Neuronal differentiation alters the ratio of Sp transcription factors recruited to the CYP46A1 promoter

CYP46A1 is a neuron-specific cytochrome P450 that plays a pivotal role in maintaining cholesterol homeostasis in the CNS. However, the molecular mechanisms underlying human CYP46A1 expression are still poorly understood, partly because of the lack of a cellular model that expresses high levels of CYP46A1. Our previous studies demonstrated that specificity protein (Sp) transcription factors control CYP46A1 expression, and are probably responsible for cell-type specificity. Herein, we have differentiated Ntera2/cloneD1 cells into post-mitotic neurons and identified for the first time a human cell model that expresses high levels of CYP46A1 mRNA. Our results show a decrease in Sp1 protein levels, concomitant with the increase in CYP46A1 mRNA levels. This decrease was correlated with changes in the ratio of Sp proteins associated to the CYP46A1 proximal promoter. To examine if the increase in (Sp3 + Sp4)/Sp1 ratio was observed in other Sp-regulated promoters, we have selected four genes - reelin, glutamate receptor subunit zeta-1, glutamate receptor subunit epsilon-1 and μ-opioid receptor - known to be expressed in the human brain and analyzed the Sp proteins binding pattern to the promoter of these genes, in undifferentiated and differentiated Ntera2/cloneD1. Our data indicate that the dissociation of Sp1 from promoter regions is a common feature amongst Sp-regulated genes that are up-regulated after neuronal differentiation. Brain cholesterol elimination depends on CYP46A1 neuronal-specific expression, yet the lack of a human cellular model which expresses high levels of CYP46A1, has hindered the study of the molecular pathways involved in neuronal cholesterol homeostasis. Herein, we demonstrated a significant increase in CYP46A1 mRNA levels after differentiation of NT2 cells. In parallel, we observed a decrease in Sp1 protein levels associated with the proximal promoter of this gene, and with the promoter regions of other Sp-regulated genes that are induced after neuronal differentiation. Our results imply that physio- or pathological conditions with alterations in Sp1 protein levels may have important consequences on neuronal gene expression and fate.