TFII-I isoforms act as a co-regulator potentiating Nurr1-induced human TH expression during dopaminergic neurogenesis

Abstract

Nuclear receptor related 1 protein (Nurr1) plays a vital role in development and maintenance of midbrain dopaminergic (mDA) neurons. Our previous study showed that Nurr1 actively represses human tyrosine hydroxylase (hTH) transcription in human neural stem cells (hNSCs), while it activates hTH expression via Nurr1 binding element (NBRE-A) in dopaminergic (DA) neuronal cells. To identify the interacting protein partners of Nurr1 to regulate hTH expression during DA neurogenesis, we performed DNA pulldown assay and identified TFII-I, a multifunctional transcription factor having four spliced isoforms. Polymerase chain reaction analysis of midbrain of embryonic mice from E9.5 to E13.5 showed that TFII-I expression switched from TFII-I∆ to TFII-Iγ isoform. TFII-IΔ preferentially interacts with SUMOylated Nurr1 and occupies hTH promoter in hNSCs, which resulted in repression of TH promoter activity. In contrast, TFII-Iγ interacts with Nurr1 on hTH promoter and enhanced hTH promoter activity in DA cells. Two TFII-I binding sites, an enhancer box (E-Box) and an Initiator element (Inr) flanking upstream and downstream of the NBRE-A respectively are present in conserved region among human, mouse and rat. In addition, ELM analysis and immunoprecipitation showed that only TFII-I∆ modified by SUMO1 at two position, K221 and K240 in putative motifs. So, we investigated the possibility whether Nurr1 and TFII-I∆ could form a repressor complex around NBRE-A on hTH promoter in hNSC-specific manner. TFII-I∆ majorly localized in nucleus while TFII-Iγ in cytoplasm of hNSCs. In addition, SUMO modified TFII-I∆ in a transcriptional complex with SUMOylated Nurr1 on the NBRE-A element, makes a complex in functional synergic control (SC) motifs and represses hTH promoter activity in hNSCs. Furthermore, SUMO deficient K221R, K240R and K221/240R forms of TFII-I Δ showed enhanced binding and resulted in hTH activation in hNSCs only. Mutation of E-box and Inr showed that TFII-I∆ represses hTH activity via Inr. Collectively, our data shows that TFII-I∆ isoform is modified by SUMO1 at SUMO consensus motifs overlapping SC motif exhibiting a strong contact with SUMO-2 modified Nurr1 leads to recruitment of synergic control factors (SCFs) and corepressors to hTH promoter resulting in repressed hTH activity in hNSCs. We further showed that de-SUMOylation did not interfere with nuclear-cytoplasmic transport rather exhibited enhanced affinity to hTH promoter and thus promoted its transactivation. Our findings contribute to understanding of specific expression of TFII-I isoforms in midbrain of mouse during embryonic development. Importantly our data established opposing role of TFII-I∆ and γ accountable for Nurr1 mediated repression and activation of hTH respectively during dopaminergic neurogenesis. Notably SUMO modified TFII-I∆ act as a critical corepressor of Nurr1 mediated hTH repression likely leading to loss of DA phenotype.