RESUMO
Mutations in the human gene ALMS1 cause Alström syndrome, a rare progressive condition characterized by neurosensory degeneration and metabolic defects. ALMS1 protein localizes to the centrosome and has been implicated in the assembly and/or maintenance of primary cilia; however its precise function, distribution within the centrosome, and mechanism of centrosomal recruitment are unknown. The C-terminus of ALMS1 contains a region with similarity to the uncharacterized human protein C10orf90, termed the ALMS motif. Here, we show that a third human protein, the candidate centrosomal protein KIAA1731, contains an ALMS motif and that exogenously expressed KIAA1731 and C10orf90 localize to the centrosome. However, based on deletion analysis of ALMS1, the ALMS motif appears unlikely to be critical for centrosomal targeting. RNAi analyses suggest that C10orf90 and KIAA1731 have roles in primary cilium assembly and centriole formation/stability, respectively. We also show that ALMS1 localizes specifically to the proximal ends of centrioles and basal bodies, where it colocalizes with the centrosome cohesion protein C-Nap1. RNAi analysis reveals markedly diminished centrosomal levels of C-Nap1 and compromised cohesion of parental centrioles in ALMS1-depleted cells. In summary, these data suggest centrosomal functions for C10orf90 and KIAA1731 and new centriole-related functions for ALMS1.
Assuntos
Centríolos/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Proteínas/fisiologia , Síndrome de Alstrom/genética , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Linhagem Celular , Centríolos/genética , Centríolos/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Sequência Conservada , Proteínas do Citoesqueleto , Deleção de Genes , Expressão Gênica , Células HEK293 , Humanos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Mutations in the human gene ALMS1 cause Alström syndrome, a disorder characterised by neurosensory degeneration, metabolic defects and cardiomyopathy. ALMS1 encodes a centrosomal protein implicated in the assembly and maintenance of primary cilia. Expression of ALMS1 varies between tissues and recent data suggest that its transcription is modulated during adipogenesis and growth arrest. However the ALMS1 promoter has not been defined. This study focused on identifying and characterising the ALMS1 proximal promoter, initially by using 5' RACE to map transcription start sites. Luciferase reporter assay and EMSA data strongly suggest that ALMS1 transcription is regulated by the ubiquitous factor Sp1. In addition, reporter assay, EMSA, chromatin immunoprecipitation and RNA interference data indicate that ALMS1 transcription is regulated by regulatory factor X (RFX) proteins. These transcription factors are cell-type restricted in their expression profile and known to regulate genes of the ciliogenic pathway. We show binding of RFX proteins to an evolutionarily conserved X-box in the ALMS1 proximal promoter and present evidence that these proteins are responsible for ALMS1 transcription during growth arrest induced by low serum conditions. In summary, this work provides the first data on transcription factors regulating general and context-specific transcription of the disease-associated gene ALMS1.
Assuntos
Síndrome de Alstrom/genética , Proteínas de Ligação a DNA/fisiologia , Regiões Promotoras Genéticas , Proteínas/genética , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Sítio de Iniciação de Transcrição , Adulto , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Mutação , Interferência de RNA , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Hypoplastic left heart (HLH) occurs in at least 1 in 10 000 live births but may be more common in utero. Its causes are poorly understood but a number of affected cases are associated with chromosomal abnormalities. We set out to localize the breakpoints in a patient with sporadic HLH and a de novo translocation. Initial studies showed that the apparently simple 1q41;3q27.1 translocation was actually combined with a 4-Mb inversion, also de novo, of material within 1q41. We therefore localized all four breakpoints and found that no known transcription units were disrupted. However we present a case, based on functional considerations, synteny and position of highly conserved non-coding sequence elements, and the heterozygous Prox1(+/-) mouse phenotype (ventricular hypoplasia), for the involvement of dysregulation of the PROX1 gene in the aetiology of HLH in this case. Accordingly, we show that the spatial expression pattern of PROX1 in the developing human heart is consistent with a role in cardiac development. We suggest that dysregulation of PROX1 gene expression due to separation from its conserved upstream elements is likely to have caused the heart defects observed in this patient, and that PROX1 should be considered as a potential candidate gene for other cases of HLH. The relevance of another breakpoint separating the cardiac gene ESRRG from a conserved downstream element is also discussed.