Abstract
Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are imprinting-related disorders associated with genetic/epigenetic alterations of the 11p15.5 region, which harbours two clusters of imprinted genes (IGs). 11p15.5 IGs are regulated by the methylation status of imprinting control regions ICR1 and ICR2. 3D chromatin structure is thought to play a pivotal role in gene expression control; however, chromatin architecture models are still poorly defined in most cases, particularly for IGs. Our study aimed at elucidating 11p15.5 3D structure, via 3C and 3D FISH analyses of cell lines derived from healthy, BWS or SRS children. We found that, in healthy cells, IGF2/H19 and CDKN1C/KCNQ1OT1 domains fold in complex chromatin conformations, that facilitate the control of IGs mediated by distant enhancers. In patient-derived cell lines, we observed a profound impairment of such a chromatin architecture. Specifically, we identified a cross-talk between IGF2/H19 and CDKN1C/KCNQ1OT1 domains, consisting in in cis, monoallelic interactions, that are present in healthy cells but lost in patient cell lines: an inter-domain association that sees ICR2 move close to IGF2 on one allele, and to H19 on the other. Moreover, an intra-domain association within the CDKN1C/KCNQ1OT1 locus seems to be crucial for maintaining the 3D organization of the region.
Highlights
Genomic imprinting is a finely tuned epigenetic process fundamental for mammalian development, whereby, through specific patterns of DNA methylation and chromatin modification, only one copy of an imprinted gene (IG) is expressed, according to its parental origin[1]
Since chromosomes are organised in topologically associating domains (TADs), and considering the linear proximity of the two IG clusters, we investigated whether the two clusters lie within the same TAD
These results indicate that the ROIs that drag the enhancers (Enh A and Enh B) into proximity with the IGF2 promoter (CTCF Up and 5′ IGF2) increase in the Beckwith-Wiedemann syndrome (BWS)-ICR1 cell line (Fig. 4a, dotted rectangle) and decrease in the Silver-Russell syndrome (SRS)-ICR1 cell line (Fig. 5a, dotted rectangle), consistent with the upregulation of IGF2 expected in BWS, and downregulation of IGF2 expected in SRS
Summary
We sought to investigate in depth the chromatin structure of chromosome region 11p15.5 and its potential deregulation in the related imprinting diseases (BWS and SRS), focusing on cis regulatory elements, including enhancers and CTCF-binding sites. The BWS-UPD cell line, which harbours methylation defects in both ICRs, due to UPD which leads to a double dosage of the paternal allele, displayed chromatin architecture alterations at both IGF2/H19 and CDKN1C/KCNQ1OT1 domains (Supplementary Fig. S7). We found alterations in the interaction profile of the CDKN1C/KCNQ1OT1 region in the BWS-ICR1 and SRS-ICR1 cell lines and, of the IGF2/H19 domain in BWS-ICR2 cells, compared to the CTRLs (Supplementary Fig. S8) These data indicate that a methylation defect at the IGF2/H19 locus can affect the chromatin conformation of the CDKN1C/KCNQ1OT1 domain and vice versa. The monoallelic ICR2-Upstream IGF2 association was still present in the BWS-ICR2 cells (Fig. 7a), and could explain the 28.73% of the cis-interactions observed in this cell line This contact most likely is engaged on the normal paternal allele. This may be because the loss of the interaction between the two domains of one allele in BWS-ICR2 cells cause the shift from +/+ to +/− and from +/− to −/− of some nuclei, maintaining unchanged the category +/−
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