| 1. |
- Bouderlique, Thibault, et al.
(författare)
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The Concerted Action of E2-2 and HEB Is Critical for Early Lymphoid Specification
- 2019
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The apparition of adaptive immunity in Gnathostomata correlates with the expansion of the E-protein family to encompass E2-2, HEB, and E2A. Within the family, E2-2 and HEB are more closely evolutionarily related but their concerted action in hematopoiesis remains to be explored. Here we show that the combined disruption of E2-2 and HEB results in failure to express the early lymphoid program in Common lymphoid precursors (CLPs) and a near complete block in B-cell development. In the thymus, Early T-cell progenitors (ETPs) were reduced and T-cell development perturbed, resulting in reduced CD4 T- and increased γδ T-cell numbers. In contrast, hematopoietic stem cells (HSCs), erythro-myeloid progenitors, and innate immune cells were unaffected showing that E2-2 and HEB are dispensable for the ancestral hematopoietic lineages. Taken together, this E-protein dependence suggests that the appearance of the full Gnathostomata E-protein repertoire was critical to reinforce the gene regulatory circuits that drove the emergence and expansion of the lineages constituting humoral immunity.
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| 2. |
- Johansson, Cecilia, et al.
(författare)
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HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation.
- 2012
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 31:14, s. 3212-27
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Tidskriftsartikel (refereegranskat)abstract
- We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.
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| 3. |
- Li, Xiaoze, et al.
(författare)
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Eight Nucleotide Substitutions Inhibit Splicing to HPV-16 3'-Splice Site SA3358 and Reduce the Efficiency by which HPV-16 Increases the Life Span of Primary Human Keratinocytes.
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:9
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Tidskriftsartikel (refereegranskat)abstract
- The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16.
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| 4. |
- Li, Xiaoze
(författare)
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Regulation of Human Papillomavirus Type 16 Late L1 mRNA Splicing
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human papillomaviruses (HPVs) cause almost half of the human cancers that are attributable to viruses. HPV type 16 is the most carcinogenic type among the HPVs and is detected in 50% of all cervical cancers. HPV-16 infects epithelial cells and HPV-16 gene expression is tightly linked to the differentiation stage of the infected cells. Early HPV-16 genes are expressed in basal layers of the epithelium whereas the late genes which encode highly immunogenic viral structural proteins are only expressed in the suprabasal layers. HPV-16 infections are normally cleared within 18-24 months, but HPV-16 can establish persistent infections that progress to cancer. Such HPV-infected cancer cells express early HPV-16 genes but never expressed the late genes. We speculate that inhibition of HPV-16 late gene expression is a prerequisite for viral persistence and progression to cervical cancer. HPV-16 uses alternative splicing to regulate expression of early and late genes. HPV RNA elements and cellular factors control the expression level of viral proteins by regulating alternative splicing. This project was carried out to enhance our understanding of the regulation of HPV-16 gene expression, in particular at the RNA splicing level. The goal of this thesis was to identify viral RNA elements and cellular factors that regulate the processing of HPV-16 early and late mRNA splicing. These studies may also contribute to the identification of diagnostic biomarkers for premalignant infections at risk of progressing to cervical cancer. We identified a splicing silencer that interacts with hnRNP D proteins and hnRNP A2/B1 to suppress HPV-16 late gene expression in mitotic cells, including cervical cancer cells. We also characterized a splicing enhancer that promotes HPV-16 early gene expression, thereby indirectly inhibiting late gene expression. Mutation in this enhancer reduced its binding to the ASF/SF2 splicing factor. This resulted in decreased expression of the viral oncogenes E6 and E7 and a reduced ability of HPV-16 to immortalize human epithelial cells, thereby, linking HPV-16 mRNA splicing regulation to its pathogenic prospects. We also identified the hnRNP G protein binding to this enhancer and has opposite effects to ASF/SF2 on splicing matched by antagonism in RNA binding.
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| 5. |
- Li, Xiaoze, et al.
(författare)
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Suppression of HPV-16 late L1 5'-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs.
- 2013
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 41:22, s. 10488-508
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Tidskriftsartikel (refereegranskat)abstract
- Human papillomavirus type 16 (HPV-16) 5'-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5'-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production.
