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- Enroth, Stefan, et al.
(author)
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Nucleosome regulatory dynamics in response to TGF beta
- 2014
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:11, s. 6921-6934
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Journal article (peer-reviewed)abstract
- Nucleosomes play important roles in a cell beyond their basal functionality in chromatin compaction. Their placement affects all steps in transcriptional regulation, from transcription factor (TF) binding to messenger ribonucleic acid (mRNA) synthesis. Careful profiling of their locations and dynamics in response to stimuli is important to further our understanding of transcriptional regulation by the state of chromatin. We measured nucleosome occupancy in human hepatic cells before and after treatment with transforming growth factor beta 1 (TGFβ1), using massively parallel sequencing. With a newly developed method, SuMMIt, for precise positioning of nucleosomes we inferred dynamics of the nucleosomal landscape. Distinct nucleosome positioning has previously been described at transcription start site and flanking TF binding sites. We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci. We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci. Depending on genomic annotation, 44-78% of them were over-represented in binding motifs for TFs. Changes in binding affinity were verified for HNF4α by qPCR. Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing.
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| 4. |
- Termén, Stefan
(author)
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Expression of cathelicidin antimicrobial peptides in man and rat
- 2004
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Doctoral thesis (other academic/artistic)abstract
- Endogenous antimicrobial peptides are effectors of innate immunity. They kill microorganisms by disrupting their cell membranes, but are also believed to have signaling functions in the immune system. They are present in epithelia, secretions and phagocytic cells. Major families of mammalian antimicrobial peptides are the defensins and the cathelicidins. This thesis describes different aspects of gene regulation of the human cathelicidin antimicrobial peptide hCAP18/LL-37 and characterization of rCRAMP, the homolog to LL-37 in the rat. Rat rCRAMP peptide was isolated from neutrophils. In addition, the peptide and the corresponding messenger RNA (mRNA) was found in tissues of the gastrointestinal tract, lung and thymus. Expression pattern, antimicrobial activity, and predicted alpha-helical structure were similar to mouse CRAMP and human LL-37. Contact with live bacteria was not required for rCRAMP expression since germ-free rats also expressed it. Phylogenetic analysis of cathelicidin preproregions confirmed homology of rCRAMP to other cathelicidins and illustrated that gene duplication in this family has been species- or lineage-specific. Characterization of rCRAMP enables use of the rat for studying responses related to cathelicidin expression. rCRAMP was also isolated from the central nervous system (CNS) of the rat, and determined to constitute a major part of the peptide/protein antimicrobial activity in rat CNS. The peptide was active against the neuropathogen Neisseria meningitidis. Regions of the CNS positive for rCRAMP both on the peptide/protein and mRNA levels were olfactory bulb, cerebellum, medulla oblongata and spinal cord. We hypothesize that rCRAMP is part of an ancient immune defense in rat CNS, but it may have additional functions. This is the first detailed report of cathelicidin expression in brain and spinal cord, and provides a basis from which to conduct functional studies of cathelicidins in the CNS. Gene regulation of LL-37 was studied with focus on the colonic epithelium. The colon harbors large quantities of bacteria and LL-37 is expressed in colonic epithelial cells as part of a defense barrier. Butyrate is produced by bacterial fermentation in the colon and this short chain fatty acid was found to induce LL-37 transcription via MAPK/ERK kinase. Further analysis of potential regulatory elements in the CAMP gene encoding LL-37 was conducted using a luciferase reporter system. This indicated presence of different silencer elements and an enhancer element in the promoter. Electrophoretic mobility shift assay indicated that a transcription factor of the Ets family likely binds to the enhancer element. An intron appeared to be involved in the inducing effect of butyrate, and an possible alternative upstream translation start codon was also identified. Increased knowledge regarding LL-37 transcription in the colon may lead to improved treatment of diseases caused by enteric pathogens.
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| 5. |
- Termén, Stefan, et al.
(author)
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p53 regulates epithelial-mesenchymal transition induced by transforming growth factor β
- 2013
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In: Journal of Cellular Physiology. - : Wiley. - 0021-9541 .- 1097-4652. ; 228:4, s. 801-813
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Journal article (peer-reviewed)abstract
- Epithelial plasticity characterizes embryonic development and diseases such as cancer. Epithelial-mesenchymal transition (EMT) is a reversible and guided process of plasticity whereby embryonic or adult epithelia acquire mesenchymal properties. Multiple signaling pathways control EMT, and the transforming growth factor β (TGFβ) pathway plays a central role as its inducer. Here, we analyzed the role of the tumor suppressor protein p53 in TGFβ-induced EMT in a well-established mammary epithelial cell model. We found that diploid NMuMG mammary cells bi-allelically express a wild type and a missense mutant (R277C) form of p53. Global reduction of both forms of p53 led to an enhanced EMT response to TGFβ. Conversely, stabilization of wild type p53 using the compound nutlin had a negative impact on EMT. After silencing both p53 forms, rescue experiments using either wild type or R277C mutant p53 revealed that wild type p53 inhibited, whereas the R277C mutant did not significantly affect, the TGFβ-driven EMT response. Under serum-free culture conditions, silencing of total p53 levels led to higher numbers of mammospheres characterized by larger size. Rescue of the silenced endogenous p53 with R277C mutant p53, in contrast, suppressed both size and numbers of the mammospheres. This work proposes that wild type p53 controls the efficiency by which mammary epithelial cells undergo EMT in response to TGFβ.
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