Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

SPAG7 is a candidate gene for the periodic fever, aphthous stomatitis, pharyngitis and adenopathy (PFAPA) syndrome.

Periodic fever, aphthous stomatitis, pharyngitis and adenopathy (PFAPA) syndrome is an auto-inflammatory disease for which a genetic basis has been postulated. Nevertheless, in contrast to the other periodic fever syndromes, no candidate genes have yet been identified. By cloning, following long insert size paired-end sequencing, of a de novo chromosomal translocation t(10;17)(q11.2;p13) in a patient with typical PFAPA syndrome lacking mutations in genes associated with other periodic fever syndromes we identified SPAG7 as a candidate gene for PFAPA. SPAG7 protein is expressed in tissues affected by PFAPA and has been functionally linked to antiviral and inflammatory responses. Haploinsufficiency of SPAG7 due to a microdeletion at the translocation breakpoint leading to loss of exons 2-7 from one allele was associated with PFAPA in the index. Sequence analyses of SPAG7 in additional patients with PFAPA point to genetic heterogeneity or alternative mechanisms of SPAG7 deregulation, such as somatic or epigenetic changes.

Functional synergism of STAT6 with either NF-kappa B or PU.1 to mediate IL-4-induced activation of IgE germline gene transcription.

Ig heavy chain class switching to IgE is directed by IL-4 and IL-13 by inducing transcription from the IgE germline promoter. A crucial transcription factor in this process is STAT6, which binds to a specific DNA element upon cytokine activation. In this paper it is shown that the B cell- and monocyte-specific factor PU.1 interacts with a closely spaced sequence in the human IgE germline promoter that overlaps with a previously described binding site for NF kappa B/rel. The authenticity of PU.1 was demonstrated by specific competition and supershifts in EMSA experiments. In addition, in vitro translated PU.1 could interact with an oligonucleotide derived from the IgE germline promoter containing the PU.1 binding site and migrated with the same mobility compared with the complex formed with nuclear extracts. Transient transfection experiments using IgE germline promoter reporter gene constructs demonstrated that mutations affecting DNA binding of PU.1 or NF kappa B/rel had no or little effect on IL-4 inducibility of these plasmids. However, point mutations that abolished binding of both factors abrogated cytokine inducibility. No strict spacing of the STAT6 and the composite PU. 1/NF-kappa B elements is required for IL-4 induction. IL-4-induced STAT6 DNA binding was retained in PU.1-/NF kappa B/rel- double mutants. The data demonstrate that cooperation of STAT6 with at least PU.1 or NF kappa B/rel is necessary for IL-4-induced activation of IgE germline gene transcription.

Cooperation of binding sites for STAT6 and NF kappa B/rel in the IL-4-induced up-regulation of the human IgE germline promoter.

Ig heavy chain class switching is directed by cytokines inducing transcription from unrearranged CH genes. Subsequently, such primed cells can undergo switch recombination to express the selected new isotype. In the case of IgE class switching, IL-4 activates the IgE germline promoter by inducing the interaction of the transcription factor STAT6 (IL-4STAT) with a responsive DNA element in the proximal region of the promoter. This study describes the characterization of two additional cis-acting elements that interact with members of the NF kappa B/rel transcription factor family in an IL-4-independent fashion. Electrophoretic mobility shift assays show that the nucleoprotein complex formed on the upstream site (NF kappa B1) contains the classical p50/p65 heterodimer. The complex on the proximal site (NF kappa B2) appears to be composed of p50 and relB. IgE germline promoter reporter gene constructs carrying point mutations in the NF kappa B2 site were largely unresponsive to IL-4 stimulation in transient transfection experiments, while plasmids with similar mutations in the NF kappa B1 site responded to cytokine stimulation better than the wild-type promoter. The NF kappa B2 effect was dependent on the presence of the STAT6 binding site, demonstrating that the NF kappa B2 motif is necessary but not sufficient for mediating cytokine up-regulation. In addition, the combination of a NF kappa B/rel binding site and the STAT6 response element conferred IL-4 inducibility to a heterologous minimal promoter, while the individual sites had no effect. The available data suggest that the NF kappa B2 nucleoprotein complex may cooperate with DNA-bound STAT6 to achieve IL-4-dependent activation of the human IgE germline gene.

Inhibition of interleukin-4- and CD40-induced IgE germline gene promoter activity by 2'-aminoethoxy-modified triplex-forming oligonucleotides.

Elevated levels of IgE are intimately associated with a number of allergic diseases, such as allergic rhinitis or asthma. Therefore, prevention of IgE production in human B-cells represents an attractive therapeutic target. IL-4-induced IgE germline gene transcription represents a crucial early step during IgE isotype switch differentiation. Gene induction is orchestrated by the coordinated action of the transcription factors STAT6 (signal transducer and activator of transcription), NF-kappaB, PU.1, and C/EBP. This study shows that 2'-aminoethoxy-modified oligonucleotides, which partially overlap with the STAT6 and the adjacent PU.1/NF-kappaB binding site, inhibit DNA binding of all three proteins with high affinity in a dose- and time-dependent fashion in vitro. Loss of protein binding correlated strongly with increasing DNA triplex formation. Importantly, the oligomers also effectively displaced pre-bound recombinant NF-kappaB p50 from double-stranded DNA in vitro. Functionally, the oligonucleotides led to a selective inhibition of IL-4-induced reporter gene activity from a construct driven by the IgE germline gene promoter in human B-cells. These data confirm the critical role of this cytokine-responsive regulatory region in IgE germline gene induction and further support the concept of specific modulation of gene expression by DNA triplex formation induced with chemically modified oligonucleotides.