Elements at the 5′ end of Xist harbor SPEN-independent transcriptional antiterminator activity

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5′ end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5′ end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

35ANALYSIS OF EPIGENETIC MODIFICATIONS AND GENOMIC IMPRINTING IN NUCLEAR TRANSFER DERIVED BOS GAURUS×B. TAURUS CONCEPTI

Somatic cell nuclear transfer in cattle is an inefficient process hindered by low pregnancy rates and fetal placental abnormalities. Improper or incomplete epigenetic reprogramming of the donor genome has been implicated as a cause for these aberrations and has been investigated extensively in mice. Here we report the use of a bovine interspecies model (Bos gaurus×B. taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in 40-day-old female nuclear transfer (NT)-derived fetuses and placentas. Previously, we identified genomic imprinting at the IGF2, GTL2 and XIST loci in the Bos gaurus×B. taurus fetuses. These results indicated maternal and paternal imprinting of the IGF2 and GTL2 loci, respectively, in the chorion, allantois, liver, lung and brain, whereas the XIST locus was maternally imprinted solely in the chorion of females. We extended this analysis to 40-day-old NT fetuses derived from a hybrid lung fibroblast cell line (female). Analysis of the donor cell line indicated conservation of imprinting of the IGF2 and GTL2 loci and bialleic expression of the XIST locus, presumably from the random patterns of X-chromosome inactivation. Analysis of three NT and three control pregnancies indicated disruption of genomic imprinting at the XIST locus in the chorions of all three clones compared to controls. In contrast, proper allelic expression of the IGF2 and GTL2 loci was observed in all fetuses and placentas. Quantification of maternal and paternal XIST transcripts in the chorion of clones and controls demonstrated a significant skewing from preferential paternal expression in controls (95.0±0.882, mean±S.E.) to mixed paternal and maternal expression in clones (73.6±5.2), (t-test; P<0.05). In an attempt to determine the cause for the abnormal allelic expression of the XIST locus in the chorion of the clones, methylation analysis of the XIST Differentially Methylated Region (DMR) was performed. Methylation-sensitive restriction digests and subsequent PCR of the XIST DMR indicated patterns were not different between controls and clones. However, when genome-wide and promoter-specific methylation analysis (bisulfite sequencing) was extended to the satellite I repeat element and epidermal cytokeratin promoter, hypermethylation was observed in the chorion of clones. These results demonstrate disruption of genomic imprinting in XIST locus in the placenta of 40-day-old clones independent of DMR methylation. They also indicate that cloning is associated with increased levels of methylation in selected genomic regions in the chorion.