komplex (Set3C) hat keine feststellbare Aktivität einer Methyltransferase, enthält jedoch zwei Histon deacetylasen (HDACs) ? eine klassische HDAC (Hos2) und eine NAD-abhängige HDAC (Hst1). Unsere funktionelle Analyse von Set3C zeigt, dass Set3C bei der Regulierung des meiotischen Genexpressionsprogramms in knospenden Hefen (S. cerevisiae) beteiligt ist. Evolutionbiologisch betrachtet, ist die Spalt-Hefe (S. pombe) sehr weit von S. cerevisiae entfernt und wird meist als ein besserer Modellorganismus fur höhere Eukaryoten angesehen. In einem Versuch, unser Wissen uber andere Organismen zu vergrößern, haben wir ähnliche Untersuchungen in S. pombe unternommen und haben herausgefunden, dass Set1C in beiden Hefen sehr stark konserviert ist. Darüberhinaus waren die Set1-Ash2 Verbindungen konserviert und wir nehmen an, dass auch in höheren Eukaryoten Set1-ahnliche Methyltransferasen Ash2-ahnliche Proteinen angehören. Dies wurde später durch mehrere Studien von anderen Gruppen bestätigt, die an Säugetieren arbeiten. Was Set3C anbelangt, wurden unsere weiteren Analysen nur durch vergleichende Proteomik beschränkt. Wir zeigen, dass der proteomische Kern von Set3C in Spalt-Hefe konserviert wird. Im Gegensatz zu Set3C in S. cerevisiae, beinhaltet diese in S. pombe nur eine HDAC, die zur Hos2 Familie gehört,. Die präsentierte Arbeit hat auch viele Auswirkungen auf die übergreifende Organisation von Proteomen. Wir beschreiben verschiedene Beispiele von gemeinsamen Komponenten zwischen unterschiedlichen Komplexen und prägen den Begriff "proteomischer Hyperlink". Wir waren in der Lage zu zeigen, dass proteomische Kerne sogar für unwesentliche Proteinkomplexe hoch konserviert sind. Die generelle proteomische Schaltung über proteomische Hyperlinks scheint jedoch verworrener und unvorhersehbar zu sein. Wir schlussfolgern, dass die Erschaffung von zuverlässigen, detailierten, proteomischen Abbildungen, welche auf dem Wissen von niederen Organismen fundieren, zur Zeit nicht möglich ist.

