Publication Date

2014-08-08

Availability

Open access

Embargo Period

2014-08-08

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Human Genetics and Genomics (Medicine)

Date of Defense

2014-05-28

First Committee Member

Katherina Walz

Second Committee Member

Carlos T Moraes

Third Committee Member

Nagi Ayad

Fourth Committee Member

Juan Young

Abstract

Structural variants are abundant in the genomes of phenotypically normal populations. However, some rare structural variants are found to be disease causing. Potocki–Lupski syndrome (PTLS) is a genomic disorder associated with an 3 Mb duplication in 17p11.2. Clinical features include leanness, intellectual disability, autistic features and developmental deficits. RAI1 gene dosage is associated with the PTLS phenotypes. To understand where and when Rai1 overexpression is detrimental, we generated a mouse that over-expresses Rai1 conditionally in forebrain neurons (I-Rai1). Phenotypic characterization of I-Rai1 mice showed leanness, hyperactivity, and impaired learning and memory ability compared with wild-type littermates. Rai1 overexpression is controlled by a “Tet-off” system which allows us to turn off the transgene expression by Doxycycline administration. When the transgene was turned off from conception to 3 months of age, no phenotypic differences were observed between I-Rai1 and their wild- type littermates, indicating that the transgene was turned off sufficiently and PTLS-like phenotypes are prevented. Interestingly, we found that turning off the transgene expression before the onset of the phenotypes (1–3 months) or after the onset of the phenotypes (3–5 months) cannot prevent nor reverse the phenotypic outcomes, indicating that Rai1 dosage in forebrain neurons is critical during brain development and function. The function of RAI1 remains largely unknown. Two domains are identified in the RAI1 protein: the N-terminal transactivational domain and the C-terminal nuclear localization domain. RAI1 is mainly bound to chromatin, indicating a possible role of RAI1 in chromatin remodeling. Smith Magenis syndrome is a reciprocal genomic disorder caused by a deletion of the same PTLS duplication region. ~10% SMS patients are carrying mutations in the RAI1 gene instead of the common deletion. We demonstrated that mutations that result in truncated RAI1 proteins lacking Nuclear Localization Signals (NLS) cause mis-localization of the protein in the cytoplasm. This result is confirmed using lymphoblastoid cells derived from an SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found mainly in the nucleus while the mutant product was only found in the cytoplasm. Missense mutations found in SMS patients do not alter RAI1 protein subcellular localization. However, the mutant proteins have impaired ability to activate the endogenous BDNF enhancer target. In summary, we demonstrated that RAI1 is a transcription factor that might be involved in chromatin remodeling and correct Rai1 dosage during early development in mice is required for normal brain development and function.

Keywords

RAI1; Potocki-Lupski Syndrome; Genomic disorder; Dosage sensitive gene; development

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