RESUMEN
By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for ß propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.
Asunto(s)
Cromosomas Humanos Par 10/genética , Proteínas de Homeodominio/genética , Hipogonadismo/genética , Síndrome de Kallmann/genética , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Pubertad/genética , Factores de Transcripción/genética , Translocación Genética/genética , Adolescente , Animales , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Hibridación in Situ , Hibridación Fluorescente in Situ , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Análisis por Micromatrices , Mutación Missense/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos Híbridos , Pez CebraRESUMEN
The Krüppel-associated box-containing zinc finger gene family (KRAB-ZNF) is one of the largest gene families of transcriptional factors in the human genome. Although the functions of most of these genes remain to be determined, it is known that KRAB-mediated transcriptional repression requires a direct interaction with the KAP1 co-repressor. By mammalian one- or two-hybrid experiments in HEK293 cells, we compared transcriptional repression activities of 61 human KRAB-ZNFs. The results showed that six SCAN-KRAB-containing ZNFs are KAP1-independent transcriptional repressors whose SCAN-KRAB domain is unable to associate with KAP1 despite retaining transcriptional repression activity. Transcriptional repression activities of the SCAN-KRAB domain of KAP1-independent KRAB-ZNFs are not influenced by depletion of endogenous KAP1 levels by small interfering RNA. Although the mechanism by which KAP1-independent KRAB-ZNFs repress transcriptional activity remains to be elucidated, it appears that there may be a pathway for transcriptional repression that does not involve KAP1. These results provide new insight into the functions of the members of the KRAB-ZNF family.
Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Transcripción Genética , Dedos de Zinc/genética , Línea Celular , Humanos , Proteínas Represoras/metabolismo , Proteína 28 que Contiene Motivos TripartitoRESUMEN
In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1,929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells. Results thus obtained have demonstrated that our Flexi ORF clones are efficient for the production of HaloTag-fusion proteins that can provide a new versatile set for a variety of functional analyses of human genes.