| 1. |
- Bugge, Anne, et al.
(författare)
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A Novel Intronic Peroxisome Proliferator-activated Receptor gamma Enhancer in the Uncoupling Protein (UCP) 3 Gene as a Regulator of Both UCP2 and-3 Expression in Adipocytes
- 2010
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:23, s. 17310-17317
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Tidskriftsartikel (refereegranskat)abstract
- Uncoupling Proteins (UCPs) are integral ion channels residing in the inner mitochondrial membrane. UCP2 is ubiquitously expressed, while UCP3 is found primarily in muscles and adipose tissue. Although the exact molecular mechanism of action is controversial, it is generally agreed that both homologues function to facilitate mitochondrial fatty acid oxidation. UCP2 and -3 expression is activated by the peroxisome proliferator-activated receptors (PPARs), but so far no PPAR response element has been reported in the vicinity of the Ucp2 and Ucp3 genes. Using genome-wide profiling of PPAR gamma occupancy in 3T3-L1 adipocytes we demonstrate that PPAR gamma associates with three chromosomal regions in the vicinity of the Ucp3 locus and weakly with a site in intron 1 of the Ucp2 gene. These sites are isolated from the nearest neighboring sites by >900 kb. The most prominent PPAR gamma binding site in the Ucp2 and Ucp3 loci is located in intron 1 of the Ucp3 gene and is the only site that facilitates PPAR gamma transactivation of a heterologous promoter. This site furthermore transactivates the endogenous Ucp3 promoter, and using chromatin conformation capture we show that it loops out to specifically interact with the Ucp2 promoter and intron 1. Our data indicate that PPAR gamma transactivation of both UCP2 and -3 is mediated through this novel enhancer in Ucp3 intron 1.
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| 2. |
- Chen, Xingqi, et al.
(författare)
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Chromatin in situ proximity (ChrISP) : Single-cell analysis of chromatin proximities at a high resolution
- 2014
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 56:3, s. 117-124
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Tidskriftsartikel (refereegranskat)abstract
- Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of similar to 170 angstrom in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA.with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.
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| 3. |
- Guibert, Sylvain, et al.
(författare)
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CTCF-binding sites within the H19 ICR differentially regulate local chromatin structures and cis-acting functions
- 2012
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Ingår i: Epigenetics. - : Informa UK Limited. - 1559-2294 .- 1559-2308. ; 7:4, s. 361-369
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Tidskriftsartikel (refereegranskat)abstract
- It is generally assumed that CTCF-binding sites are synonymous with the demarcation of expression domains by promoting the formation of chromatin loops. We have proposed earlier, however, that such features may be context-dependent. In support of this notion, we show here that chromatin loop structures, impinging on CTCF-binding sites 1/2 and 3/4 at the 5' and 3'-ends, respectively, within the maternal allele of the H19 imprinting control region (ICR), differ significantly. Although abrogation of CTCF binding to the maternal H19 ICR allele results in loss of chromatin loops in the 3'-region, there is a dramatic gain of long-range chromatin loops impinging on the 5'-region. As the degree of occupancy of its four CTCF-binding sites discriminates between the chromatin insulator and replication timing functions, we submit that the CTCF-binding sites within the H19 ICR are functionally diverse and organize context-dependent higher order chromatin conformations.
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| 4. |
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| 5. |
- Göndör, Anita, et al.
