Deformed epidermal autoregulatory factor 1 (DEAF1) is a transcription factor that binds to (T/C)TCG(G/T) half-sites and has been shown to be involved in human diseases of cancer, diabetes, depression and intellectual disorders. We used chromatin immunoprecipitation assays to assess endogenous levels of DEAF1 and RNA polymerase II occupancy on the promoter and 5'UTR of the DEAF1 gene. In exponentially growing HEK293 cells, low levels of DEAF1 bind to sequences between -718 and +232, with +1 marking the start of translation. Within 0.5 hr of treating the cells with 500 µM H2O2, DEAF1 occupancy is increased between 7-18 fold at B (-718/-569), -577/-444, C (-432/-299), D (-205/-112) and E(-97/17). There were no statistically significant changes in either RNA polymerase II phospho-serine 5 (RNA PolII pS5) or RNA polymerase II phospho-serine 2 (RNA PolII pS2) binding with H2O2 treatment compared to control. With media change, there is an increase in RNA PolII pS2 and pS5 occupancy at both a distal site -1462/-1326 and in the coding region at 133/232, while no significant change in DEAF1 occupancy was detected. DEAF1 occupancy at the DEAF1 promoter and 5'UTR are inversely correlated with RNA polymerase II occupancy, however, there were no measurable differences in DEAF1 RNA levels at 0.5 hr and 1 hr time points. In summary, these data indicate that there is increased occupancy of DEAF1 at its own promoter following stress, which inversely affects occupancy of RNA polymerase at proximal promoter and 5'UTR sites of the DEAF1 gene.

Deformed epidermal autoregulatory factor 1 (DEAF1) encodes a transcription factor essential in early embryonic and neuronal development. In humans, mutations in the DNA binding domain of DEAF1 cause intellectual disability together with clinical characteristics collectively termed DEAF1-associated neurodevelopmental disorders (DAND). The objective of this study is to 1) assess the pathogenicity of newly identified variants using established functional assays, and 2) confirm and map the interaction domain of DEAF1 with HDAC1 and evaluate the importance of DEAF1-HDAC1 interaction on DEAF1-mediated transcriptional repression. Exome sequencing analysis identified six de novo DEAF1 mutations (p.D200Y, p.S201R, p.K250E, p.D251N, p.K253E, and p.F297S). Promoter activity experiments indicate DEAF1 transcriptional repression activity was altered by p.K250E, p.K253E, and p.F297S. Transcriptional activation activity was altered by p.K250E, p.K253E, p.F297S, and p.D251N. Combined with clinical phenotype of the patients, this work establishes the pathogenicity of new DEAF1 variants. Previous studies identified a potential protein interaction between DEAF1 and several proteins of the nucleosome remodeling and deacetylating (NuRD) complex including Histone Deacetylase 1 (HDAC1), Retinoblastoma Binding Protein 4 (RBBP4), Methyl CpG Binding Domain Protein 3 (MBD3). GST pull-down and co-immunoprecipitation (CoIP) assays confirmed and mapped the interaction with HDAC1 between amino acids 113 – 176 of DEAF1. To determine whether DEAF1-mediated repression requires HDAC1 activity, HEK293t wild type or CRISPR/Cas9-mediated DEAF1 knockout cells were treated with the HDAC inhibitor Trichostatin A (TSA). Interestingly, this study demonstrates that the requirement of HDAC1 activity on DEAF1-mediated transcriptional repression activity is target gene specific and expands our understanding of DEAF1 mediated transcriptional repression.

DEAF1 is a transcription factor linked to suicide and depression and is recurrently mutated in non-syndromic intellectual disorder (ID). In humans with major depressive disorder (MDD), DEAF1 is reported to have altered expression in the prefrontal cortex and the dorsal raphe nucleus of females but not males, and therefore may function in sex-specific depression. Standard (whole body) knockout of Deaf1 has been reported to alter 5-hydroxytryptamine (serotonin) receptor 1A (Htr1a) levels in the frontal cortex and dorsal raphe nucleus. We hypothesized that mice with targeted deletion of Deaf1 in brain would produce behavior phenotypes analogous to human MDD and ID, and that changes in Deaf1-target gene expression would be associated with behavior changes. To test this hypothesis, we produced a mouse line to allow conditional gene targeting of exons 2-5 (Deaf1-flox). Deaf1-flox mice were bred to congenic status onto a C57BL/6 background, and were then bred to mice transgenic for the Nestin-Cre gene to produce embryonic knockout of Deaf1 in neuronal precursor cells. Adult mice were tested for anxiety behavior using the Elevated Plus Maze and Open Field Exploration tests, and were tested for depression-like behavior using the Porsolt forced swim and sucrose preference tests. Relative to control mice, both male and female mice with homozygous deletion of Deaf1 in brain displayed increased anxiety measured by the anxiety tests, and no differences were measured in tests for depression-like behavior. Rotarod testing showed no deficits in motor skills of male mice. Learning and memory in mice were tested using Morris Water Maze (MWM) and fear conditioning. No change in long-term memory in male mice was observed in MWM, but both male and female mice lacking Deaf1 displayed severe deficits in fear conditioning tests. Eif4g3 and Tmem80 are target genes of Deaf1 and decreased mRNA levels were observed for Eif4g3 and Tmem80 throughout the brain of Deaf1 knockout mice, with no change in Htr1a mRNA levels. Our results demonstrate that conditional knockout of Deaf1 in neuronal precursors produces anxiety behavior and deficits in learning and memory in adult mice, potentially without changes in Htr1a mRNA levels, and that this mouse model may be useful in understanding the molecular mechanisms underlying MDD and ID in humans.