Developmentally Regulated Xist Promoter Switch Mediates Initiation of X Inactivation

Abstract

Developmental regulation of the mouse Xist gene at the onset of X chromosome inactivation is mediated by RNA stabilization. Here, we show that alternate promoter usage gives rise to distinct stable and unstable RNA isoforms. Unstable Xist transcript initiates at a novel upstream promoter, whereas stable Xist RNA is transcribed from the previously identified promoter and from a novel downstream promoter. Analysis of cells undergoing X inactivation indicates that a developmentally regulated promoter switch mediates stabilization and accumulation of Xist RNA on the inactive X chromosome.

Characterization of the Genomic Xist Locus in Rodents Reveals Conservation of Overall Gene Structure and Tandem Repeats but Rapid Evolution of Unique Sequence

“…The mouse putative early promoter P 0 , active in undifferentiated ES cells, was mapped within this region (Johnston et al 1998), but we were not able to identify its vole homolog.…”

“…Transcription of mouse Xist RNA is initiated from multiple start sites, with the major transcript in somatic cells being initiated at the P 2 site (Brockdorff et al 1992;Johnston et al 1998). The positions of putative P 1 and P 2 start sites in voles were established initially by comparison of vole and mouse sequences.…”

“…Nuclease protection was performed using the S1 Nuclease Protection Assay Kit (Ambion) with modifications described previously (Johnston et al 1998). Probes used were M. rossiaemeridionalis VR1 ‫,)562+-662מ(‬ VR2 ( +1174-+1678), VR3 ( +15881-+16160), and VR4 ( +18229-+18523).…”

“…Antisense transcription initiates ∼15 kb 3Ј of Xist at the Tsix promoter . Initial studies indicated that unstable sense transcript is driven by an upstream promoter P 0 located ‫5.6מ‬ kb from the P 1 initiation site of mouse Xist (Johnston et al 1998). However, subsequent work has shown that this is unlikely to be the case and has suggested unstable sense transcripts are initiated from the major somatic promoters P 1 /P 2 .…”

Use of terminal transferase-dependent antisense RNA amplification to determine the transcription start site of the Snrpn gene in individual neurons

“…Variation in promoter usage plays a wide role in gene expression of both prokaryotes and eukaryotes (reviewed in 1) and recent data suggests that it may be involved in the mechanism of X chromosome inactivation (2) and imprinting (3,4). For genes that are expressed or imprinted at different stages in development or in different cell types, determination of transcription start sites may require methods sensitive enough for detection in one or a few cells.…”