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| 6. |
- Mota, Ana, et al.
(författare)
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FRET-FISH probes chromatin compaction at individual genomic loci in single cells
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells.
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| 7. |
- Ólafsson, Einar B., et al.
(författare)
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Convergent Met and voltage-gated Ca2+ channel signaling drives hypermigration of Toxoplasma-infected dendritic cells
- 2021
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 134:5
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Tidskriftsartikel (refereegranskat)abstract
- Ras–Erk MAPK signaling controls many of the principal pathways involved in metazoan cell motility, drives metastasis of multiple cancer types and is targeted in chemotherapy. However, its putative roles in immune cell functions or in infections have remained elusive. Here, using primary dendritic cells (DCs) in an infection model with the protozoan Toxoplasma gondii, we show that two pathways activated by infection converge on Ras–Erk MAPK signaling to promote migration of parasitized DCs. We report that signaling through the receptor tyrosine kinase Met (also known as HGF receptor) contributes to T. gondii-induced DC hypermotility. Furthermore, voltage-gated Ca2+ channel (VGCC, subtype CaV1.3) signaling impacted the migratory activation of DCs via calmodulin–calmodulin kinase II. We show that convergent VGCC signaling and Met signaling activate the GTPase Ras to drive Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively) phosphorylation and hypermotility of T. gondii-infected DCs. The data provide a molecular basis for the hypermigratory mesenchymal-to-amoeboid transition (MAT) of parasitized DCs. This emerging concept suggests that parasitized DCs acquire metastasis-like migratory properties that promote infection-related dissemination.
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| 8. |
- Pena-Perez, Lucia, et al.
(författare)
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FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs.
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| 9. |
- Somberg, Monika, 1979-, et al.
(författare)
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Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism
- 2011
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Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 92:10, s. 2411-2421
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Tidskriftsartikel (refereegranskat)abstract
- Two splice sites on the human papillomavirus type 16 (HPV-16) genome are used exclusively by the late capsid protein L1 mRNAs: SD3632 and SA5639. These splice sites are suppressed in mitotic cells. This study showed that serine/arginine-rich protein 30c (SRp30c), also named SFRS9, activated both SD3632 and SA5639 and induced production of L1 mRNA. Activation of HPV-16 L1 mRNA splicing by SRp30c required an intact arginine/serine-repeat (RS) domain of SRp30c. In addition to this effect, SRp30c could enhance L1 mRNA production indirectly by inhibiting the early 3′-splice site SA3358, which competed with the late 3′-splice site SA5639. SRp30c bound directly to sequences downstream of SA3358, suggesting that SRp30c inhibited the enhancer at SA3358 and caused a redirection of splicing to the late 3′-splice site SA5639. This inhibitory effect of SRp30c was independent of its RS domain. These results suggest that SRp30c can activate HPV-16 L1 mRNA expression via a bimodal mechanism: directly by stimulating splicing to late splice sites and indirectly by inhibiting competing early splice sites.
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| 10. |
- Yu, Haoran, et al.
(författare)
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HnRNP G prevents inclusion on the HPV16 L1 mRNAs of the central exon between splice sites SA3358 and SD3632
- 2018
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Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 99:3, s. 328-343
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Tidskriftsartikel (refereegranskat)abstract
- HPV16 late L1 mRNAs encode a short central exon that is located between HPV16 3′-splice site SA3358 and HPV16 5′-splice site SD3632. While SA3358 is used to produce both HPV16 early mRNAs encoding the E6 and E7 oncogenes, and late mRNAs encoding E4, L1 and L2, SD3632 is used exclusively to produce late L1 mRNA. We have previously identified an 8-nucleotide regulatory RNA element that is required for inclusion of the exon between SA3358 and SD3632 to produce L1 mRNAs at the expense of mRNAs polyadenylated at the HPV16 early polyadenylation signal pAE. Here we show that this HPV16 8-nucleotide splicing enhancer interacts with hnRNP G. Binding of hnRNP G to this element prevents inclusion of the exon between SA3358 and SD3632 on the HPV16 late L1 mRNAs. We concluded that hnRNP G has a splicing inhibitory role and that hnRNP G can control HPV16 mRNA splicing.
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