Background & Aims Proprotein convertase subtilisin/kexin type 9 (PCSK9) controls blood cholesterol levels by fostering the LDL receptor (LDLR) degradation in hepatocytes. Additionally, PCSK9 has been suggested to participate in immunoregulation by modulating cytokine production. We studied the immunological role of PCSK9 in Streptococcus pneumoniae bacteraemia in vivo and in a human hepatocyte cell line. Methods CRISPR/Cas9 mutagenesis was utilized to create pcsk9 knock‐out (KO) zebrafish, which were infected with S pneumoniae to assess the role of PCSK9 for the survival of the fish and in the transcriptomic response of the liver. The direct effects of PCSK9 on the expression of acute‐phase reaction (APR) genes were studied in HepG2 cells. Results The pcsk9 KO zebrafish lines (pcsk9tpu‐13 and pcsk9tpu‐2,+15) did not show developmental defects or gross phenotypical differences. In the S pneumoniae infected zebrafish, the mortality of pcsk9 KOs was similar to the controls. A liver‐specific gene expression analysis revealed that a pneumococcal challenge upregulated pcsk9, and that the pcsk9 deletion reduced the expression of APR genes, including hepcidin antimicrobial peptide (hamp) and complement component 7b (c7b). Accordingly, silencing PCSK9 in vitro in HepG2 cells using small interfering RNAs (siRNAs) decreased HAMP expression. Conclusions We demonstrate that PCSK9 is not critical for zebrafish survival in a systemic pneumococcal infection. However, PCSK9 deficiency was associated with the lower expression of APR genes in zebrafish and altered the expression of innate immunity genes in a human hepatocyte cell line. Overall, our data suggest an evolutionarily conserved function for PCSK9 in APR in the liver.
The proprotein convertase subtilisin/kexins (PCSKs) regulate biological actions by cleaving immature substrate proteins. The archetype PCSK, FURIN, promotes the pathogenicity of viruses by proteolytically processing viral proteins. FURIN has also important regulatory functions in both innate and adaptive immune responses but its role in the CD8 + CTLs remains enigmatic. We used a T-cell-specific FURIN deletion in vivo to demonstrate that FURIN promotes host response against the CTL-dependent lymphocytic choriomeningitis virus by virtue of restricting viral burden and augmenting interferon gamma (IFNG) production. We also characterized Furin KO CD8 + T cells ex vivo, including after their activation with FURIN regulating cytokines IL12 or TGFB1. Furin KO CD8 + T cells show an inherently activated phenotype characterized by the upregulation of effector genes and increased frequencies of CD44 + , TNF + , and IFNG + cells. In the activated CTLs, FURIN regulates the productions of IL2, TNF, and GZMB and the genes associated with the TGFBRsignaling pathway. FURIN also controls the expression of Eomes, Foxo1, and Bcl6 and the levels of ITGAE and CD62L, which implies a role in the development of CTL memory. Collectively, our data suggest that the T-cell expressed FURIN is important for host responses in viral infections, CTL homeostasis/activation, and memory development.