Der SET-Bereich befindet sich unter den verschiedenen Proteinsequenzbereichen, die mit epigentischer Regulation hauptsächlich durch die Präsenz von Trihtorax (trxG) und Polycomb (PcG) Gruppen von Chromatinmodifikatoren in Zusammenhang gebracht werden. Nach der Entdeckung des ersten SET-Bereichs vor einigen Jahren, welcher die Histon-Lysin-Methyltransferase (Su(var)39) enthält, wurde den Proteinen mit SET-Bereich sehr viel Aufmerksamkeit geschenkt. Obwohl die Histon-Lysin-Methylierung schon länger als 30 Jahre bekannt ist, war ihre Funktion vor diesem überragenden Ergebnis größten Teils unbekannt. In meiner Arbeit beschreibe ich die kombinatorische und funktionale Charakterisierung von 3 Hefe Proteinkomplexen durch die Anwendung von proteomischer SEAM (Sequential Epitope Tagging and Mass Spectrometry). Zwei dieser Komplex enthalten einen SET-Bereich und die Dritte ist der Rad6 Komplex aus S. pombe (Sp_Rad6C). Der Set1 Komplex (Set1C) beinhaltet 8 Bausteine, methylisiert Lysin 4 in Histon H3 und ist die erste, entdeckte Histone H3 Lysin 4 Methyltransferase. Es beinhaltet ein Ash2 Homolog (Bre2), einen bekannten Baustein von trxG. Kürzlich wurden Rad6 beinhaltende Komplexe gezeigt, die in engem Bezug zu der H3-K4 und H3-K79 Methylation durch ubiquitinierung von Histon H2B und die Etablierung von trans-histonen Signalwegen stehen. Unsere Analysen von Sp-Rad6C führten zu mehreren interessanten Ansichten. Der Set3 Komplex (Set3C) hat keine feststellbare Aktivität einer Methyltransferase, enthält jedoch zwei Histon deacetylasen (HDACs) ? eine klassische HDAC (Hos2) und eine NAD-abhängige HDAC (Hst1). Unsere funktionelle Analyse von Set3C zeigt, dass Set3C bei der Regulierung des meiotischen Genexpressionsprogramms in knospenden Hefen (S. cerevisiae) beteiligt ist. Evolutionbiologisch betrachtet, ist die Spalt-Hefe (S. pombe) sehr weit von S. cerevisiae entfernt und wird meist als ein besserer Modellorganismus fur höhere Eukaryoten angesehen. In einem Versuch, unser Wissen uber andere Organismen zu vergrößern, haben wir ähnliche Untersuchungen in S. pombe unternommen und haben herausgefunden, dass Set1C in beiden Hefen sehr stark konserviert ist. Darüberhinaus waren die Set1-Ash2 Verbindungen konserviert und wir nehmen an, dass auch in höheren Eukaryoten Set1-ahnliche Methyltransferasen Ash2-ahnliche Proteinen angehören. Dies wurde später durch mehrere Studien von anderen Gruppen bestätigt, die an Säugetieren arbeiten. Was Set3C anbelangt, wurden unsere weiteren Analysen nur durch vergleichende Proteomik beschränkt. Wir zeigen, dass der proteomische Kern von Set3C in Spalt-Hefe konserviert wird. Im Gegensatz zu Set3C in S. cerevisiae, beinhaltet diese in S. pombe nur eine HDAC, die zur Hos2 Familie gehört,. Die präsentierte Arbeit hat auch viele Auswirkungen auf die übergreifende Organisation von Proteomen. Wir beschreiben verschiedene Beispiele von gemeinsamen Komponenten zwischen unterschiedlichen Komplexen und prägen den Begriff "proteomischer Hyperlink". Wir waren in der Lage zu zeigen, dass proteomische Kerne sogar für unwesentliche Proteinkomplexe hoch konserviert sind. Die generelle proteomische Schaltung über proteomische Hyperlinks scheint jedoch verworrener und unvorhersehbar zu sein. Wir schlussfolgern, dass die Erschaffung von zuverlässigen, detailierten, proteomischen Abbildungen, welche auf dem Wissen von niederen Organismen fundieren, zur Zeit nicht möglich ist.

Cellular and tissue characteristics are results of dynamic regulation of gene expression. DNA wrapped into proteins, referred to as chromatin, requires involvement of mechanisms guiding accessibility of specific sequences. In higher organisms, chromatin remodeling proteins are indispensable in regulating chromatin structure including ISWI ATPase SMARCA5. SMARCA5 is involved in almost any transaction on DNA including transcription, however precise in vivo role of SMARCA5 in these processes remains unknown. To advance understanding of specific role of SMARCA5 in the development of chromatin structure during transcription we devised cellular model in which SMARAC5 level is manipulated while chromatin structure development and transcriptional response are monitored. Our data indicate that the transfected DNA template that is transcribed is enriched with histone H3 and its specific methylation of Histone H3 lysine (K) 4, a mark of active chromatin structure. Overexpression of SMARCA5 results within the reporter gene coding sequence in ~2,5-3 fold increase of both H3 occupancy an its modification H3K4Me3. Increased DNA template commitment into chromatinization is associated with repression of reporter gene expression. These results are supported by studies indicating dynamic development of nucleosomal...

