Differential 3D genome architecture and imprinted gene expression: cause or consequence?

Imprinted genes provide an attractive paradigm to unravel links between transcription and genome architecture. The parental allele-specific expression of these essential genes - which are clustered in chromosomal domains - is mediated by parental methylation imprints at key regulatory DNA sequences. Recent chromatin conformation capture (3C)-based studies show differential organization of topologically associating domains between the parental chromosomes at imprinted domains, in embryonic stem and differentiated cells. At several imprinted domains, differentially methylated regions show allelic binding of the insulator protein CTCF, and linked focal retention of cohesin, at the non-methylated allele only. This generates differential patterns of chromatin looping between the parental chromosomes, already in the early embryo, and thereby facilitates the allelic gene expression. Recent research evokes also the opposite scenario, in which allelic transcription contributes to the differential genome organization, similarly as reported for imprinted X chromosome inactivation. This may occur through epigenetic effects on CTCF binding, through structural effects of RNA Polymerase II, or through imprinted long non-coding RNAs that have chromatin repressive functions. The emerging picture is that epigenetically-controlled differential genome architecture precedes and facilitates imprinted gene expression during development, and that at some domains, conversely, the mono-allelic gene expression also influences genome architecture.

Exploring chromatin structural roles of non-coding RNAs at imprinted domains.

Different classes of non-coding RNA (ncRNA) influence the organization of chromatin. Imprinted gene domains constitute a paradigm for exploring functional long ncRNAs (lncRNAs). Almost all express an lncRNA in a parent-of-origin dependent manner. The mono-allelic expression of these lncRNAs represses close by and distant protein-coding genes, through diverse mechanisms. Some control genes on other chromosomes as well. Interestingly, several imprinted chromosomal domains show a developmentally regulated, chromatin-based mechanism of imprinting with apparent similarities to X-chromosome inactivation. At these domains, the mono-allelic lncRNAs show a relatively stable, focal accumulation in cis. This facilitates the recruitment of Polycomb repressive complexes, lysine methyltranferases and other nuclear proteins - in part through direct RNA-protein interactions. Recent chromosome conformation capture and microscopy studies indicate that the focal aggregation of lncRNA and interacting proteins could play an architectural role as well, and correlates with close positioning of target genes. Higher-order chromatin structure is strongly influenced by CTCF/cohesin complexes, whose allelic association patterns and actions may be influenced by lncRNAs as well. Here, we review the gene-repressive roles of imprinted non-coding RNAs, particularly of lncRNAs, and discuss emerging links with chromatin architecture.

Oscillatory expression of Hes1 regulates cell proliferation and neuronal differentiation in the embryonic brain.

The expression of the transcriptional repressor Hes1 oscillates in many cell types, including neural progenitor cells (NPCs), but the significance of Hes1 oscillations in development is not fully understood. To examine the effect of altered oscillatory dynamics of Hes1, we generated two types of Hes1 knock-in mice, a shortened (type-1) and an elongated (type-2) Hes1 gene, and examined their phenotypes focusing on neural development. Although both mutations affected Hes1 oscillations, the type-1 mutation dampened Hes1 oscillations more severely, resulting in much lower amplitudes. The average levels of Hes1 expression in type-1 mutant NPCs were also lower than in wild-type NPCs but similar to or slightly higher than those in Hes1 heterozygous mutant mice, which exhibit no apparent defects. Whereas type-2 mutant mice were apparently normal, type-1 mutant mice displayed smaller brains than wild-type mice and upregulated proneural gene expression. Furthermore, proliferation of NPCs decreased and cell death increased in type-1 mutant embryos. When Hes3 and Hes5 were additionally deleted, neuronal differentiation was also accelerated, leading to microcephaly. Thus, robust Hes1 oscillations are required for maintenance and proliferation of NPCs and the normal timing of neurogenesis, thereby regulating brain morphogenesis.

Role of the imprinted allele of the Cdkn1c gene in mouse neocortical development.

Imprinted genes are expressed from only one allele in a parent of origin-specific manner. The cyclin-dependent kinase inhibitor p57kip2 is encoded by an imprinted gene Cdkn1c, with the paternal allele being silenced. The possible expression and function of the paternal allele of Cdkn1c have remained little studied, however. We now show that the paternal allele of the Cdkn1c gene is expressed at a low level in the developing mouse neocortex. Surprisingly, the central nervous system-specific conditional deletion of the paternal allele (pat cKO) at the Cdkn1c locus resulted in a marked reduction in brain size. Furthermore, pat cKO gradually reduced the number of neural stem-progenitor cells (NPCs) during neocortical development, and thus reduced the number of upper-layer neurons, which were derived from late-stage NPCs. Our results thus show that the paternal allele of the Cdkn1c locus plays a key role in maintenance of NPCs during neocortical development.