RESUMO
The regulation of nucleosome positioning and composition by ATP-dependent chromatin remodeling enzymes and their associated binding partners plays important biological roles in mammals. CECR2 is a binding partner to the ISWI (imitation switch) ATPase SNF2L/SMARCA1 and is involved in neural tube closure and inner ear development; however, its functions in adult tissues have not been examined. Here, we report that CECR2 contributes to spermatogenesis and forms a complex that includes the other ISWI ATPase SNF2H/SMARCA5 in the testis. Cecr2 mutant males non-penetrant for neural tube defects sired smaller litters than wild-type males. Strikingly, while we found that Cecr2 mutants have normal seminiferous epithelium morphology, sperm count, motility, and morphology, the mutant spermatozoa were compromised in their ability to fertilize oocytes. Investigation of CECR2/ISWI complexes in the testis showed that SNF2H interacted with CECR2, and this interaction was also observed in embryonic stem cells, suggesting that CECR2 may interact with SNF2H or SNF2L depending on the cell type. Finally, we found that Cecr2 mutants exhibit misregulation of the homeobox transcription factor Dlx5 in the testis, suggesting that CECR2 complexes may regulate gene expression during spermatogenesis. Taken together, our results demonstrate a novel role of CECR2-containing complexes in spermatogenesis and show that CECR2 interacts predominantly with SNF2H instead of SNF2L in the testis.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Espermatogênese , Testículo/metabolismo , Adenosina Trifosfatases/genética , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Epitélio Seminífero/citologia , Epitélio Seminífero/metabolismo , Contagem de Espermatozoides , Motilidade dos Espermatozoides , Espermatozoides/citologia , Espermatozoides/metabolismo , Testículo/citologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Over 200 mouse genes are associated with neural tube defects (NTDs), including Cecr2, the bromodomain-containing subunit of the CERF chromatin remodeling complex. METHODS: Gene-trap mutation Cecr2(Gt45Bic) results in 74% exencephaly (equivalent of human anencephaly) on the BALB/c strain. Gene expression altered during cranial neural tube closure by the Cecr2 mutation was identified through microarray analysis of 11-14 somites stage Cecr2(Gt45Bic)embryos. RESULTS: Analysis of Affymetrix Mouse 430 2.0 chips detected 60 transcripts up-regulated and 54 transcripts down-regulated in the Cecr2(Gt45Bic) embryos (fold > 1.5, p < 0.05). The Cecr2 transcript was reduced only approximately 7- to 14-fold from normal levels, suggesting the Cecr2(Gt45Bic) is a hypomorphic mutation. We therefore generated a novel Cecr2 null allele (Cecr2 (tm1.1Hemc)). Resulting mutants displayed a stronger penetrance of exencephaly than Cecr2(Gt45Bic) in both BALB/c and FVB/N strains, in addition to midline facial clefts and forebrain encephalocele in the FVB/N strain. The Cecr2 transcript is reduced 260-fold in the Cecr2(tm1.1Hemc) line. Subsequent qRT-PCR using Cecr2 (tm1.1Hemc) mutant heads confirmed downregulation of transcription factors Alx1/Cart1, Dlx5, Eya1, and Six1. CONCLUSIONS: As both Alx1/Cart1 and Dlx5 mouse mutations result in exencephaly, we hypothesize that changes in expression of these mesenchymal/ectodermal transcription factors may contribute to NTDs associated with Cecr2.
