CRISPR/dCas9-mediated Transcriptional Inhibition Ameliorates the Epigenetic Dysregulation at D4Z4 and Represses DUX4-fl in FSH Muscular Dystrophy

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent myopathies, affecting males and females of all ages. Both forms of the disease are linked by epigenetic derepression of the D4Z4 macrosatellite repeat array at chromosome 4q35, leading to aberrant expression of D4Z4-encoded RNAs in skeletal muscle. Production of full-length DUX4 (DUX4-fl) mRNA from the derepressed D4Z4 array results in misexpression of DUX4-FL protein and its transcriptional targets, and apoptosis, ultimately leading to accumulated muscle pathology. Returning the chromatin at the FSHD locus to its nonpathogenic, epigenetically repressed state would simultaneously affect all D4Z4 RNAs, inhibiting downstream pathogenic pathways, and is thus an attractive therapeutic strategy. Advances in CRISPR/Cas9-based genome editing make it possible to target epigenetic modifiers to an endogenous disease locus, although reports to date have focused on more typical genomic regions. Here, we demonstrate that a CRISPR/dCas9 transcriptional inhibitor can be specifically targeted to the highly repetitive FSHD macrosatellite array and alter the chromatin to repress expression of DUX4-fl in primary FSHD myocytes. These results implicate the promoter and exon 1 of DUX4 as potential therapeutic targets and demonstrate the utility of CRISPR technology for correction of the epigenetic dysregulation in FSHD.

Figure 1

Recruitment of dCas9 and VP64 to the DUX4 promoter or exon 1 activates DUX4-fl in facioscapulohumeral muscular dystrophy (FSHD) myocytes. (a) Schematic diagram of the FSHD locus at chromosome 4q35, with distances shown relative to the DUX4 MAL start codon (*). For simplicity, only the distal D4Z4 repeat unit of the macrosatellite array is depicted below. DUX4 exons 1 and 2 are located within the D4Z4 repeat, and exon 3 lies in the distal subtelomeric sequence. In FSHD skeletal myocytes, DUX4-fl mRNA from the distal repeat is stabilized by a polyadenylation signal in exon 3 that is present in disease-permissive haplotypes of 4qA. The p13-E11 diagnostic probe region^, and the NDE (non-deleted element)^, lie proximal to the D4Z4 array. The locations of sgRNA target sequences used in this study (#1–11) are indicated. Positions of chromatin immunoprecipitation amplicons are shown as unlabeled black bars (in order from 5' to 3': p13-E11, DUX4 exon 1, intron 1, and exon 3). Refer to text for more details. (b) Effects of targeting dCas9 and VP64 to the FSHD locus on DUX4-fl expression. FSHD myogenic cultures were subjected to four serial coinfections with lentiviral supernatants expressing either components of the SunTag system encoding dCas9 and VP64 (ST), a SunTag variant lacking VP64 (ST[CTL]) or individual sgRNAs (#1–9). After the final round of infection, cells were induced to differentiate and harvested ~48 hours later for analysis of DUX4-fl expression by quantitative reverse transcriptase polymerase chain reaction. Data are plotted as the mean + standard deviation (SD) value of three to five independent experiments, with relative mRNA expression for the mock-infected cells set to 1.

Figure 2

Recruitment of dCas9-KRAB to the DUX4 promoter or exon 1 represses DUX4-fl in facioscapulohumeral muscular dystrophy (FSHD) myocytes. (a) Effects of targeting dCas9-KRAB to the FSHD locus on DUX4-fl expression. FSHD myogenic cultures were subjected to four serial co-infections with lentiviral supernatants expressing either dCas9-KRAB, a dCas9 variant lacking an effector domain (dCas9[CTL]), or individual sgRNAs (#1–11). After the final round of infection, cells were induced to differentiate and harvested ~40 hours later for analysis of DUX4-fl expression by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). (b) Recruitment of dCas9-KRAB to the FSHD locus does not impair skeletal myocyte differentiation. Expression of the terminal muscle differentiation marker Myosin heavy chain (MyHC) was assessed by qRT-PCR in the cultures described in Figure 2a. (c) Recruitment of dCas9-KRAB to the FSHD locus does not repress expression of FRG1 and FRG2. Levels of FSHD candidate genes FRG1 and FRG2 were measured by qRT-PCR in the cultures described in Figure 2a. For a–c, data are plotted as the mean + SD value of at least three independent experiments, with relative mRNA expression for the mock-infected cells set to 1. *P < 0.05 (Student's t-test).

Figure 3

Recruitment of dCas9-KRAB to the DUX4 promoter or exon 1 represses DUX4-FL target genes in facioscapulohumeral muscular dystrophy myocytes. Levels of the DUX4-FL target genes TRIM43, ZSCAN4, and MBD3L2 were assessed by quantitative reverse transcriptase polymerase chain reaction in the cultures described in Figure 2a. Data are plotted as the mean + SD value of at least three independent experiments, with relative mRNA expression for the mock-infected cells set to 1. *P < 0.05; **P < 0.01 (Student's t-test).

Figure 4

Targeting a transcriptional effector to the DUX4 promoter or exon 1 has no effect on expression of several off-target genes. Levels of Jumonji or KLF14 and UBR4 were assessed by quantitative reverse transcriptase polymerase chain reaction in mock-infected cultures or in cultures expressing the SunTag activator system alone or with sgRNA #3 or #6 (as in Figure 1b, lanes 1, 2, 5, and 8). Jumonji contains an off-target match (12-bp seed + PAM) to sgRNA #3 in intron 7. KLF14 and UBR4 lie 28 and 76 kb downstream of off-target matches (9 bp seed + PAM) to sgRNA #6. Refer to text for more details. Data are plotted as the mean + SD value of at least three independent experiments, with relative mRNA expression for the mock-infected cells set to 1.

Figure 5

Recruitment of dCas9-KRAB to the DUX4 promoter or exon 1 represses the D4Z4 locus in facioscapulohumeral muscular dystrophy (FSHD) myocytes. Chromatin immunoprecipitation (ChIP) assays were performed using FSHD myogenic cultures infected with combinations of lentiviral supernatants expressing either dCas9-KRAB or individual sgRNAs targeting the DUX4 promoter (#6–8) or exon 1 (#3–5). Following infection, cells were induced to differentiate for ~40 hours, as in Figures 2 and 3. Chromatin was immunoprecipitated using antibodies specific for (a) KAP1, (b) HP1α, (c) HP1β, (d) H3K27ac, or (e) the elongating form of RNA Pol II (Pol II-PS2), and analyzed by qPCR using primers to the (f) p13-E11 region of 4q35 or exon 1, intron 1, or exon 3 of DUX4. Location of primers is shown in Figure 1a. In cases where enrichment of the specific factor was observed across the DUX4 locus, an off-target region was also assessed. Data are presented as fold enrichment of the target region by each specific antibody normalized to α-histone H3, with enrichment for the mock-infected cells set to 1. For all panels, each bar represents the average of at least three independent ChIP experiments.