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1.
iScience ; 26(10): 107823, 2023 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-37744032

RESUMO

Double homeobox (DUX) genes are unique to eutherian mammals, expressed transiently during zygotic genome activation (ZGA) and involved in facioscapulohumeral muscular dystrophy (FSHD) and cancer when misexpressed. We evaluate the 3 human DUX genes and the ancestral single homeobox gene sDUX from the non-eutherian mammal, platypus, and find that DUX4 cytotoxicity is not shared with DUXA or DUXB, but surprisingly is shared with platypus sDUX, which binds DNA as a homodimer and activates numerous ZGA genes and long terminal repeat (LTR) elements. DUXA, although transcriptionally inactive, has DNA binding overlap with DUX4, and DUXA-VP64 activates DUX4 targets and is cytotoxic. DUXA competition antagonizes the activity of DUX4 on its target genes, including in FSHD patient cells. Since DUXA is a DUX4 target gene, this competition potentiates feedback inhibition, constraining the window of DUX4 activity. The DUX gene family therefore comprises antagonistic members of opposing function, with implications for their roles in ZGA, FSHD, and cancer.

2.
bioRxiv ; 2023 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-36711898

RESUMO

Double homeobox (DUX) genes are unique to eutherian mammals and normally expressed transiently during zygotic genome activation. The canonical member, DUX4, is involved in facioscapulohumeral muscular dystrophy (FSHD) and cancer, when misexpressed in other contexts. We evaluate the 3 human DUX genes and the ancestral single homeobox gene sDUX from the non-eutherian mammal, platypus, and find that DUX4 activities are not shared with DUXA or DUXB, which lack transcriptional activation potential, but surprisingly are shared with platypus sDUX. In human myoblasts, platypus sDUX drives cytotoxicity, inhibits myogenesis, and induces DUX4 target genes, particularly those associated with zygotic genome activation (ZGA), by binding DNA as a homodimer in a way that overlaps the DUX4 homeodomain crystal structure. DUXA lacks transcriptional activity but has DNA-binding and chromatin accessibility overlap with DUX4 and sDUX, including on ZGA genes and LTR elements, and can actually be converted into a DUX4-like cytotoxic factor by fusion to a synthetic transactivation domain. DUXA competition antagonizes the activity of DUX4 on its target genes, including in FSHD patient cells. Since DUXA is an early DUX4 target gene, this activity potentiates feedback inhibition, constraining the window of DUX4 activity. The DUX gene family therefore comprises cross-regulating members of opposing function, with implications for their roles in ZGA, FSHD, and cancer. HIGHLIGHTS: Platypus sDUX is toxic and inhibits myogenic differentiation.DUXA targets overlap substantially with those of DUX4.DUXA fused to a synthetic transactivation domain acquires DUX4-like toxicity.DUXA behaves as a competitive inhibitor of DUX4.

3.
Int J Mol Sci ; 23(4)2022 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-35216102

RESUMO

FSHD is caused by loss of silencing of the DUX4 gene, but the DUX4 protein has not yet been directly detected immunohistologically in affected muscle, raising the possibility that DUX4 expression may occur at time points prior to obtaining adult biopsies for analysis, with consequent perturbations of muscle being responsible for disease progression. To test the extent to which muscle can regenerate following DUX4-mediated degeneration, we employed an animal model with reversible DUX4 expression, the iDUX4pA;HSA mouse. We find that muscle histology does recover substantially after DUX4 expression is switched off, with the extent of recovery correlating inversely with the duration of prior DUX4 expression. However, despite fairly normal muscle histology, and recovery of most cytological parameters, the fibroadipogenic progenitor compartment, which is significantly elevated during bouts of fiber-specific DUX4 expression, does not return to basal levels, even many weeks after a single burst of DUX4 expression. We find that muscle that has recovered from a DUX4 burst acquires a propensity for severe fibrosis, which can be revealed by subsequent cardiotoxin injuries. These results suggest that a past history of DUX4 expression leads to maintained pro-fibrotic alterations in the cellular physiology of muscle, with potential implications for therapeutic approaches.


Assuntos
Fibrose/genética , Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Adipogenia/genética , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Músculo Esquelético/patologia
4.
Oncogenesis ; 10(10): 68, 2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34642317

RESUMO

CIC-DUX4 sarcoma (CDS) is a highly aggressive and metastatic small round type of predominantly pediatric sarcoma driven by a fusion oncoprotein comprising the transcriptional repressor Capicua (CIC) fused to the C-terminal transcriptional activation domain of DUX4. CDS rapidly develops resistance to chemotherapy, thus novel specific therapies are greatly needed. We demonstrate that CIC-DUX4 requires P300/CBP to induce histone H3 acetylation, activate its targets, and drive oncogenesis. We describe the synthetic route to a selective and highly potent P300/CBP inhibitor named iP300w and related stereoisomers, and find that iP300w efficiently suppresses CIC-DUX4 transcriptional activity and reverses CIC-DUX4 induced acetylation. iP300w is active at 100-fold lower concentrations than related stereoisomers or A-485. At low doses, iP300w shows specificity to CDS cancer cell lines, rapidly inducing cell cycle arrest and preventing growth of established CDS xenograft tumors when delivered in vivo. The effectiveness of iP300w to inactivate CIC-DUX4 highlights a promising therapeutic opportunity for CDS.

