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A feedback loop between nonsense-mediated decay and the retrogene DUX4 in facioscapulohumeral muscular dystrophy.

Feng Q, Snider L, Jagannathan S, Tawil R, van der Maarel SM, Tapscott SJ, Bradley RK - Elife (2015)

Bottom Line: DUX4 is a double homeobox transcription factor that is normally expressed in the testis and causes apoptosis and FSHD when misexpressed in skeletal muscle.The mechanism(s) of DUX4 toxicity in muscle is incompletely understood.We report that DUX4-triggered proteolytic degradation of UPF1, a central component of the nonsense-mediated decay (NMD) machinery, is associated with profound NMD inhibition, resulting in global accumulation of RNAs normally degraded as NMD substrates.

View Article: PubMed Central - PubMed

Affiliation: Computational Biology Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States.

ABSTRACT
Facioscapulohumeral muscular dystrophy (FSHD) is a muscular dystrophy caused by inefficient epigenetic repression of the D4Z4 macrosatellite array and somatic expression of the DUX4 retrogene. DUX4 is a double homeobox transcription factor that is normally expressed in the testis and causes apoptosis and FSHD when misexpressed in skeletal muscle. The mechanism(s) of DUX4 toxicity in muscle is incompletely understood. We report that DUX4-triggered proteolytic degradation of UPF1, a central component of the nonsense-mediated decay (NMD) machinery, is associated with profound NMD inhibition, resulting in global accumulation of RNAs normally degraded as NMD substrates. DUX4 mRNA is itself degraded by NMD, such that inhibition of NMD by DUX4 protein stabilizes DUX4 mRNA through a double-negative feedback loop in FSHD muscle cells. This feedback loop illustrates an unexpected mode of autoregulatory behavior of a transcription factor, is consistent with 'bursts' of DUX4 expression in FSHD muscle, and has implications for FSHD pathogenesis.

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DUX4 and NMD form a feedback loop.(A) Schematic of potential double-negative feedback loop betweenDUX4 and NMD, in which DUX4 inhibits NMD and NMD degradesDUX4 mRNA. (B) Levels of endogenously transcribed DUX4 mRNAfollowing control treatment or ectopic DUX4 expression,measured 2 days after the initiation of myogenesis in FSHD1 (54-2) and FSHD2(MB200) cells. (C) Relative levels of transcripts from theGl-DUX4-Intron2(+) and Gl-DUX4-Intron2(−) constructs followingcontrol treatment or ectopic DUX4 expression in normalmyoblasts (54-1 cells). For each construct, data are normalized such that thesiControl treatment is 1. Error bars, standard deviation. (D)Schematic of potential model of interactions between DUX4 and NMD in healthy(top) and FSHD (bottom) muscle cells. In healthy cells, DUX4 mRNA isefficiently degraded by NMD; in FSHD cells, DUX4 triggers proteolyticdegradation of UPF1 and inhibits NMD, resulting in the accumulation of DUX4mRNA and protein.DOI:http://dx.doi.org/10.7554/eLife.04996.007
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fig4: DUX4 and NMD form a feedback loop.(A) Schematic of potential double-negative feedback loop betweenDUX4 and NMD, in which DUX4 inhibits NMD and NMD degradesDUX4 mRNA. (B) Levels of endogenously transcribed DUX4 mRNAfollowing control treatment or ectopic DUX4 expression,measured 2 days after the initiation of myogenesis in FSHD1 (54-2) and FSHD2(MB200) cells. (C) Relative levels of transcripts from theGl-DUX4-Intron2(+) and Gl-DUX4-Intron2(−) constructs followingcontrol treatment or ectopic DUX4 expression in normalmyoblasts (54-1 cells). For each construct, data are normalized such that thesiControl treatment is 1. Error bars, standard deviation. (D)Schematic of potential model of interactions between DUX4 and NMD in healthy(top) and FSHD (bottom) muscle cells. In healthy cells, DUX4 mRNA isefficiently degraded by NMD; in FSHD cells, DUX4 triggers proteolyticdegradation of UPF1 and inhibits NMD, resulting in the accumulation of DUX4mRNA and protein.DOI:http://dx.doi.org/10.7554/eLife.04996.007

Mentions: As DUX4 expression inhibits NMD and NMD degrades DUX4 mRNA, wehypothesized that DUX4 and the NMD pathway might participate in adouble-negative feedback loop (Figure 4A). Thisfeedback loop predicts that DUX4 will indirectly stabilize its own mRNA by inhibitingNMD. To test this, we ectopically expressed DUX4 in FSHD1 and FSHD2 myotubes andmeasured levels of endogenously transcribed DUX4 mRNA. Ectopic DUX4 expression led to anapproximately fivefold increase in endogenously transcribed DUX4 mRNA levels (Figure 4B). We next tested whetherDUX4's spliced 3′ UTR, which is important for NMD-mediateddegradation of DUX4 mRNA, contributed to this increase. We transfected our chimericβ-globin + DUX4 3′ UTR reporters into normalmyoblasts and ectopically expressed DUX4. Levels of the NMD-susceptibleconstruct containing the complete DUX4 3′ UTR increased1.43-fold following ectopic DUX4 expression, while levels of the construct without thesecond intron of the DUX4 3′ UTR exhibited a more modestincrease of 1.08-fold (Figure 4C). As with theUPF1 KD experiments, the chimeric construct exhibited more modesteffect sizes in these feedback loop experiments than we observed for the endogenous DUX4mRNA itself.10.7554/eLife.04996.007Figure 4.DUX4 and NMD form a feedback loop.


