Cloning and characterization of a novel alternatively spliced transcript of the human CHD7 putative helicase

Abstract Background The CHD7 (Chromodomain Helicase DNA binding protein 7) gene encodes a member of the chromodomain family of ATP-dependent chromatin remodeling enzymes. Mutations in the CHD7 gene are found in individuals with CHARGE, a syndrome characterized by multiple birth malformations in several tissues. CHD7 was identified as a binding partner of PBAF complex (Polybromo and BRG Associated Factor containing complex) playing a central role in the transcriptional reprogramming process associated to the formation of multipotent migratory neural crest, a transient cell population associated with the genesis of various tissues. CHD7 is a large gene containing 38 annotated exons and spanning 200 kb of genomic sequence. Although genes containing such number of exons are expected to have several alternative transcripts, there are very few evidences of alternative transcripts associated to CHD7 to date indicating that alternative splicing associated to this gene is poorly characterized. Findings Here, we report the cloning and characterization by experimental and computational studies of a novel alternative transcript of the human CHD7 (named CHD7 CRA_e), which lacks most of its coding exons. We confirmed by overexpression of CHD7 CRA_e alternative transcript that it is translated into a protein isoform lacking most of the domains displayed by the canonical isoform. Expression of the CHD7 CRA_e transcript was detected in normal liver, in addition to the DU145 human prostate carcinoma cell line from which it was originally isolated. Conclusions Our findings indicate that the splicing event associated to the CHD7 CRA_e alternative transcript is functional. The characterization of the CHD7 CRA_e novel isoform presented here not only sets the basis for more detailed functional studies of this isoform, but, also, contributes to the alternative splicing annotation of the CHD7 gene and the design of future functional studies aimed at the elucidation of the molecular functions of its gene products.

We are deeply grateful to Zizi de Medonça, Sandra Regina de Souza, Débora Cristina da Costa, Ricardo Krett de Oliveira, Helena Medeiros and Priscila Baptista Audine for excellent technical assistance and to our lab colleagues for discussions and criticisms. We also thank Dr. Juan Carlos Bustos Valenzuela for generously providing the modified pLVEGFP lentiviral vector and Dr. Ana Cláudia Oliveira Carreira and her undergraduate trainee Fernanda Marques Câmara Sodré for helping with the NOTCH2 RTPCR.

We are deeply grateful to Zizi de Medonça, Sandra Regina de Souza, Débora Cristina da Costa, Ricardo Krett de Oliveira, Helena Medeiros and Priscila Baptista Audine for excellent technical assistance and to our lab colleagues for discussions and criticisms. We also thank Dr. Juan Carlos Bustos Valenzuela for generously providing the modified pLV-EGFP lentiviral vector and Dr. Ana Cláudia Oliveira Carreira and her undergraduate trainee Fernanda Marques Câmara Sodré for helping with the NOTCH2 RT-PCR.

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP) and the Universtity of São Paulo (USP). MAAD holds a post-doctoral fellowship from the Brazilian National Research Council (CNPq) and FST held an undergraduate trainee fellowship from the São Paulo State Research Foundation (FAPESP).

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP) and the Universtity of São Paulo (USP). MAAD holds a postdoctoral fellowship from the Brazilian National Research Council (CNPq) and FST held an undergraduate trainee fellowship from the São Paulo State Research Foundation (FAPESP).

The funding sources had no involvement in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

The funding sources had no involvement in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.