RESUMO
BACKGROUND: Positioning of nucleosomes along DNA is an integral regulator of chromatin accessibility and gene expression in diverse cell types. However, the precise nature of how histone demethylases including the histone 3 lysine 4 (H3K4) demethylase, KDM5B, impacts nucleosome positioning around transcriptional start sites (TSS) of active genes is poorly understood. RESULTS: Here, we report that KDM5B is a critical regulator of nucleosome positioning in embryonic stem (ES) cells. Micrococcal nuclease sequencing (MNase-Seq) revealed increased enrichment of nucleosomes around TSS regions and DNase I hypersensitive sites in KDM5B-depleted ES cells. Moreover, depletion of KDM5B resulted in a widespread redistribution and disorganization of nucleosomes in a sequence-dependent manner. Dysregulated nucleosome phasing was also evident in KDM5B-depleted ES cells, including asynchronous nucleosome spacing surrounding TSS regions, where nucleosome variance was positively correlated with the degree of asynchronous phasing. The redistribution of nucleosomes around TSS regions in KDM5B-depleted ES cells is correlated with dysregulated gene expression, and altered H3K4me3 and RNA polymerase II occupancy. In addition, we found that DNA shape features varied significantly at regions with shifted nucleosomes. CONCLUSION: Altogether, our data support a role for KDM5B in regulating nucleosome positioning in ES cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Epigênese Genética , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Nucleossomos/químicaRESUMO
Recessive SLC25A46 mutations cause a spectrum of neurodegenerative disorders with optic atrophy as a core feature. We report a patient with optic atrophy, peripheral neuropathy, ataxia, but not cerebellar atrophy, who is on the mildest end of the phenotypic spectrum. By studying seven different nontruncating mutations, we found that the stability of the SLC25A46 protein inversely correlates with the severity of the disease and the patient's variant does not markedly destabilize the protein. SLC25A46 belongs to the mitochondrial transporter family, but it is not known to have transport function. Apart from this possible function, SLC25A46 forms molecular complexes with proteins involved in mitochondrial dynamics and cristae remodeling. We demonstrate that the patient's mutation directly affects the SLC25A46 interaction with MIC60. Furthermore, we mapped all of the reported substitutions in the protein onto a 3D model and found that half of them fall outside of the signature carrier motifs associated with transport function. We thus suggest that there are two distinct molecular mechanisms in SLC25A46-associated pathogenesis, one that destabilizes the protein while the other alters the molecular interactions of the protein. These results have the potential to inform clinical prognosis of such patients and indicate a pathway to drug target development.
Assuntos
Ataxia/genética , Proteínas Mitocondriais/genética , Atrofia Óptica/genética , Doenças do Sistema Nervoso Periférico/genética , Proteínas de Transporte de Fosfato/genética , Polimorfismo de Nucleotídeo Único , Criança , Estudos de Associação Genética , Humanos , Masculino , Dinâmica Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Linhagem , Proteínas de Transporte de Fosfato/química , Proteínas de Transporte de Fosfato/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Dominant optic atrophy (DOA) and axonal peripheral neuropathy (Charcot-Marie-Tooth type 2, or CMT2) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively. In yeast, homologs of OPA1 (Mgm1) and MFN2 (Fzo1) work in concert with Ugo1, for which no human equivalent has been identified thus far. By whole-exome sequencing of patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein mitofilin (Fcj1). Loss of function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2 while exemplifying a new class of modified solute transporters linked to mitochondrial dynamics.