Cornelia de Lange Syndrome (CdLS) is a multisystem genetic disorder caused by mutations in the cohesin complex. It is believed that cohesin is able to regulate gene expression with CTCF by holding chromatin in topological complexes, such as active chromatin hubs, and that CdLS is caused by loss of these complexes causing aberrant gene expression. In order to determine if loss of these complexes in CdLS resulted in a general change in the compaction of chromatin, I undertook a series of analyses of the nucleus in CdLS patient lymphoblastoid cell lines (LCLs), compared to wildtype, and later in RNAi knockdown models of CdLS. By fluorescent in situ hybridisation (FISH) I studied the chromatin compaction of different regions of the genome, and found that in some, but not all, CdLS cell lines, gene-rich regions have less compact chromatin compared to wildtype. RNAi knockdown of two proteins that are mutated in CdLS, NIPBL and SMC1, also resulted in decompaction of regions of the genome, however these were different regions than in the patient LCLs, perhaps due to variation between cell lines. This change was not due to the interaction between cohesin and CTCF, as I found that knockdown of CTCF did not result in changes in chromatin compaction. I have also looked at the published data for gene expression in CdLS, and in mouse and Drosophila models of CdLS, and have found no correlation between the genes misexpressed in CdLS in the three species, nor between three cell lines of the same species. These data suggest that the variation in chromatin compaction observed in CdLS may not be due to an interaction between cohesin and CTCF, and that cohesin can act independently of CTCF to regulate gene expression.

Eukaryote DNA is organised into the more compact nucleosome by wrapping 147bp of DNA around a histone octamer core. The N-terminal tails of the histones protrude through the DNA and can be modified by a variety of enzymes. Acetylation of Histone 4 Lysine 16 (H4K16ac) is an important modification associated with an increase in transcription, and in flies is an important component of the doseage compensation system. It is also unique amongst histone modifications in that it has been directly associated with chromatin decompaction. H4K16ac has been linked to development through its Histone Acetyltransferase, MOF. Deletion of MOF in mice leads to mass chromatin defects, and embryonic lethality prior to the blastocyst stage. I set out to understand the role of H4K16ac in differentiating Embryonic Stem cells (ES cells) and chromatin compaction in vivo. I generated a ChIP-seq profile for H4K16ac in undifferentiated ES cells, and after 3 days of retinoic acid (RA) differentiation. This revealed an association of H4K16ac with the promoters of transcribed genes in pluripotent ES cells, followed by loss H4K16ac on ES cell specific genes and gain of the modification on differentiation specific genes. There were some silent genes in ES cells, however, which were acetylated on their promoters. Through this study I also found that H4K16ac and MOF mark active enhancers in ES cells, along with H3K4me1 and H3K27Ac and p300. H4K16ac did not mark a known regulatory region in limb cells, and it is possible that it marks active enhancers only of ES cells. Furthermore, I looked at the compaction state large regions (>100kb) which lost H4K16ac upon differentiation by FISH, to determine if loss of H4K16ac could predict compaction. The regions selected showed no change in compaction state between UD and D3 cells, meaning that loss of H4K16ac does not directly lead to chromatin compaction in vivo. However loss of H4K16ac may be necessary for any subsequent compaction, or the change in compaction may take place at nucleosomal level. Finally, I attempted both to overexpress and reduce the level of MOF in ES cells. I was unable to manipulate the level of MOF in this cell type in either direction; expression of endogenous MOF was silenced after very little time, and stable MOF shRNA cell lines showed no reduction in levels of MOF. Therefore, potentially, dosage of MOF/H4K16ac in this cell type is critical. This study may help to understand the significance of H4K16ac in ES cell differentiation and chromatin compaction.