5.
J Clin Invest ; 130(5): 2465-2477, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32250341

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is caused by loss of repression of the DUX4 gene; however, the DUX4 protein is rare and difficult to detect in human muscle biopsies, and pathological mechanisms are obscure. FSHD is also a chronic disease that progresses slowly over decades. We used the sporadic, low-level, muscle-specific expression of DUX4 enabled by the iDUX4pA-HSA mouse to develop a chronic long-term muscle disease model. After 6 months of extremely low sporadic DUX4 expression, dystrophic muscle presented hallmarks of FSHD histopathology, including muscle degeneration, capillary loss, fibrosis, and atrophy. We investigated the transcriptional profile of whole muscle as well as endothelial cells and fibroadiopogenic progenitors (FAPs). Strikingly, differential gene expression profiles of both whole muscle and, to a lesser extent, FAPs, showed significant overlap with transcriptional profiles of MRI-guided human FSHD muscle biopsies. These results demonstrate a pathophysiological similarity between disease in muscles of iDUX4pA-HSA mice and humans with FSHD, solidifying the value of chronic rare DUX4 expression in mice for modeling pathological mechanisms in FSHD and highlighting the importance FAPs in this disease.


Assuntos
Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/metabolismo , Transcrição Gênica , Animais , Modelos Animais de Doenças , Células Progenitoras Endoteliais/patologia , Feminino , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/patologia
6.
Sci Adv ; 5(9): eaaw7781, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31535023

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) results from mutations causing overexpression of the transcription factor, DUX4, which interacts with the histone acetyltransferases, EP300 and CBP. We describe the activity of a new spirocyclic EP300/CBP inhibitor, iP300w, which inhibits the cytotoxicity of the DUX4 protein and reverses the overexpression of most DUX4 target genes, in engineered cell lines and FSHD myoblasts, as well as in an FSHD animal model. In evaluating the effect of iP300w on global histone H3 acetylation, we discovered that DUX4 overexpression leads to a dramatic global increase in the total amount of acetylated histone H3. This unexpected effect requires the C-terminus of DUX4, is conserved with mouse Dux, and may facilitate zygotic genome activation. This global increase in histone H3 acetylation is reversed by iP300w, highlighting the central role of EP300 and CBP in the transcriptional mechanism underlying DUX4 cytotoxicity and the translational potential of blocking this interaction.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Mioblastos/patologia , Acetilação , Animais , Morte Celular , Células Cultivadas , Modelos Animais de Doenças , Proteína p300 Associada a E1A/genética , Feminino , Histonas/genética , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/metabolismo , Mioblastos/metabolismo , Processamento de Proteína Pós-Traducional
7.
Sci Rep ; 8(1): 16957, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30446688

RESUMO

Loss of silencing of the DUX4 gene on chromosome 4 causes facioscapulohumeral muscular dystrophy. While high level DUX4 expression induces apoptosis, the effects of low level DUX4 expression on human myogenic cells are not well understood. Low levels and sporadic expression of DUX4 have been reported in FSHD biopsy samples and myoblast cultures. Here, we show that a large set of human myogenic genes is rapidly deregulated by DUX4, including MYOD1 and MYF5, which are efficiently repressed even by low, non-toxic levels of DUX4. Human myoblasts modified to express low nontoxic levels of DUX4 were significantly impaired from differentiating into myotubes in vitro. Surprisingly, inhibition of differentiation does not require the transcriptional activation domain, thus is likely a feature of all mammalian DUX genes. DUX4 does not bind near the MYF5 gene, but has a prominent ChIP-seq peak within the MYF5 -118 kb enhancer. We find that when DUX4 binds at this site, it directs enhancer activity towards a nearby transcriptional start site for a noncoding nonfunctional RNA we name DIME (DUX4-induced MYF5 enhancer) transcript. These data highlight the anti-myogenic properties of DUX4 in human myogenic progenitor cells, and provide an example of enhancer disruption in the downregulation of MYF5.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Sequência de Bases , Proteínas de Homeodomínio/metabolismo , Humanos , Fibras Musculares Esqueléticas/citologia , Proteína MyoD/genética , Mioblastos/citologia , Fator Regulador Miogênico 5/genética
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