| 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. |
- Fernandez Woodbridge, Alejandro
(författare)
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Bioinformatic analyses of the structural and functional complexity in chromosomal interactomes
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Evolution requires information storage systems with different demands with respect to persistence. While the genome provides a mechanism for long term, static and accurate information storage, it is incapable of mediating adaptation to short term changes in the environment. Chromatin, however, constitutes a dynamic, reprogrammable memory with different levels of persistence. Moreover, chromatin states carry information not only in 2D, i.e. in the structure of the primary chromatin fibre, but also in the 3D organization of the genome in the nuclear space. The following thesis delves into the new bioinformatic and wet lab protocols developed to map, quantitative and functionally analyze the 3D architecture of chromatin. The chromatin insulator protein CTCF is a major factor underlying the 3D organization of the epigenome. We have uncovered, however, that CTCF binding sites within a regulatory region have multiple functions that are influenced by the chromatin environment and possibly the combinatorial usage of the 11 Zn-fingers of CTCF (Paper I). This observation exemplifies that understanding the function of dynamic and transient chromatin fibre interactions requires novel technology that enables the detection of 3D chromatin folding with high resolution in single cells and in small cell populations. We therefore set out to devise a novel method for the visualization of higher order chromatin structures by combining the strengths of both DNA Fluorescent In Situ Hybridization (FISH) and In Situ Proximity Ligation Assay (ISPLA) technologies (Paper II). The resulting Chromatin in Situ Proximity (ChrISP) assay thus takes advantage of the direct contact detection of ISPLA and the locus-specific nature of FISH and uncovered the existence of compact chromatin structures at the nuclear envelope with unprecedented resolution. To complement ChrISP with a high throughput method capable of quantitatively recovering chromatin fibre contacts in small cell populations, we furthermore innovated the Nodewalk assay (Paper III). The protocol builds on existing ligation based chromosome conformation capture methods, but features significant reduction in the random ligation event frequency, inclusion of negative and positive ligation controls, iterative template resampling, increased signal to noise ratio and improved sensitivity. Using this technique, we have uncovered a cancer cell-specific, productive chromatin fibre interactome connecting the promoter and enhancer of c-MYC to a network of enhancers and super-enhancers. Underpinning this new protocol, I have developed the Nodewalk Analysis Pipeline (NAP) (Paper IV). This suite of tools consists of preprocessing, analysis and post-processing modules designed specifically for the rapid and efficient analysis of Nodewalk datasets through an interactive and user-friendly web based interface. Overall the work described in this thesis advances our understanding of the role of CTCF in nuclear organization and provides innovative wet lab techniques along with specialized software tools. Moreover, this work is an example of an emerging trend where the challenge of understanding chromatin dynamics within the 3D nuclear architecture demands a close synergistic collaboration between the fields of biology, biotechnology and bioinformatics.
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| 3. |
- 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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| 4. |
- Johansson, Henrik J., et al.
(författare)
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Breast cancer quantitative proteome and proteogenomic landscape
- 2019
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.
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| 5. |
- Orre, Lukas Minus, et al.
(författare)
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SubCellBarCode : Proteome-wide Mapping of Protein Localization and Relocalization
- 2019
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 73:1, s. 166-182.e7
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Tidskriftsartikel (refereegranskat)abstract
- Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.
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| 6. |
- Zhu, Yafeng, et al.
(författare)
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Discovery of coding regions in the human genome by integrated proteogenomics analysis workflow
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Proteogenomics enable the discovery of novel peptides (from unannotated genomic protein-coding loci) and single amino acid variant peptides (derived from single-nucleotide polymorphisms and mutations). Increasing the reliability of these identifications is crucial to ensure their usefulness for genome annotation and potential application as neoantigens in cancer immunotherapy. We here present integrated proteogenomics analysis workflow (IPAW), which combines peptide discovery, curation, and validation. IPAW includes the SpectrumAI tool for automated inspection of MS/MS spectra, eliminating false identifications of single-residue substitution peptides. We employ IPAW to analyze two proteomics data sets acquired from A431 cells and five normal human tissues using extended (pH range, 3-10) high-resolution isoelectric focusing (HiRIEF) pre-fractionation and TMT-based peptide quantitation. The IPAW results provide evidence for the translation of pseudogenes, lncRNAs, short ORFs, alternative ORFs, N-terminal extensions, and intronic sequences. Moreover, our quantitative analysis indicates that protein production from certain pseudogenes and lncRNAs is tissue specific.
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