Chromatin structure and epigenetic mechanisms play an important role in initiating and maintaining the intricate patterns of gene expression required for embryonic development. One such mechanism, DNA methylation (5mC), involves the chemical modification of cytosine bases in DNA and is implicated in maintaining patterns of transcription. However, many fundamental aspects of DNA methylation are not fully understood, including the mechanisms by which it influences transcriptional states. Recent data suggest functional links between DNA methylation and a second epigenetic mechanism that has important roles in transcriptional repression, the polycomb group (PcG) repressor system. Here, I suggest that an intact DNA methylation system is required for the repression of many PcG target genes by influencing the genomic targeting of the polycomb repressor 2 complex (PRC2) and its signature histone modification, H3K27me3 (K27me3). I demonstrate differential genomic localisation of K27me3 at gene promoter regions in hypomethylated mouse embryonic fibroblast (MEF) cells deficient for the major maintenance DNA methyltransferase, Dnmt1. Globally, Dnmt1-/- MEFs have a higher level of the K27me3 mark than controls, as assessed by western blot and immunofluorescence. I observe increased K27me3 at a relatively small number of gene promoters in Dnmt1-/- MEFs that often are associated with high levels of DNA methylation in wildtype MEFs, consistent with the notion that DNA methylation is capable of antagonising PRC2 binding at certain loci. Conversely, I show that a large number of developmentally important genes that are normally repressed and highly bound by K27me3, including classic polycomb targets, the Hox genes, display dramatically reduced association with K27me3 in Dnmt1-/- MEFs. Many of these genes, but not all, show reciprocal increases in promoter H3K4me3 modification and are transcriptionally de-repressed in Dnmt1-/- MEFs. I suggest that these genes are mostly associated with CpG-rich promoters with low levels of DNA methylation in wildtype cells, implying that their silencing is not dependent on the canonical role of DNA methylation. Consistent with the findings of recently published work, I suggest a working model where PRC2 binding in wildtype cells is restricted by CpG methylation. According to this model, the differential genomic location of K27me3 in hypomethylated Dnmt1-/- MEFs is explained by a redistribution of PRC2 to normally DNA methylated, unbound loci, resulting in a titration effect and coincident loss of K27me3 from normal targets. It was also apparent that certain PRC2-target genes, including the developmentally important Hox gene clusters, are strongly affected in Dnmt1-/- MEFs, displaying striking loss of K27me3. As intergenic transcription has been implicated in relief from polycomb silencing and abundant intergenic transcription has been reported within Hox clusters, I measured RNA expression at Hox clusters and a small number of other PcG target genes in Dnmt1-/- MEFs using highdensity tiling arrays. In Dnmt1-deficient MEFs, widespread increases in intergenic transcription were observed within Hox clusters. In addition, mapping of the elongatingpolymerase- associated H3K36me3 histone modification showed widespread increases in this mark at intergenic and promoter regions in Dnmt1-/- MEFs. Increased local intergenic RNA and H3K36me3 were found to correlate with K27me3 loss for this cohort of genes. I suggest a working model where increased intergenic transcription and H3K36me3 in Dnmt1-/- MEFs leads to accelerated loss of K27me3 at certain loci, including Hox clusters. Taken together with recently published data, this work suggests that a major role of DNA methylation is in shaping the PRC2/K27me3 landscape. The potential implications of this putative role for DNA methylation are widespread, including our knowledge of how DNA methylation influences transcriptional regulation, and the consequence of rearranged DNA methylation patterns that are observed in many diseases including cancers.

The centromere is a specialized chromatin domain that serves as the assembly site for the mitotic kinetochore structure, thereby playing a fundamental role in facilitating the maintenance of the genetic information. A histone H3 variant termed CENP-A is specifically found at all active centromeres. Beyond this, however, little is known about how and to which extent the chromatin environment of centromeres modulates and contributes towards centromere identity and function. Here, I have employed a novel Human Artificial Chromosome (HAC), the centromere of which can be targeted by fusions to the tet repressor, to characterize the chromatin environment underlying active kinetochores, as well as to specifically probe the role of this environment in the maintenance of kinetochore structure and function. My data demonstrate that centromeric chromatin resembles the downstream regions of actively transcribed genes. This includes the previously unrecognized presence of histone H3 nucleosomes methylated at lysine 36 within the chromatin underlying functional kinetochores. Targeted manipulation of this chromatin through tethering of a heterochromatin-seeding transcriptional repressor results in the inactivation of HAC kinetochore function concomitant with a hierarchical disassembly of the structure. Through an even more selective engineering of the HAC centromere chromatin, I have provided evidence supporting a critical role for nucleosomes dimethylated at lysine 4 on histone H3 in facilitating local transcription of the underlying DNA. Tethering of different chromatin-modifying activities into the HAC kinetochore collectively reveals a critical role for both, histone H3 dimethylated on lysine 4 and low-level, non-coding transcription in the maintenance of the CENP-A chromatin domain. On one hand, repression of centromeric transcription negatively correlates with the maintenance of CENP-A and ultimately results in the loss of kinetochore function. On the other hand, increasing kinetochore-associated RNA polymerase activity to within physiological levels for euchromatin is associated with rapid loss of CENP-A from the HAC centromere. Together, my data point towards the requirement for a delicate balance of transcriptional activity that is required to shape and maintain the chromatin environment of active centromeres.