DNA methylation has proven to be powerful for brain tumor characterization and diagnostic classification. To obtain information about the oncogenic role of DNA methylation, we analyzed medulloblastoma, choroid plexus, and atypical teratoid/rhabdoid tumors (AT/RTs) with public data from 450K-methylation arrays (N=584) and gene-expression arrays (N=110). In addition, two AT/RTs, five choroid plexus tumors and three medulloblastomas were analyzed by using reduced representation bisulfite sequencing, exome sequencing, and RNA-sequencing of matched samples. Only few somatic alterations in addition to SMARCB1 deletion were present in our AT/RTs. DNA methylation analysis generated 2325-5739 and 17175-25187 differentially methylated regions (DMRs) between tumor types in 450K array and RRBS sequencing data, respectively. AT/RTs harbored generally higher DNA methylation levels than the other tumor types. Next, DNA methylation differences were integrated with gene expression data. Surprisingly, only eight genes showed cancer-specific association between differential DNA methylation and an opposite expression change at promoter or linked enhancer in both public and in-house data. There were 44 cancer-specific genes with expression-methylation association when DNA methylation analysis was extended to genomic neighborhoods. To gain information about changes in epigenetic regulation between tumor types, we studied which previously experimentally validated transcription factor (TF) binding sites are enriched in cancer specific DMRs. Several TFs known to promote neural development, such as NEUROG2 and NEUROD1, were enriched in regions hypermethylated in AT/RT, whereas TFs, such as SMAD2, involved in the inhibition of neural development were associated with regions hypermethylated in medulloblastoma. This suggests that DNA methylation is regulating especially the target sites for neural regulators in AT/RT tumors, thus inhibiting neural development. Expression differences did not explain the predicted decreased activity of most of these neural TFs. Low number of genes with cancer-specific expression and methylation change is at least partly explained by the different gene expression patterns in medulloblastomas and choroid plexus tumors, thus providing different references for comparison. Also differences in the measurement techniques contribute to this. Taken together, these results suggest that DNA methylation has a role as an epigenetic regulator for the oncogenesis of AT/RTs. Citation Format: Kirsi Johanna Granberg, Joonas Tuominen, Kristiina Nordfors, Meeri Pekkarinen, Ville Kytölä, Sergei Häyrynen, Ebrahim Afyounian, Olli Lohi, Pauli Helen, Juha Kesseli, Joonas Haapasalo, Hannu Haapasalo, Matti Nykter. DNA methylation analysis reveals epigenetic regulation of neural differentiation in AT/RTs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-173.
Understanding oncogenic epigenetic mechanisms in brain tumors is crucial for improved diagnosis and treatment. Recently DNA methylation has proven to be powerful for brain tumor characterization and diagnostic classification. To evaluate tumor type specific features, we compared atypical teratoid/rhabdoid tumors (AT/RTs), medulloblastomas (MBs), and choroid plexus tumors with each other by integrating DNA methylation (507 samples), gene expression (120 samples), and transcription factor (TF) -binding data. Different tumor entities were used to find unique changes affecting each of the entities and further to identify functions driven by these changes. Our results provide insight on how the aberrant DNA methylation induces oncogenesis of AT/RTs. These tumors are known for their aggressiveness and exceptionally low mutation rates. Our results suggest that in AT/RT, elevated DNA methylation masks the binding sites of TFs such as NEUROD1, ASCL1 and MYCN driving neural development. DNA methylation in AT/RTs is also associated with reduced gene expression for specific neural regulators such as NEUROG1 and NEUROD2. For MBs, DNA methylation patterns predict a more advanced differentiation state. In MB, we found masked TF binding sites for TFs such as REST and ZEB1 that normally inhibit neural differentiation. We then wanted to further characterize DNA methylation and compared these tumors to pluripotent stem cells (PSCs) and normal fetal brain samples. As a result, we were able to find two different regulatory programs in AT/RTs: One in which DNA methylation is similar to PSCs and which harbors mostly neural TF binding sites. Second program has AT/RT-specific DNA methylation, and these sites are uniquely associated with polycomb repressive complex 2 members. However, this second program also covers neural TF binding sites and is likely to have relevance in oncogenic regulation.
Understanding oncogenic epigenetic mechanisms in brain tumors is crucial for improved diagnosis and treatment. To evaluate tumor type-specific features, we compared atypical teratoid/rhabdoid tumors (AT/RTs), medulloblastomas, and choroid plexus tumors with each other by integrating DNA methylation (507 samples), gene expression (120 samples), and transcription factor (TF) -binding data. Our results suggest that aberrant DNA methylation plays a vital role in the oncogenesis of AT/RTs, which are known for their aggressiveness and exceptionally low mutation rates. In AT/RT, elevated DNA methylation masks the binding sites of TFs driving neural development and is associated with reduced gene expression for specific neural regulators, whereas DNA methylation patterns predict a more advanced differentiation state for MB. By analyzing the data together with pluripotent stem cells, we found two regulatory programs in AT/RT: pluripotent stem cell -like DNA methylation patterns and AT/RT-specific DNA methylation uniquely associated with polycomb repressive complex 2 (PRC2) members. Both methylation patterns cover neural TF binding sites in a TF-specific manner, suggesting their relevance for oncogenic regulation.
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