Asymmetric Bidirectional Transcription from the FSHD-Causing D4Z4 Array Modulates DUX4 Production

Facioscapulohumeral Disease (FSHD) is a dominantly inherited progressive myopathy associated with aberrant production of the transcription factor, Double Homeobox Protein 4 (DUX4). The expression of DUX4 depends on an open chromatin conformation of the D4Z4 macrosatellite array and a specific haplotype on chromosome 4. Even when these requirements are met, DUX4 transcripts and protein are only detectable in a subset of cells indicating that additional constraints govern DUX4 production. Since the direction of transcription, along with the production of non-coding antisense transcripts is an important regulatory feature of other macrosatellite repeats, we developed constructs that contain the non-coding region of a single D4Z4 unit flanked by genes that report transcriptional activity in the sense and antisense directions. We found that D4Z4 contains two promoters that initiate sense and antisense transcription within the array, and that antisense transcription predominates. Transcriptional start sites for the antisense transcripts, as well as D4Z4 regions that regulate the balance of sense and antisense transcripts were identified. We show that the choice of transcriptional direction is reversible but not mutually exclusive, since sense and antisense reporter activity was often present in the same cell and simultaneously upregulated during myotube formation. Similarly, levels of endogenous sense and antisense D4Z4 transcripts were upregulated in FSHD myotubes. These studies offer insight into the autonomous distribution of muscle weakness that is characteristic of FSHD

Human Gene Targeting by Adeno-Associated Virus Vectors Is Enhanced by DNA Double-Strand Breaks

The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications

Transcription from D4Z4 is dynamically switching directions.

<p>(A) Human myoblasts were infected with <i>eGFP</i>←D4Z4→<i>dsRED</i>. After expression was observed (arbitrarily designated day 0) cells were sorted by flow cytometry to purify dsRED-GFP(+) cells (red box; R1) or GFP(+) cells (green box; R2). Gates were set one log above cut-off thresholds established in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035532#pone-0035532-g001" target="_blank">figure 1</a>. dsRED(−)GFP(+) and GFP(+) cells were seeded into separate dishes and cultured until dishes were 85% of confluence. Cells were collected and analyzed by flow cytometry after 7 days of culture. The fluorescence intensity of cells collected from R1 or R2 are shown as a scatter plot and the percentage of cells meeting criteria for dsRED-GFP(+) or GFP(+) are labeled. (B) HEK-293T cells were infected with <i>eGFP</i>←D4Z4→<i>dsRED</i>, sorted by flow cytometry to obtain dsRED-GFP(+) cells and seeded to culture dishes as single cells. Single cell derived colonies were photographed at 20× magnification and clonally isolated for analysis by flow cytometry; scale bar = 50 µm. (C) WA09 Human ES cells were infected with <i>eGFP</i>←D4Z4→<i>dsRED</i> as a single cell suspension and cultured under conditions that prevent differentiation. Colonies containing red cells (∼1/250 GFP(+)) were isolated using glass cloning rings and transferred to new culture wells. To ensure clonal growth, the cells were subcloned four times by repeatedly isolating single-cell-derived colonies. The resulting population was less than 0.5% double positive for dsRED and GFP (red box). (D) Cells from (C) were sorted for dsRED-GFP(+) fluorescence and expanded as colonies that were scored for <i>dsRED</i> and <i>eGFP</i> expression by fluorescence microscopy; Scale bar = 50 µm.</p

Reporter constructs and their activity in human and mouse myoblasts and myotubes.

<p>(A) The distal end of chromosome 4 is shown above a schematic of an <i>Eco</i>RI fragment cloned from an FSHD-affected individual (λ42). λ42 contains two full D4Z4 units (green and pink rectangles), and two partial D4Z4 units on either end. The <i>DUX4</i> open reading frame is shown as a yellow colored rectangle with homeoboxes shown as black boxes within each D4Z4 repeat. The approximate location of the TATA box (TACAA) and the transcription start site (bent arrow) are indicated. The restriction enzymes <i>Sfo</i>I and <i>Apo</i>I were used to clone the non-coding region. The location of previously identified miRNA fragments from D4Z4 are shown as blue lines <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035532#pone.0035532-Snider1" target="_blank">[3]</a> (B) The non-coding region of D4Z4 was isolated from the second repeat of λ42 and placed upstream or downstream of the indicated reporter genes (D4Z4→<i>eGFP</i>, sense promoter driving <i>eGFP</i>; <i>eGFP</i>←D4Z4, antisense promoter driving <i>eGFP</i>; <i>eGFP</i>←D4Z4→<i>dsRED</i>, <i>DUX4</i> promoter driving <i>dsRED</i> in the sense direction and <i>eGFP</i> in the antisense direction. (C) Control and FSHD Human myoblasts were differentiated into myotubes and assayed for <i>DUX4</i> expression by RT-PCR. Locations of primers 1 and 2 to detect <i>DUX4</i> transcripts are shown along with common splice sites within the <i>DUX4</i> transcripts. (D) Mouse and Human myoblasts transduced with a lentivirus vector encoding D4Z4→<i>eGFP</i> were sorted by flow cytometry, and expanded in culture. The cells were seeded at equal densities, switched to either myoblast proliferation medium or myotube differentiation medium for 72 hours, and imaged by fluorescence microscopy. Scale bars = 50 µm. Images were taken at the same time with the same exposure settings.</p

Antisense transcripts begin in a GC-rich region distal to the <i>DUX4</i> ORF.

<p>(A) 5′ RACE was performed on cells transduced with <i>eGFP</i>←D4Z4 lentivirus using primers based within the <i>eGFP</i> open reading frame. The <i>eGFP</i>←D4Z4 reporter construct is shown with the previously identified sense transcription start site indicated by a bent arrow. The <i>eGFP</i> gene and poly-adenylation signal (box with A) is shown upside down and backwards to indicate reporting transcription in the antisense direction. The primer used for cDNA synthesis with reverse transcriptase is shown as a red arrow with the location of the 5′ end of the antisense transcript indicated as a question mark. (B) Nucleotide sequence of the 5′ end of six separate isolates cloned from the 5′RACE reaction. The sequence is shown as a 3′-5′ strand to depict the antisense transcript. (C) Antisense-strand-specific RT-PCR strategy is diagramed. <i>DUX4</i> is indicated as yellow rectangle with black boxes showing the location of the homeobox motifs and is drawn adjacent to the antisense promoter region. <i>Tth</i>111I and <i>Kpn</i>I sites are shown pointing to their approximate location at the 5′ end of the antisense promoter. The strand specific RT primer contains a linker sequence (red line) that is complementary to primers used in subsequent PCR reactions (red arrows). Primers complementary to the antisense strand upstream of the <i>Tth</i>111I site are shown as black arrows. <i>In vitro</i> transcribed RNA was used as a positive control and H₂O as a negative control. (D) Human control and FSHD myoblasts and myotubes were assayed by strand-specific RT-PCR for the antisense transcript, quantified by densitometry, and normalized to GAPDH transcripts in the same RNA preparations. N = 4. * p<0.05. (E) A plasmid containing <i>eGFP</i>←D4Z4(Δ<i>Spe</i>I-<i>Tth</i>111I)→<i>dsRED</i> containing a deletion of the start site cluster upstream of the restriction enzyme site <i>Tth</i>111I was transfected into C2C12 cells and fluorescence measured by flow cytometry. The graph shows the ratio of dsRED-GFP(+) cells to dsRED-GFP(+)+GFP(+) cells shown as a percentage. Error bars = SD of the mean of three experiments that measured duplicate samples. Lower panel: flow diagrams of the corresponding deletions.</p