SNARE [soluble NSF ( N -ethylmaleimide-sensitive factor) attachment protein receptor] proteins are essential for membrane fusion but their regulation is not yet fully understood. We have previously shown that the amino-terminal Longin domain of the v-SNARE TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein)/VAMP7 plays an inhibitory role in neurite outgrowth. The goal of this study was to investigate the regulation of TI-VAMP as a model of v-SNARE regulation. We show here that the Longin domain (LD) plays a dual role. First, it negatively regulates the ability of TI-VAMP and of a Longin/Synaptobrevin chimera to participate in SNARE complexes. Second, it interacts with the adaptor complex AP-3 and this interaction targets TI-VAMP to late endosomes. Accordingly, in mocha cells lacking AP-3δ, TI-VAMP is retained in an early endosomal compartment. Furthermore, TI-VAMPc, an isoform of TI-VAMP lacking part of the LD, does not interact with AP-3, and therefore is not targeted to late endosomes; however, this shorter LD still inhibits SNARE-complex formation. These findings support a mechanism controlling both localization and function of TI-VAMP through the LD and clathrin adaptors. Moreover, they point to the amino-terminal domains of SNARE proteins as multifunctional modules responsible for the fine tuning of SNARE function.

On the human long-arm pseudoautosomal region (XqPAR), genes that are subject to inactivation are closely linked with those that escape. Genes subject to inactivation are not only silenced on the inactive X in females, but they are also inactivated on the Y chromosome in males. One of the genes subject to this unusual inactivation pattern is the synaptobrevin-like 1 gene (SYBL1). Previously we showed that its silencing on the inactive X and the Y allele involves DNA methylation. This study explores the molecular events associated with SYBL1 silencing and investigates their relationship. Promoter DNA methylation profiles were determined by bisulfite sequencing and immunoprecipitation experiments demonstrate that chromatin on the repressed Xi and the Y alleles has underacetylated histones H3 and H4 and H3-lysine 9 methylation. In addition, the inactive X and the Y allele were found to have a condensed chromatin conformation. In contrast, the expressed allele shows H3 and H4 acetylation, H3-lysine 4 methylation and a less compacted chromatin conformation. In ICF syndrome, a human disease affecting DNA methylation, SYBL1 escapes from silencing and this correlates with altered patterns of histone methylation and acetylation. Combined, our data suggest that specific combinations of histone methylation and acetylation are involved in the somatic maintenance of permissive and repressed chromatin states at SYBL1. Although it is unclear at present how this allele-specific silencing comes about, the data also indicate that the epigenetic features of the ‘Y inactivation’ of SYBL1 are mechanistically similar to those associated with X-chromosome inactivation.

Rett Syndrome (RTT) is an X‐linked dominant neurodevelopmental disorder, which almost exclusively affects girls, with an estimated prevalence of one in 10,000–15,000 female births. Mutations in the methyl CpG binding protein 2 gene (MECP2) have been identified in roughly 75% of classical Rett girls. The vast majority of Rett cases (99%) are sporadic in origin, and are due to de novo mutations. We collected DNA samples from 50 Italian classical Rett girls, and screened the MECP2 coding region for mutations by denaturing high‐performance liquid chromatography (DHPLC) and subsequent direct sequencing. DHPLC is a recently developed method for mutation screening which identifies heteroduplexes formed in DNA samples containing mismatches between wild type and mutant DNA strands, combining high sensitivity, reduced cost per run, and high throughput. In our series, 19 different de novo MECP2 mutations, eight of which were previously unreported, were found in 35 out of 50 Rett girls (70%). Seven recurrent mutations were characterized in a total of 22 unrelated cases. Initial DHPLC screening allowed the identification of 17 out of 19 different mutations (90%); after optimal conditions were established, this figure increased to 100%, with all recurrent MECP2 mutations generating a characteristic chromatographic profile. Detailed clinical data were available for 27 out of 35 mutation carrying Rett girls. Milder disease was detectable in patients carrying nonsense mutation as compared to patients carrying missense mutations, although this difference was not statistically significant (P = 0.077). Hum Mutat 18:132–140, 2001.