We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549-BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto-inhibitory domains of BRAF and RAF1, which are replaced in-frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low-grade gliomas.

BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a “cancer-testis” antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function.

BackgroundThe discovery of novel anti-viral restriction factors illuminates unknown aspects of innate sensing and immunity. We identified RNA-associated Early-stage Anti-viral Factor (REAF) using a whole genome siRNA screen for restriction factors to human immunodeficiency virus (HIV) that act in the early phase of viral replication.ResultsWe observed more than 50 fold rescue of HIV-1 infection, using a focus forming unit (FFU) assay, following knockdown of REAF by specific siRNA. Quantitative PCR was used to show that REAF knockdown results in an increase of early and late reverse transcripts which impacts the level of integration. REAF thus appears to act at an early stage of the viral life cycle during reverse transcription. Conversely when REAF is over-expressed in target cells less infected cells are detectable and fewer reverse transcripts are produced. Human REAF can also inhibit HIV-2 and simian immunodeficiency virus (SIV) infection. REAF associates with viral nucleic acids and may act to prevent reverse transcription.ConclusionsThis report firmly places REAF alongside APOBECs and SAMHD1 as a potent inhibitor of HIV replication acting early in the replication cycle, just after cell entry. We propose that REAF is part of an anti-viral surveillance system destroying incoming retroviruses. This novel mechanism could apply to invasion of cells by any intracellular pathogen.

Activation of the major histocompatibility complex (MHC) by interferon-gamma (IFN−γ) is a fundamental step in the adaptive immune response to pathogens. Here, we show that reorganization of chromatin loop domains in the MHC is evident within the first 30 min of IFN−γ treatment of fibroblasts, and that further dynamic alterations occur up to 6 h. These very rapid changes occur at genomic sites which are occupied by CTCF and are close to IFN−γ-inducible MHC genes. Early responses to IFN−γ are thus initiated independently of CIITA, the master regulator of MHC class II genes and prepare the MHC for subsequent induction of transcription.