Transcriptional control of human T‐BET expression: The role of Sp1

Abstract

Murine T-bet (T-box expressed in T cells) is a master regulator of IFN-c gene expressionin NK and T cells. T-bet also plays a critical role in autoimmunity, asthma and other diseases. However, cis elements or trans factors responsible for regulating T-bet expression remain largely unknown. Here, we report on our discovery of six Sp1-binding sites within the proximal human T-BET promoter that are highly conserved among mammalian species. Electrophoretic mobility shift assays demonstrate a physical association between Sp1 and the proximal T-BET promoter with a direct dose response between Sp1 expression and T-BET promoter activity. Ectopic overexpression of Sp1 also enhanced T-BET expression and cytokine-induced IFN-c secretion in NK cells and T cells. Mithramycin A, which blocks the binding of Sp1 to the T-BET promoter, diminished both T-BET expression and IFN-c protein production in monokine-stimulated primary human NK cells. Collectively, our results suggest that Sp1 is a positive transcriptional regulator of T-BET. As T-BET and IFN-c are critically important in inflammation, infection, and cancer, targeting Sp1, possibly with mithramycin A, may be useful for preventing and/ or treating diseases associated with aberrant T-BET or IFN-c expression.

Integration of Distinct Intracellular Signaling Pathways at Distal Regulatory Elements Directs T-bet Expression in Human CD4+ T Cells

“…This finding is consistent with a recent genome-wide analysis using high-throughput sequencing and wholegenome tiled array strategies to identify HS within unfractionated human CD4 ϩ T cells, which revealed accessibility around the mRNA start site of the T-bet gene (28). Similar to the promoter region of the human IL12RB2 gene, this region is highly GC rich, contains several binding sites for the ubiquitously expressed transcription factor Sp1, and displays promoter activity in reporter gene assays in cell lines of hematopoietic and nonhematopoietic origin (1,10). The region corresponding to HS3 may therefore contain the minimal promoter of the T-bet gene.…”

“…Further studies reported that T-bet expression is strongly induced by IFN-␥ signaling via STAT1 activation (8 -11). More recent studies have invoked a role for Notch signaling and the transcription factors Sp1 and STAT1/STAT4 in the regulation of T-bet expression in the mouse (10,12,13). T-bet has also been shown to play an important role in human Th1 cell differentiation, as has been demonstrated by retroviral transduction (14 -16).…”

TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia

“…Data were analyzed as previously described. 25,26 All primer and probe sequences are available on request.…”

“…[25][26][27][28][29] Briefly, Phoenix cells were transiently transfected with TSC-22-pMSCV or the vector alone and supernatants were collected 48 hours after transfection and used for 3 cycles of infections. Upon infection, cell lines (eg, YAC-1 and L1210) were sorted by FACSVantage for green fluorescent protein (GFP) expression to at least 99% purity.…”

“…26 The antibodies used were as follows: rabbit anti-human TSC-22 polyclonal antibody (a kind gift of Drs Hitoshi Kawamata [Dokkyo University School of Medicine, Tochigi, Japan] and Marco G. Cecchini [University of Bern, Bern, Switzerland]); mouse anti-MYC-tag monoclonal antibody (Cell Signaling Technology, Beverly, MA); goat polyclonal anti-␤-ACTIN antibody (Santa Cruz Biotechnology, Santa Cruz, CA).…”