Identification of a CTCF-independent insulator that delimits X-inactivated from expressed transcripts within the UBA1 gene
Open Access
- Author:
- Prothero, Katie Eva
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 12, 2010
- Committee Members:
- Laura Carrel, Dissertation Advisor/Co-Advisor
Laura Carrel, Committee Chair/Co-Chair
Sergei A Grigoryev, Committee Member
Ira Joseph Ropson, Committee Member
Jiyue Xhu, Committee Member - Keywords:
- DNA methylation
X chromosome inactivation
epigenetics - Abstract:
- In female mammals, X chromosome inactivation (XCI) transcriptionally silences most genes on one X. Nonetheless, in humans, 15% of genes escape XCI and are expressed from both active and inactive Xs. Many of these escape genes are clustered, suggesting their expression is coordinately regulated. An attractive model is that these clustered escape genes are flanked by regulatory sequences such as insulators. To identify sequences that control XCI, I focused on a cluster of human escape genes that includes the Ubiquitin Activating Enzyme E1 gene, UBA1. UBA1 has a distinctive gene structure with five alternative promoters and 5’ untranslated exons that each splice to a single exon 2. Inactive X expression is also unique; two upstream exons are X-inactivated whereas two downstream exons assayed escape XCI. The 2.1 kb between X-inactivated and expressed exons is the smallest inactive X expression boundary and is a tractable region to identify sequences that regulate inactive X expression. To test the prediction that escape domains are regulated by chromatin insulators, eight overlapping constructs within the UBA1 boundary were tested; a 330 bp sequence demonstrated insulator function. Despite similar gene structure and high sequence conservation, all mouse Uba1 transcripts are X-inactivated and the locus lacks insulator activity. The UBA1 insulator is novel; while the insulator-binding protein CTCF has been identified at one XCI expression boundary, CTCF binds the UBA1 locus downstream of the insulator and appears unrelated to inactive X regulation. That the insulator is functional in XCI gene regulation is suggested by epigenetic modifications that specifically demarcate this sequence on the inactive X. Chromatin immunoprecipitation demonstrated that the UBA1 insulator segregates heterochromatin from the euchromatic escape domain. Additionally, DNA methylation differences distinguish the human insulator; this sequence is hypomethylated on the inactive X, whereas the active X is heavily methylated, as is the orthologous sequence that lacks boundary activity on both Xs in mouse. The identification of a novel insulator at the UBA1 expression boundary strengthens the model that escape domains are regulated by chromatin boundaries and pinpoints a region that can be further dissected to identify novel factors that bind and regulate XCI escape.