A feedback loop between nonsense-mediated decay and the retrogene DUX4 in facioscapulohumeral muscular dystrophy.

Feng Q, Snider L, Jagannathan S, Tawil R, van der Maarel SM, Tapscott SJ, Bradley RK - Elife (2015)

DUX4 and NMD form a feedback loop.(A) Schematic of potential double-negative feedback loop betweenDUX4 and NMD, in which DUX4 inhibits NMD and NMD degradesDUX4 mRNA. (B) Levels of endogenously transcribed DUX4 mRNAfollowing control treatment or ectopic DUX4 expression,measured 2 days after the initiation of myogenesis in FSHD1 (54-2) and FSHD2(MB200) cells. (C) Relative levels of transcripts from theGl-DUX4-Intron2(+) and Gl-DUX4-Intron2(−) constructs followingcontrol treatment or ectopic DUX4 expression in normalmyoblasts (54-1 cells). For each construct, data are normalized such that thesiControl treatment is 1. Error bars, standard deviation. (D)Schematic of potential model of interactions between DUX4 and NMD in healthy(top) and FSHD (bottom) muscle cells. In healthy cells, DUX4 mRNA isefficiently degraded by NMD; in FSHD cells, DUX4 triggers proteolyticdegradation of UPF1 and inhibits NMD, resulting in the accumulation of DUX4mRNA and protein.DOI:http://dx.doi.org/10.7554/eLife.04996.007
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Related In: Results  -  Collection

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fig4: DUX4 and NMD form a feedback loop.(A) Schematic of potential double-negative feedback loop betweenDUX4 and NMD, in which DUX4 inhibits NMD and NMD degradesDUX4 mRNA. (B) Levels of endogenously transcribed DUX4 mRNAfollowing control treatment or ectopic DUX4 expression,measured 2 days after the initiation of myogenesis in FSHD1 (54-2) and FSHD2(MB200) cells. (C) Relative levels of transcripts from theGl-DUX4-Intron2(+) and Gl-DUX4-Intron2(−) constructs followingcontrol treatment or ectopic DUX4 expression in normalmyoblasts (54-1 cells). For each construct, data are normalized such that thesiControl treatment is 1. Error bars, standard deviation. (D)Schematic of potential model of interactions between DUX4 and NMD in healthy(top) and FSHD (bottom) muscle cells. In healthy cells, DUX4 mRNA isefficiently degraded by NMD; in FSHD cells, DUX4 triggers proteolyticdegradation of UPF1 and inhibits NMD, resulting in the accumulation of DUX4mRNA and protein.DOI:http://dx.doi.org/10.7554/eLife.04996.007
Mentions: As DUX4 expression inhibits NMD and NMD degrades DUX4 mRNA, wehypothesized that DUX4 and the NMD pathway might participate in adouble-negative feedback loop (Figure 4A). Thisfeedback loop predicts that DUX4 will indirectly stabilize its own mRNA by inhibitingNMD. To test this, we ectopically expressed DUX4 in FSHD1 and FSHD2 myotubes andmeasured levels of endogenously transcribed DUX4 mRNA. Ectopic DUX4 expression led to anapproximately fivefold increase in endogenously transcribed DUX4 mRNA levels (Figure 4B). We next tested whetherDUX4's spliced 3′ UTR, which is important for NMD-mediateddegradation of DUX4 mRNA, contributed to this increase. We transfected our chimericβ-globin + DUX4 3′ UTR reporters into normalmyoblasts and ectopically expressed DUX4. Levels of the NMD-susceptibleconstruct containing the complete DUX4 3′ UTR increased1.43-fold following ectopic DUX4 expression, while levels of the construct without thesecond intron of the DUX4 3′ UTR exhibited a more modestincrease of 1.08-fold (Figure 4C). As with theUPF1 KD experiments, the chimeric construct exhibited more modesteffect sizes in these feedback loop experiments than we observed for the endogenous DUX4mRNA itself.10.7554/eLife.04996.007Figure 4.DUX4 and NMD form a feedback loop.

Bottom Line: DUX4 is a double homeobox transcription factor that is normally expressed in the testis and causes apoptosis and FSHD when misexpressed in skeletal muscle.The mechanism(s) of DUX4 toxicity in muscle is incompletely understood.We report that DUX4-triggered proteolytic degradation of UPF1, a central component of the nonsense-mediated decay (NMD) machinery, is associated with profound NMD inhibition, resulting in global accumulation of RNAs normally degraded as NMD substrates.

View Article: PubMed Central - PubMed

Affiliation: Computational Biology Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, United States.

ABSTRACT
Facioscapulohumeral muscular dystrophy (FSHD) is a muscular dystrophy caused by inefficient epigenetic repression of the D4Z4 macrosatellite array and somatic expression of the DUX4 retrogene. DUX4 is a double homeobox transcription factor that is normally expressed in the testis and causes apoptosis and FSHD when misexpressed in skeletal muscle. The mechanism(s) of DUX4 toxicity in muscle is incompletely understood. We report that DUX4-triggered proteolytic degradation of UPF1, a central component of the nonsense-mediated decay (NMD) machinery, is associated with profound NMD inhibition, resulting in global accumulation of RNAs normally degraded as NMD substrates. DUX4 mRNA is itself degraded by NMD, such that inhibition of NMD by DUX4 protein stabilizes DUX4 mRNA through a double-negative feedback loop in FSHD muscle cells. This feedback loop illustrates an unexpected mode of autoregulatory behavior of a transcription factor, is consistent with 'bursts' of DUX4 expression in FSHD muscle, and has implications for FSHD pathogenesis.

Show MeSH
Related in: MedlinePlus