Neuronal chromatin undergoes dynamic changes in compaction and organization during post-natal neuronal maturation (38
), but little is known about chromatin changes to specific loci. Here we have shown visual evidence for a highly orchestrated chromatin decondensation of essential clusters of snoRNAs during neuronal maturation. Although previous research has shown visual evidence of transcriptionally induced changes to chromatin at specific loci in non-neuronal cells (5
), our results appear to represent the most dramatic chromatin decondensation of an endogenous locus currently reported in interphase mammalian nuclei.
We have shown that allele- and neuron-specific chromatin decondensation of imprinted snoRNA loci correlates with and is required for increased nucleolar size during neuronal maturation. Why this high level of allele-specific chromatin decondensation appears limited to the only two imprinted snoRNA cluster loci is presumably due to high expression of large clusters of snoRNAs and specific events required for snoRNA processing and biogenesis. Two proteins known to associate with biogenesis of C/D box snoRNAs, Tip49a and Tip49b, are involved in an ATP-dependent chromatin remodeling complex which catalyzes ATP-dependent nucleosome sliding (41
). Perhaps these or other snoRNA processing factors are brought to the site of transcription to process snoRNAs and remodel the surrounding chromatin. Lack of one of these snoRNA clusters (HBII-85) is necessary to cause PWS (33
), yet little is known of the function of these snoRNAs within neuronal nuclei. Our results suggest that one function of neuron-specific snoRNAs may be to modify rRNA, thereby increasing nucleolar size during neuronal maturation.
Recent publications point to the HBII-85/52 snoRNA clusters as being central to the PWS phenotype and 15q duplication syndrome. Two individuals have now been found with partial overlapping HBII-85 deletions and PWS-like phenotypes (33
), whereas overexpression of MBII-52 in a Snprn-Ube3a
locus paternal duplication mouse appears to contribute to social and behavioral abnormalities (43
). In addition, recent human patients with and without PWS have suggested an exclusion of upstream genes MKRN3
from a causal role in the PWS phenotype (44
). Our results showing imprinted snoRNA chromatin decondensation impacting nucleolar size are consistent with the essential role of the HBII-85/52 locus in post-natal maturation of mammalian neurons.
While individual snoRNA genes are found in all eukaryotic genomes, snoRNA gene organization and regulation has changed considerably throughout evolution, favoring the colonization of introns to exploit host gene promoters to transcribe multiple snoRNAs from a single promoter (45
). The presence of two eutherian-specific C/D snoRNA gene clusters at two evolutionarily distinct chromosomal locations (human 15q and 14q) has been suggested to be a potential evolutionary link to the recent emergence of parental imprinting of these loci (28
). Furthermore, human chromosomes 14 and 15 are both acrocentric in humans, encoding repetitive rRNA genes at their p arms, suggesting a potential evolutionary link to localizing snoRNA genes near their target genes for optimal perinucleolar organization. In a recent comparison of gene neighborhood differences between human and chimpanzee, the 15q11–q13 locus was among the most highly divergent in gene neighborhood, suggesting a recent evolutionary advantage in the primate lineage to altered chromosome neighborhood particularly for genes specifically expressed in brain (46
). The neuron specificity of the snoRNA chromatin decondensation observed in our study is consistent with a role for the 15q11–q13 snoRNAs in post-natal brain development, a mammalian occurrence under positive selection in the primate lineage.
Epigenetic regulation of both mammalian snoRNA loci is highly complex, requiring imprinting, ncRNA and chromatin remodeling for properly modulated allele- and neuron-specific expression. Homologous trans
interactions between maternal and paternal alleles have been reported for both of these loci adding to the complex epigenetic mechanisms required for proper temporal and spatial transcription of these snoRNA loci (47
). Our DNA FISH results in a transgenic mouse line harboring two additional transcriptionally active copies of the PWS-IC through MBII-85 at another genetic locus revealed that transcriptional activity alone was insufficient to induce full chromatin decondensation, further implicating the importance of snoRNA cluster genomic neighborhood in chromatin remodeling and decondensation.
In the primate lineage, chromosome 15q11–q13 is enriched in segmental low copy repeats or duplicons (49
) that predispose it to a series of common breakpoints (50
). These multiple breakpoints give rise not only to the large deletions and duplications seen in AS, PWS and 15q duplication, but also smaller 15q deletions and duplications distal to the snoRNA cluster found in other neurodevelopmental disorders, including epilepsy (52
), intellectual disability (54
), schizophrenia (56
) and autism (53
). Our results suggest the possibility that imprinted mammalian snoRNA chromatin decondensation during neuronal maturation could exert neighborhood effects on the phenotypic manifestations of copy number variations common to 15q11–q13 in humans.