A potential role for long, non-coding RNAs in regulating escape domain boundaries on the inactive X chromosome
Open Access
- Author:
- Young, Heather Marie
- Graduate Program:
- Biomedical Sciences
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- Laura Carrel, Thesis Advisor/Co-Advisor
- Keywords:
- X inactivation
X chromosome
lncRNA
AK148627
Kdm5c
boundary regulation
escape domain - Abstract:
- In mammalian females, dosage compensation occurs by transcriptionally silencing one X chromosome. While most genes are silenced some genes, such as Kdm5c, escape inactivation. At least four escapees in mouse are flanked by long, non-coding RNAs (lncRNAs) that also escape X inactivation, raising the possibility that lncRNAs may be involved in regulating escape domains on the inactive X. X-linked bacterial artificial chromosome (BAC) transgene approaches have shown that Kdm5c ectopically escapes inactivation, suggesting that sequences contained within the BAC contribute to Kdm5c escape. Truncated transgenes lacking sequences distal of Kdm5c, including the lncRNA escapee AK148627 adjacent to Kdm5c, leads to an escape domain expansion that extends into normally inactivated loci. These results indicate that sequences distal to Kdm5c are necessary for X inactivation boundary regulation. Since lncRNAs can be cis-acting regulators within the genome, our goal is to determine whether the lncRNA juxtaposed to Kdm5c has a functional role in regulating escape, specifically in silencing adjacent genes. AK148627 maps 2.6 kb downstream from Kdm5c and is transcribed in the same orientation. The close juxtaposition prompted us to test whether they are two distinct transcripts. While robust AK148627 transcription can be detected from both undifferentiated (prior to X-chromosome inactivation (pre-XCI)) and somatic (post-XCI) cells, transcription is not apparent immediately upstream of the annotated transcription start. Transcription extending from annotated Kdm5c exons to AK148627 is not detectable. Sequences upstream of the annotated lncRNA transcription start site show weak promoter activity by luciferase evaluation. Altogether, these data argue that AK148627 is indeed a separate transcript from Kdm5c. To evaluate a possible cis-regulatory function for AK148627, a luciferase reporter vector containing AK148627 and its regulatory sequences was used. Inclusion of AK148627 sequences decreased luciferase reporter activity suggesting that expression of the lncRNA from these sequences represses adjacent gene expression. Additionally, vectors containing a 5’ truncated lncRNA lacking the transcription start site did not reduce luciferase levels, nor could AK148627 transcripts be detected. Interestingly, AK148627 is expressed from the luciferase vector in HEK293 cells in which AK148627 is not conserved in the human genome. This suggests that the RNA transcript itself is important for the suppression seen. Importantly, AK148627 is expressed pre-XCI at levels that are roughly three times higher than post-XCI levels in mouse ES cells supporting a regulatory role at critical time points during inactivation. These results suggest that the RNA transcript is expressed at a biologically relevant time point in pre-XCI cells and are consistent with an X-chromosome inactivation regulatory role as predicted by previous transgene studies that require the lncRNA to silence adjacent genes.