Date of Graduation


Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Eric J Wagner, PhD.

Committee Member

Joseph Alcorn, PhD.

Committee Member

Swathi Arur, PhD.

Committee Member

Thomas Cooper, MD.

Committee Member

Anil Sood, MD.

Committee Member

Ambro van Hoof, PhD.


Double Homeobox 4, Dux4, is the leading candidate gene for Facioscapulohumeral Dystrophy (FSHD). FSHD is the third most common muscular dystrophy, and is characterized by progressive muscle weakness primarily in the upper body. In individuals diagnosed with FSHD, Dux4 is inappropriately expressed in somatic cells due to two conditions. The first is hypomethylation of the subtelomeric D4Z4 repeats on chromosome 4. Each D4Z4 repeat on chromosome 4 is 3.3kb in length and contains the open reading frame for Dux4. Hypomethylation of the D4Z4 repeats primarily occurs due to contraction of the repeats from 11-100 (typical numbers in the healthy population) to between 1 and 10 repeats. Concomitant with the hypomethylation of the D4Z4 repeats on chromosome 4 is a single nucleotide polymorphism in the flanking DNA that generates a non-consensus polyadenylation signal (PAS). This PAS allows for the productive transcription of a polyadenylated Dux4 mRNA from the terminal D4Z4 repeat. Dux4 is anemically expressed in patient somatic cells, but contributes to FSHD pathology due to Dux4-dependent cellular reprogramming.

We aim to understand what regulatory elements facilitate the cleavage and polyadenylation (CPA) of the Dux4 mRNA beyond the non-consensus PAS and to determine if inefficient CPA underlies the poor expression of Dux4 in patient cells. We designed a transcriptional read-through reporter to assay cleavage and polyadenylation in cells and confirm that additional cis elements are required for CPA of Dux4 besides the non-consensus PAS. This element is located outside the region where cis regulatory elements for CPA are usually present. Moreover, the element which lies downstream of the PAS, is within a degenerate repeat region, called β-satellite DNA. Using the knowledge gained from characterizing Dux4 mRNA 3′end formation, we designed antisense oligonucleotides (ASOs) to impair the production of polyadenylated Dux4. Prior to antagonizing Dux4 CPA, we demonstrate, in proof of principle experiments that ASOs directed toward required CPA regulatory elements can impair gene expression, and may redirect polyadenylation. Finally, the work presented here lays the foundation for us to impair Dux4 CPA in reporter driven assays and patient cells; and to exploit currently available deep sequencing technology to determine the specificity of PAS-directed ASOs.


cleavage and polyadenylation, Facioscapulohumeral Dystrophy, antisense oligonucleotides