ABSTRACT DNA in the eukaryotic nucleus is complexed with histone and non-histone proteins into chromatin. Nucleosomes, the basic repeating unit of chromatin, not only package DNA but are also intimately involved the regulation of gene expression. All DNA transactions including replication, transcription, recombination and repair take place in such a chromatin environment. Access to packaged nucleosomal DNA in vivo is mediated at least in part by protein complexes that modify or remodel chromatin. Buried sequences in nucleosomes can also transiently become accessible to DNA binding proteins during cycles of partial, reversible unwrapping of nucleosomal DNA from the histone octamer. We have investigated the ability of the human, bifunctional DNA glycosylase, endonuclease III (hNTH1), to initiate base excision repair (BER) of discretely positioned oxidative lesions in model nucleosomes. hNTH1 was able to process a thymine glycol (Tg) lesion almost as efficiently as naked DNA, when the minor groove of the lesion faced away from the histone octamer. Lesion processing did not require or result in detectable nucleosome disruption, as assayed in gel mobility-shift experiments. Instead, hNTH1 formed a slower migrating enzyme-nucleosome ternary complex that was found to contain processed DNA. Processing of an inward-facing Tg residue located just 5 bp away from the outward-facing lesion was much reduced and processing of a sterically occluded Tg residue positioned closer to the dyad center of the nucleosome was even more reduced. Notably, processing of both inward-facing lesions was found to increase as a function of enzyme concentration. Restriction enzyme protection studies indicated that access to these inward-facing lesions did not entail nucleosomal translocation or sliding. Collectively, these observations are consistent with a model in which hNTH1 binds to lesions during cycles of reversible, partial unwrapping of nucleosomal DNA from the histone octamer core. To further investigate this partial unwrapping hypothesis, we studied the kinetics of hNTH1 processing of sterically occluded lesions in greater detail. Our results suggest that efficiency of processing of inward-facing lesions is a function of both DNA unwrapping and rewrapping rates, and enzyme affinity for the lesion. In addition, we determined that APE1 which catalyzes the second step in BER, exhibited an increasing capacity to process inward-facing furan residues as its concentration was increased. Thus as with hNTH1, we hypothesize that APE1 can capture occluded furan residues during cycles of partial DNA unwrapping. We propose that cellular regulatory factors benefit from this intrinsic, periodic exposure of nucleosomal DNA exposure in vivo, which may be amplified by the downstream recruitment of remodeling and / or modifying proteins to facilitate DNA transactions in the cell.

<p>The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.</p><p>One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes. </p><p>I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity. </p><p>One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.</p><p>In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.</p> / Dissertation

Currently research has focused on insulators from non-plant species such as the fruit fly, Drosophila melanogaster. The accumulated data suggests that many different insulator sequences exist in D. melanogaster, each one containing its own different primary binding protein, while sharing similar secondary binding proteins. Together, they produce chromatin loops separating enhancers and promoters into distinct domains preventing cross-talk between them. Is this the case in plants? To approach this question, we have investigated enhancer-blocking insulators in the model plant Arabidopsis thaliana using two unrelated approaches. Firstly, we have developed an assay for the direct selection of insulators in Arabidopsis thaliana using a random oligonucleotide library. This assay helped us to define four novel insulator sequences named InI-3, InII-12, InIII-50, and InIII-78. Secondly, we have used genetic analyses to characterize potential insulator sequences originally from three non-plant species: UASrpg from the fungus Ashbya gossypii, BEAD1c from human T-cell receptors, and gypsy from D. melanogaster, that have been reported to function in A. thaliana. Our findings suggest that non-plant insulators and their protein binding sites function in plants and support the model of multiple, functional, different insulator sequences as was found in D. melanogaster. They also argue for the conservation of insulator mechanisms across species.