| 1. |
- 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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| 2. |
- Reinius, Björn, et al.
(författare)
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Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse
- 2010
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 11:1, s. 614-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Sexual dimorphism in brain gene expression has been recognized in several animal species.However, the relevant regulatory mechanisms remain poorly understood. To investigatewhether sex-biased gene expression in mammalian brain is globally regulated or locallyregulated in diverse brain structures, and to study the genomic organisation of brain-expressedsex-biased genes, we performed a large scale gene expression analysis of distinct brainregions in adult male and female mice. Results: This study revealed spatial specificity in sex-biased transcription in the mouse brain, andidentified 173 sex-biased genes in the striatum; 19 in the neocortex; 12 in the hippocampusand 31 in the eye. Genes located on sex chromosomes were consistently over-represented inall brain regions. Analysis on a subset of genes with sex-bias in more than one tissue revealedY-encoded male-biased transcripts and X-encoded female-biased transcripts known to escapeX-inactivation. In addition, we identified novel coding and non-coding X-linked genes withfemale-biased expression in multiple tissues. Interestingly, the chromosomal positions of allof the female-biased non-coding genes are in close proximity to protein-coding genes thatescape X-inactivation. This defines X-chromosome domains each of which contains a codingand a non-coding female-biased gene. Lack of repressive chromatin marks in non-codingtranscribed loci supports the possibility that they escape X-inactivation. Moreover, RNADNAcombined FISH experiments confirmed the biallelic expression of one such noveldomain. Conclusion: This study demonstrated that the amount of genes with sex-biased expression variesbetween individual brain regions in mouse. The sex-biased genes identified are localized onmany chromosomes. At the same time, sexually dimorphic gene expression that is common toseveral parts of the brain is mostly restricted to the sex chromosomes. Moreover, the studyuncovered multiple female-biased non-coding genes that are non-randomly co-localized onthe X-chromosome with protein-coding genes that escape X-inactivation. This raises thepossibility that expression of long non-coding RNAs may play a role in modulating geneexpression in domains that escape X-inactivation in mouse.
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| 3. |
- Sandhu, Kuljeet Singh, et al.
(författare)
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Nonallelic transvection of multiple imprinted loci is organized by the H19 imprinting control region during germline development
- 2009
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 23:22, s. 2598-2603
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Tidskriftsartikel (refereegranskat)abstract
- Recent observations highlight that the mammalian genome extensively communicates with itself via long-range chromatin interactions. The causal link between such chromatin cross-talk and epigenetic states is, however, poorly understood. We identify here a network of physically juxtaposed regions from the entire genome with the common denominator of being genomically imprinted. Moreover, CTCF-binding sites within the H19 imprinting control region (ICR) not only determine the physical proximity among imprinted domains, but also transvect allele-specific epigenetic states, identified by replication timing patterns, to interacting, nonallelic imprinted regions during germline development. We conclude that one locus can directly or indirectly pleiotropically influence epigenetic states of multiple regions on other chromosomes with which it interacts.
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| 4. |
- Shi, Chengxi
(författare)
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Epigenetic regulation of higher order chromatin conformations and networks
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Higher order chromatin conformations result from the packaging of the genome into the physical confines of the cell nucleus. Structural hallmarks of the nucleus influence the spatio-temporal behavior of genome underlying the regulation of genomic functions. Moreover, accumulated data show that the physical proximities between interphase chromatin fibers significantly contribute to the regulation of genomic transcription, replication and repair. The dynamic patterns of spatial crosstalk between genomic regions are, moreover, controlled by environmental cues to fine-tune gene transcription. The studies in this thesis focus on the nature of higher order chromatin conformations and networks and their developmental regulation in mouse and human model systems. The thesis also includes the description of a novel technique that enables the visualization of higher order chromatin proximities in single cells at a resolution far beyond that of the microscope. Specifically, we identified developmentally regulated genome-wide chromosomal interactomes impinging on the H19 imprinting control region (ICR) in embryonic stem (ES) cells and derived embryoid bodies (EBs). The chromosomal interactomes appear poorly conserved between mouse and human. We further constructed chromosomal interaction networks with crosswise interacting pattern and present the modular topology of the human networks. The molecular glue connecting chromosomes to each other was identified as poly(ADP-ribose). TGFβ signaling was shown to rapidly rewire the chromosomal interaction networks by targeting a CTCF-PARP1 feed-back loop to decrease poly(ADP-ribose) levels in the nucleus. We further captured a developmentally conserved imprinted interaction network, which is dependent on CTCF binding sites on the maternal H19 ICR allele. This network was shown to function as a vehicle to transfer epigenetic states from H19/Igf2 domain to other imprinted domains it interacts with. We propose the principle of non-allelic transvection of epigenetic states as a notable functional outcome of the physical contacts between chromatin fibers. Finally, we invented Chromatin In Situ Proximity (ChrISP), which is a novel technique to identify and visualize proximities between chromatin fibers or between chromatin fiber as well as structural hallmarks in single cells at a high resolution. By employing the ChrISP technique we demonstrated that modification of epigenetic marks by environmental cues triggers large-scale changes in chromosome conformations. It is concluded that higher order chromatin conformations and networks are epigenetically regulated by environmental cues and significantly contribute to the regulation of genomic functions during developmental and pathological processes.
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