Gametic imprinting is a developmental process that induces parental-specific expression or repression of autosomal and X-chromosome-linked genes. The mouse Igf2r gene (encoding the receptor for insulin- like growth factor type-2) is imprinted and is expressed from the maternal allele after embryonic implantation. We previously proposed that methylation of region 2, a region rich in cytosine-guanine doublets (a 'CpG island') in the second intron of Igf2r, is the imprinting signal that maintains expression of the maternal allele. Here we use mouse transgenes to test the role of region 2 and the influence of chromosome location on Igf2r imprinting. Yeast artificial chromosome transgenes successfully reproduced the imprinted methylation and expression pattern of the endogenous Igf2r gene; deletion of region 2 from these transgenes caused a loss of imprinting and restored biallelic Igf2r expression. These results define a primary role for region 2 and a negligible role for chromosomal location in Igf2r imprinting; they also show that methylation imprints can maintain allelic expression. Short transgenes containing only region 2 and yeast artificial chromosome transgenes with an inactive Igf2r promoter do not attract parental-specific methylation. All transgenes showing paternal-specific repression of Igf2r produced an antisense RNA whose transcription was dependent on region 2. The production of an antisense RNA by the repressed parental allele is reminiscent of the imprinting of the Igf2/H19 gene pair and may indicate that expression competition could play a general role in imprinting.

Immunotherapeutic approaches are currently in the spotlight for their potential as disease-modifying treatments for neurodegenerative disorders. The discovery that α-synuclein (α-syn) can transmit from cell to cell in a prion-like fashion suggests that immunization might be a viable option for the treatment of synucleinopathies. This possibility has been bolstered by the development of next-generation active vaccination technology with short peptides-AFFITOPEs® (AFF) that do not elicit a α-syn specific T-cell response. This approach allows the generation of long-term sustained, more specific, non-cross reacting antibodies suitable for the treatment of synucleinopathies such as Parkinson’s disease (PD). In this context, we screened a large library of peptides that mimic the c-terminus region of α-syn and discovered a novel set of AFF that identified α-syn oligomers. Next, the peptide that elicited the most specific response against α-syn (AFF 1) was selected for immunizing two different transgenic mouse models of PD and Dementia with Lewy bodies (DLB), the PDGF- and the mThy1-α-syn tg mice. Vaccination with AFF 1 resulted in high antibody titers in CSF and plasma, which crossed into the CNS and recognized α-syn aggregates. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn oligomers in axons and synapses that was accompanied by reduced degeneration of TH fibers in the caudo-putamen nucleus and by improvements in motor and memory deficits in both in vivo models. Clearance of α-syn involved activation of microglia and increased anti-inflammatory cytokine expression, further supporting the efficacy of this novel active vaccination approach for synucleinopathies.

Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21 WAF1/CIP1 levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21 WAF1/CIP1 induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21 WAF1/CIP1 . These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53.

The human IGF2R gene has been reported to be either biallelically or very rarely monoallelically expressed, in contrast to the maternally expressed mouse counterpart. We describe here an analysis of the 5' portion of the human IGF2R gene and show that it contains a maternally methylated CpG island in the second intron. A similar maternally methylated intronic element has been proposed to be the imprinting box for the mouse gene and although the relevance of this element has yet to be directly demonstrated, methylation has been reported to be essential to maintain allele-specific expression of imprinted genes. Allelic expression analysis of human IGF2R in 70 lymphoblastoid cell lines identified only one line showing monoallelic expression. Thus, in this tissue monoparental methylation of the IGF2R gene does not correlate with allele-specific expression. We also confirm here that the human IGF2R gene is located in an asynchronously replicating chromosomal region, as are all other imprinted genes so far analyzed. The mouse and human IGF2R intronic CpG islands both contain numerous large direct repeats that are methylated following maternal, but not paternal, transmittance. Thus features that attract maternal-specific methylation are conserved between the mouse and human genes. Since these intronic CpG islands share organizational rather than sequence homology, this suggests that secondary DNA structure may play a role in attracting a maternal methylation imprint.