(författare)
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Chromatin insulators and cohesins
- 2008
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Ingår i: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 9:4, s. 327-329
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Göndör, Anita, 1977-
(författare)
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Epigenetic Regulation of Higher Order Chromatin Conformations and Gene Transcription
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epigenetic states constitute heritable features of the chromatin to regulate when, where and how genes are expressed in the developing conceptus. A special case of epigenetic regulation, genomic imprinting, is defined as parent of origin-dependent monoallelic expression. The Igf2-H19 locus is considered as paradigm of genomic imprinting with a growth-promoting gene, Igf2, expressed paternally and a growth antagonist, H19 encoding a non-coding transcript, expressed only from the maternal allele. The monoallelic expression patterns are regulated by the epigenetic status at an imprinting control region (ICR) in the 5´-flank of the H19 gene. The chromatin insulator protein CTCF interacts with only the maternal H19 ICR allele to prevent downstream enhancers to communicate with the Igf2 promoters. Mutations of these CTCF binding sites lead to biallelic Igf2 expression, increased size of the conceptus and predisposition for cancer. Reasoning that these effects cannot be explained by the regulation of Igf2 expression alone, a technique was invented to examine long-range chromatin interactions without prior knowledge of the interacting partners. Applying the circular chromosomal conformation capture (4C) technique to mouse neonatal liver cells, it was observed that 114 unique sequences interacted with the H19 ICR. A majority of these interactors was in complex with only the maternal H19 ICR allele and depended on the presence of functional CTCF binding sites. The functional consequence of chromosomal networks was demonstrated by the observation that the maternal H19 ICR allele regulated the transcription of two genes on another chromosome. As the chromosomal networks underwent reprogramming during the maturation of embryonic stem cells, attention was turned to human cancer cells, displaying features common with mouse embryonic stem cells. Subsequently, chromatin folding at the human H19 ICR suggested that stable chromatin loops were organized by synergistic interactions within and between baits and interactors. The presence of these interactions was linked to DNA methylation patterns involving repeat elements. A "flower" model of chromatin networks was formulated to explain these observations. This thesis has unravealed a novel feature of the epigenome and its functions to regulate gene expression in trans. The identified roles for CTCF as an architectural factor in the organization of higher order chromatin conformations may be of importance in understanding development and disease ontogeny from novel perspectives.
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| 7. |
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| 8. |
- Göndör, Anita, et al.
(författare)
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High-resolution circular chromosome conformation capture assay
- 2008
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Ingår i: Nature protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 3:2, s. 303-313
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Tidskriftsartikel (refereegranskat)abstract
- The pioneering chromosome conformation capture (3C) method provides the opportunity to study chromosomal folding in the nucleus. It is based on formaldehyde cross-linking of living cells followed by enzyme digestion, intramolecular ligation and quantitative (Q)-PCR analysis. However, 3C requires prior knowledge of the bait and interacting sequence (termed interactor) rendering it less useful for genome-wide studies. As several recent reports document, this limitation has been overcome by exploiting a circular intermediate in a variant of the 3C method, termed 4C (for circular 3C). The strategic positioning of primers within the bait enables the identification of unknown interacting sequences, which form part of the circular DNA. Here, we describe a protocol for our 4C method, which produces a high-resolution interaction map potentially suitable for the analysis of cis-regulatory elements and for comparison with chromatin marks obtained by chromatin immunoprecipitation (ChIP) on chip at the sites of interaction. Following optimization of enzyme digestions and amplification conditions, the protocol can be completed in 2-3 weeks.
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| 9. |
- Göndör, Anita, et al.
(författare)
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Transcription in the loop.
- 2006
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Ingår i: Nat Genet. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 38:11, s. 1229-30
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Tidskriftsartikel (refereegranskat)
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| 10. |
- Göndör, Anita, et al.
(författare)
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Window into the Complexities of Chromosome Interactomes
- 2010
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Ingår i: Cold Spring Harbor Symposia on Quantitative Biology. - : Cold Spring Harbor Laboratory. - 0091-7451 .- 1943-4456. ; 75, s. 493-500
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Tidskriftsartikel (refereegranskat)abstract
- DNA is folded into increasingly complex yet highly mobile structures to organize the chromosomes. In the interphase nucleus, chromosomes or part of the chromosomes encounter one another preferentially at the boundaries between chromosomal territories. Although this situation implies that the preferred chromosomal neighborhood is a key determinant of interactions between chromosomes, what this means in functional terms is currently not well understood. Using the H19 imprinting control region as a window, it has been demonstrated that epigenetic information of the primary chromatin fiber has dual functions. Thus, epigenetic marks not only influence the proximity between chromatin fibers but also transfer epigenetic states between chromatin fibers both in cis and in trans. High-throughput sequence and DNA fluorescence it situ hybridization (FISH) analyses reveal that these features require chromatin movements that are restricted in space and time. The mechanisms involved in the establishment of chromosome interactomes may provide insight of fundamental importance into pivotal regulatory processes in the nucleus, such as the coordination of transcriptional programs and replication timing.
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