Deformed epidermal autoregulatory factor -1 (DEAF-1) is a transcription factor mapping to the chromosomal region 11p15.5, a region associated with loss of heterozygosity (LOH) in human cancers. Potential DEAF-1 binding motifs were identified in the PTEN promoter and the ability of DEAF-1 to regulate PTEN gene expression was investigated. DEAF-1 increased transcription 10-14 fold with PTEN sequences between -429 and -221, while mutations in the DNA binding domain (DEAF-ADWA) and nuclear localization signal of DEAF-1 abolished this increase. DEAF-1 was shown to bind sequences between -339 and -233. Rabdomyosarcoma (RD) stable cell lines with inducible expression of DEAF-1 and DEAF-1-ADWA were produced. Increased DEAF-1 expression had no significant effects on PTEN RNA expression or cell proliferation when compared to controls, but did increase susceptibility to UV-induced apoptosis. These studies suggest that DEAF-1 may contribute to the regulation of PTEN gene expression, but other factors may play a more significant role.

Deformed Epidermal Autoregulatory Factor-1 (DEAF-1) is a transcription factor that binds TTCG motifs and has roles in fetal development, clinical depression and cancer. In order to further our understanding of the DEAF-1 protein, this study characterizes previously unidentified DEAF-1 interacting proteins and post-translational modifications of DEAF-1. A region encompassing the DNA binding domain of DEAF-1 interacts with the C-terminal Bax interacting domain of the Ku70 subunit of the DNA-PK holoenzyme. Ku70 acts as an anti-apoptotic protein through C-terminal domain and so DEAF-1 was assessed for its ability to influence apoptosis after various stimuli. DEAF-1 acted as a pro-apoptotic protein after intrinsic stimuli. Apoptotic activities occurred through a nuclear, DNA independent mechanism and a mutation that eliminated Ku70 interactions also inhibited DEAF-1 pro-apoptotic activities. Analysis of mammalian purified DEAF-1 indicated a number of phosphorylation sites and also a methylated arginine residue. Various assays were performed on mutated forms of DEAF-1 to determine the significance of the modified sites on DEAF-1 functions and properties. Lysine mutation of the methylated arginine site appeared to augment protein-protein interactions with itself and also Ku70. Alanine mutations at three of the identified phosphorylation sites increased DEAF-1 pro-apoptotic activities. In vitro kinase assays identified CDK5 as potential kinase that can phosphorylate DEAF-1. These studies provide new insight into potential functions, properties, and regulation of DEAF-1.

The transcription factor DEAF-1 is the mammalian homologue of a critical Drosophila developmental gene and is essential for neonatal survival in mice. Haploinsufficiency of Deaf-1 in the testis of adult mice was initially thought to cause loss of spermatogenesis and disrupted morphology of the seminiferous tubules, and this heterozygosity was thought to be sufficient to disrupt epigenetic programming in the developing sperm to produce inheritance of testicular defects in both heterozygous and genotypically normal offspring. Although Deaf-1 knockout mice do display disrupted testis structure, infertility at advanced age, hyperproliferation of early germ cells, and abnormal staging of seminiferous tubules, this phenotype was also observed in normal mouse strains that were born in the SIUC vivarium. Mice ordered from a vendor and raised at SIUC did not show testicular defects. This suggests an environmental factor at the SIUC vivarium may act as an endocrine disruptor during embryonic testicular development. Deaf-1-/- mice die soon after birth, often as the result of exencephaly, a gross neural tube defect (NTD). Unlike many mouse models, exencephalic Deaf-1-/- mice do not display a higher incidence of NTDs in females as compared to their male littermates. DEAF-1 promotes Bax-mediated apoptosis; studies using terminal UTP nick-end labeling (TUNEL) suggest a global increase in apoptosis in both exencephalic and normal Deaf-1-/- fetuses during neurulation as compared to their Deaf-1+/+ littermates. This indicates that Deaf-1 is crucial for correct apoptotic patterning during development, which, in turn, is essential for neural tube closure. Finally, cDNA microarray comparison of e14.5 Deaf-1 knockout and wildtype fetuses reveals expression of translation initiation factor 4g3 (Eif4g3) to be downregulated in Deaf-1-/- fetuses. Electrophoretic mobility shift assay using recombinant DEAF-1, and chromatin immunoprecipitation assay of a human cell line confirmed DEAF-1 could bind the eIF4G3 promoter both in vitro and in vivo. Additionally, transcription of the Deaf-1 Antisense Transcript (Das) was found to be significantly downregulated in both e14.5 fetuses and e18.5 fetal brains from Deaf-1-/- mice, suggesting that either lack of Deaf-1 protein or lack of exons 2 through 5 in Deaf-1 knockout mice causes changes in levels of the noncoding RNA that shares Deaf-1's promoter in the mouse.