| 1. |
- Birney, Ewan, et al.
(författare)
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Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 447:7146, s. 799-816
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Tidskriftsartikel (refereegranskat)abstract
- We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.
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| 2. |
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| 3. |
- Bogaert, Annelies, et al.
(författare)
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N-terminal proteoforms may engage in different protein complexes
- 2023
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Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:8
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Tidskriftsartikel (refereegranskat)abstract
- Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared with their canonical counterparts. Such proteoforms can have altered localizations, stabilities, and functions. Although proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. In addition, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues, as well as tissue-specific expression, highlighting their biological relevance. Protein–protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared with their canonical counterparts, thus further expanding the functional diversity of proteomes.
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| 4. |
- Bäckström, Anna, et al.
(författare)
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A Sample Preparation Protocol for High Throughput Immunofluorescence of Suspension Cells on an Adherent Surface
- 2020
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 68:7, s. 473-489
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Tidskriftsartikel (refereegranskat)abstract
- Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial informationin situat single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.
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| 5. |
- Danielsson, Frida, et al.
(författare)
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RNA Deep Sequencing as a Tool for Selection of Cell Lines for Systematic Subcellular Localization of All Human Proteins
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:1, s. 231-239
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Tidskriftsartikel (refereegranskat)abstract
- One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner.
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| 6. |
- Hjelm, Barbara, et al.
(författare)
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Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
- 2011
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 20:11, s. 1824-1835
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Tidskriftsartikel (refereegranskat)abstract
- A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.
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| 7. |
- Huyghe, Jeroen R., et al.
(författare)
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Discovery of common and rare genetic risk variants for colorectal cancer
- 2019
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Ingår i: Nature Genetics. - : Nature Publishing Group. - 1061-4036 .- 1546-1718. ; 51:1, s. 76-
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Tidskriftsartikel (refereegranskat)abstract
- To further dissect the genetic architecture of colorectal cancer (CRC), we performed whole-genome sequencing of 1,439 cases and 720 controls, imputed discovered sequence variants and Haplotype Reference Consortium panel variants into genome-wide association study data, and tested for association in 34,869 cases and 29,051 controls. Findings were followed up in an additional 23,262 cases and 38,296 controls. We discovered a strongly protective 0.3% frequency variant signal at CHD1. In a combined meta-analysis of 125,478 individuals, we identified 40 new independent signals at P < 5 x 10(-8), bringing the number of known independent signals for CRC to similar to 100. New signals implicate lower-frequency variants, Kruppel-like factors, Hedgehog signaling, Hippo-YAP signaling, long noncoding RNAs and somatic drivers, and support a role for immune function. Heritability analyses suggest that CRC risk is highly polygenic, and larger, more comprehensive studies enabling rare variant analysis will improve understanding of biology underlying this risk and influence personalized screening strategies and drug development.
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| 8. |
- Joshi, Rubin Narayan, et al.
(författare)
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TcellSubC : An Atlas of the Subcellular Proteome of Human T Cells
- 2019
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Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- We have curated an in-depth subcellular proteomic map of primary human CD4+ T cells, divided into cytosolic, nuclear and membrane fractions generated by an optimized fractionation and HiRIEF-LC-MS/MS workflow for limited amounts of primary cells. The subcellular proteome of T cells was mapped under steady state conditions, as well as upon 15 min and 1 h of T cell receptor (TCR) stimulation, respectively. We quantified the subcellular distribution of 6,572 proteins and identified a subset of 237 potentially translocating proteins, including both well-known examples and novel ones. Microscopic validation confirmed the localization of selected proteins with previously known and unknown localization, respectively. We further provide the data in an easy-to-use web platform to facilitate re-use, as the data can be relevant for basic research as well as for clinical exploitation of T cells as therapeutic targets.
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| 9. |
- Kalderén, Christina, et al.
(författare)
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CCL2 mediates anti-fibrotic effects in human fibroblasts independently of CCR2
- 2014
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Ingår i: International Immunopharmacology. - : Elsevier BV. - 1567-5769 .- 1878-1705. ; 20:1, s. 66-73
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Tidskriftsartikel (refereegranskat)abstract
- CCL2 is known for its major role as a chemoattractant of monocytes for immunological surveillance and to site of inflammation. CCL2 acts mainly through the G-protein-coupled receptor CCR2 but has also been described to mediate its effects independently of this receptor in vitro and in vivo. Emerging pieces of evidence indicate that the CCL2/CCR2 axis is involved in fibrotic diseases, such as increased plasma levels of CCL2 and the presence of CCL2-hyperresponsive fibroblasts explanted from patients with systemic sclerosis and idiopathic pulmonary fibrosis. One of the profibrotic key mediators is the myofibroblast characterized by overexpression of alpha-smooth muscle actin and collagen I. However, the correlation between the CCL2/CCR2 axis and the activation of fibroblasts is not yet fully understood. We have screened human fibroblasts of various origins, human pulmonary fibroblasts (HPF), human fetal lung fibroblasts (HFL-1) and primary preadipocytes (SPF-1) in regard to CCL2 stimulated fibrotic responses. Surprisingly we found that CCL2 mediates anti-fibrotic effects independently of CCR2 in human fibroblasts of different origins.
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| 10. |
- Kitazawa, Taro, et al.
(författare)
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A unique bipartite Polycomb signature regulates stimulus-response transcription during development.
- 2021
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 53:3, s. 379-391
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Tidskriftsartikel (refereegranskat)abstract
- Rapid cellular responses to environmental stimuli are fundamental for development and maturation. Immediate early genes can be transcriptionally induced within minutes in response to a variety of signals. How their induction levels are regulated and their untimely activation by spurious signals prevented during development is poorly understood. We found that in developing sensory neurons, before perinatal sensory-activity-dependent induction, immediate early genes are embedded into a unique bipartite Polycomb chromatin signature, carrying active H3K27ac on promoters but repressive Ezh2-dependent H3K27me3 on gene bodies. This bipartite signature is widely present in developing cell types, including embryonic stem cells. Polycomb marking of gene bodies inhibits mRNA elongation, dampening productive transcription, while still allowing for fast stimulus-dependent mark removal and bipartite gene induction. We reveal a developmental epigenetic mechanism regulating the rapidity and amplitude of the transcriptional response to relevant stimuli, while preventing inappropriate activation of stimulus-response genes.
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