SAF-A mutants disrupt chromatin structure through dominant negative effects on RNAs associated with chromatin.

Here we provide a brief review of relevant background before presenting results of our investigation into the interplay between scaffold attachment factor A (SAF-A), chromatin-associated RNAs, and DNA condensation. SAF-A, also termed heterogenous nuclear protein U (hnRNP U), is a ubiquitous nuclear scaffold protein that was implicated in XIST RNA localization to the inactive X-chromosome (Xi) but also reported to maintain open DNA packaging in euchromatin. Here we use several means to perturb SAF-A and examine potential impacts on the broad association of RNAs on euchromatin, and on chromatin compaction. SAF-A has an N-terminal DNA binding domain and C-terminal RNA binding domain, and a prominent model has been that the protein provides a single-molecule bridge between XIST RNA and chromatin. Here analysis of the impact of SAF-A on broad RNA-chromatin interactions indicate greater biological complexity. We focus on SAF-A's role with repeat-rich C0T-1 hnRNA (repeat-rich heterogeneous nuclear RNA), shown recently to comprise mostly intronic sequences of pre-mRNAs and diverse long non-coding RNAs (lncRNAs). Our results show that SAF-A mutants cause dramatic changes to cytological chromatin condensation through dominant negative effects on C0T-1 RNA's association with euchromatin, and likely other nuclear scaffold factors. In contrast, depletion of SAF-A by RNA interference (RNAi) had no discernible impact on C0T-1 RNA, nor did it cause similarly marked chromatin changes as did three different SAF-A mutations. Overall results support the concept that repeat-rich, chromatin-associated RNAs interact with multiple RNA binding proteins (RBPs) in a complex dynamic meshwork that is integral to larger-scale chromatin architecture and collectively influences cytological-scale DNA condensation.

Stable C0T-1 repeat RNA is abundant and is associated with euchromatic interphase chromosomes.

Recent studies recognize a vast diversity of noncoding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using C0T-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (C0T-1 RNA), including LINE-1. C0T-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The C0T-1 RNA territory resists mechanical disruption and fractionates with the nonchromatin scaffold but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. C0T-1 RNA has several properties similar to XIST chromosomal RNA but is excluded from chromatin condensed by XIST. These findings impact two "black boxes" of genome science: the poorly understood diversity of noncoding RNA and the unexplained abundance of repetitive elements.

Translating dosage compensation to trisomy 21.

Down's syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down's syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a 'chromosome 21 Barr body'. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of 'chromosome therapy'.

miR-21 represses Pdcd4 during cardiac valvulogenesis.

The discovery of small non-coding microRNAs has revealed novel mechanisms of post-translational regulation of gene expression, the implications of which are still incompletely understood. We focused on microRNA 21 (miR-21), which is expressed in cardiac valve endothelium during development, in order to better understand its mechanistic role in cardiac valve development. Using a combination of in vivo gene knockdown in zebrafish and in vitro assays in human cells, we show that miR-21 is necessary for proper development of the atrioventricular valve (AV). We identify pdcd4b as a relevant in vivo target of miR-21 and show that protection of pdcd4b from miR-21 binding results in failure of AV development. In vitro experiments using human pulmonic valve endothelial cells demonstrate that miR-21 overexpression augments endothelial cell migration. PDCD4 knockdown alone was sufficient to enhance endothelial cell migration. These results demonstrate that miR-21 plays a necessary role in cardiac valvulogenesis, in large part due to an obligatory downregulation of PDCD4.