Expression of human gene coding RORγT receptor depends on the Sp2 transcription factor

Abstract

Th17 cells are involved in the immune response against pathogens, autoimmunity, and tumor progression. The differentiation of human Th17 cells requires the upregulation of RORγT, which in human cells is still not well understood. We identified 2 putative binding motifs for specificity protein transcription factors from the specificity protein/Kruppel-like factor family in the promoter of human RORγT and investigated the involvement of specificity proteins in the transcriptional regulation of this gene. To this end, a human lymphocytic cell line and in vitro-differentiated Th17 cells were used in promoter activity assays, in situ mutagenesis, chromatin immunoprecipitation, and real-time RT-PCR assays. In some experiments, specificity protein expression and activity was inhibited by siRNA and mithramycin A. The results showed that the transcription factor specificity protein 2 recognized binding motifs in the human RORγT promoter, which was critical for maintaining expression. Furthermore, specificity protein 2 was necessary for maximum IL-17 expression in in vitro-differentiated Th17 cells. These observations demonstrate the significant role of specificity protein 2 in the regulation of the Th17 signature transcription factor RORγT and the maintenance of the Th17 phenotype. The findings also suggest that specificity protein 2 plays a role in Th17-dependent physiologic and pathologic immune responses and might serve as a potential novel target for their modulation.

Functional Analysis of the rs774872314, rs116171003, rs200231898 and rs201107751 Polymorphisms in the Human RORγT Gene Promoter Region

“…The specificity of the observed bands was confirmed using the competition assay with excess unlabeled wild-type probe (see Figure 3). We detected two bands that were previously identified to be occupied by Sp1 (upper) and Sp2 proteins (lower) [13]. We observed elimination (Sp1) and reduction (Sp2) of binding to the p1, p2 and p4 probes.…”

“…Furthermore, site-directed mutagenesis suggested that promoter function is dependent on the cooperative interactions of these two boxes [13]. Analyzing public resources for single nucleotide polymorphism data, we found that several naturally occurring polymorphisms are located within one of the GC-boxes (GC-box 2).…”

“…All promoter constructs for the RORγT gene were described in our previous works [3,13], with the exception of the newly constructed phRORγTp1(−180/+78)Luc, phRORγTp2(−180/+78)Luc, phRORγTp3(−180/+78)Luc, and phRORγTp4(−180/+78)Luc, which were transfected into Jurkat T cells with FuGENE HD (Roche, Basel, Switzerland). The luciferase activity in the cells was measured in 96-well white plates on Infinite ® 200 PRO (Tecan, Männedorf, Switzerland).…”

“…Processes that are responsible for the observed pattern of expression include epigenetic DNA modifications [12] and direct interactions of transcription factors with cis-elements within the 5′-flanking region of the gene. Previously, we cloned the human promoter region of RORγT and identified several elements important for its expression, including four E-boxes capable of binding upstream stimulatory factor(USF) transcription factors [3] and two GC-boxes that are able to interact with Sp2 and, to a lesser extent, Sp1 [13]. Both identified GC-boxes seem to be crucial for the activity of the promoter and the expression of the gene in human lymphocytes [13].…”

“…Previously, we cloned the human promoter region of RORγT and identified several elements important for its expression, including four E-boxes capable of binding upstream stimulatory factor(USF) transcription factors [3] and two GC-boxes that are able to interact with Sp2 and, to a lesser extent, Sp1 [13]. Both identified GC-boxes seem to be crucial for the activity of the promoter and the expression of the gene in human lymphocytes [13]. After analyzing the National Center for Biotechnology Information (NCBI) Variation Resources [14], we found that four single nucleotide polymorphisms (SNPs) (rs774872314, rs116171003, rs200231898, rs201107751) are located within GC-box 2 of the RORγT gene.…”