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Serine Arginine-Rich Splicing Factor 1 (SRSF1) Contributes to the Transcriptional Activation of CD3ζ in Human T Cells.

Moulton VR, Gillooly AR, Perl MA, Markopoulou A, Tsokos GC - PLoS ONE (2015)

Bottom Line: T lymphocytes from many patients with systemic lupus erythematosus (SLE) express decreased levels of the T cell receptor (TCR)-associated CD3 zeta (ζ) signaling chain, a feature directly linked to their abnormal phenotype and function.Chromatin immunoprecipitation assays show that SRSF1 is recruited to the CD3ζ promoter.Thus our study identifies a novel mechanism whereby SRSF1 regulates CD3ζ expression in human T cells and may contribute to the T cell defect in SLE.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston MA, United States of America.

ABSTRACT
T lymphocytes from many patients with systemic lupus erythematosus (SLE) express decreased levels of the T cell receptor (TCR)-associated CD3 zeta (ζ) signaling chain, a feature directly linked to their abnormal phenotype and function. Reduced mRNA expression partly due to defective alternative splicing, contributes to the reduced expression of CD3ζ chain. We previously identified by oligonucleotide pulldown and mass spectrometry approaches, the serine arginine-rich splicing factor 1 (SRSF1) binding to the 3' untranslated region (UTR) of CD3ζ mRNA. We showed that SRSF1 regulates alternative splicing of the 3'UTR of CD3ζ to promote expression of the normal full length 3`UTR over an unstable splice variant in human T cells. In this study we show that SRSF1 regulates transcriptional activation of CD3ζ. Specifically, overexpression and silencing of SRSF1 respectively increases and decreases CD3ζ total mRNA and protein expression in Jurkat and primary T cells. Using promoter-luciferase assays, we show that SRSF1 enhances transcriptional activity of the CD3ζ promoter in a dose dependent manner. Chromatin immunoprecipitation assays show that SRSF1 is recruited to the CD3ζ promoter. These results indicate that SRSF1 contributes to transcriptional activation of CD3ζ. Thus our study identifies a novel mechanism whereby SRSF1 regulates CD3ζ expression in human T cells and may contribute to the T cell defect in SLE.

No MeSH data available.


Related in: MedlinePlus

SRSF1 regulates transcriptional activity of the CD3ζ promoter in 293T cells.(A) Schematic showing luciferase constructs with different lengths of the CD3ζ promoter. (B) 293T cells were co-transfected with an empty pGL2-basic plasmid or the indicated CD3ζ promoter-luciferase constructs and empty vector (pcDNA) or SRSF1 expression vector (pSRSF1) in increasing concentrations such that the ratio of the pcDNA or pSRSF1 (i.e., effector) plasmid to the luciferase (i.e., reporter) plasmid was 2:1 or 3:1. 24hours post transfection, cells were lysed, and luciferase activity measured using the dual-luciferase assay. Graphs show average values from at least three independent experiments and error bars represent SEM. Asterisks indicate p <0.05; NS = not significant.
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pone.0131073.g003: SRSF1 regulates transcriptional activity of the CD3ζ promoter in 293T cells.(A) Schematic showing luciferase constructs with different lengths of the CD3ζ promoter. (B) 293T cells were co-transfected with an empty pGL2-basic plasmid or the indicated CD3ζ promoter-luciferase constructs and empty vector (pcDNA) or SRSF1 expression vector (pSRSF1) in increasing concentrations such that the ratio of the pcDNA or pSRSF1 (i.e., effector) plasmid to the luciferase (i.e., reporter) plasmid was 2:1 or 3:1. 24hours post transfection, cells were lysed, and luciferase activity measured using the dual-luciferase assay. Graphs show average values from at least three independent experiments and error bars represent SEM. Asterisks indicate p <0.05; NS = not significant.

Mentions: In order to examine if SRSF1 can control transcriptional activity of CD3ζ, we performed promoter-luciferase reporter assays. We used three promoter-luciferase constructs (zeta-2, zeta-3 and zeta-4) with varying lengths (-160/+58, -307/+58 and -485/+58) of the CD3ζ promoter as shown in schematic in Fig 3A, according to the numbering published previously [26]. 293T cells were transfected with the CD3ζ promoter-luciferase ‘reporter’ construct and co-transfected with increasing amounts of the SRSF1 or empty ‘effector’ constructs such that effector: reporter plasmid ratios were 2:1, or 3:1, and promoter activity measured using dual-luciferase assays. As seen in Fig 3B, SRSF1 induced a dose dependent increase in CD3ζ promoter activity with all three lengths of the CD3ζ promoter (Fig 3B). As control, an empty luciferase construct (pGL2-basic) was co-transfected with increasing amounts of SRSF1. Data show average values from at least three independent experiments, error bars represent SEM Data for control vs SRSF1-transfected cells for the pGL2-basic construct were 1 ± 0 vs 1.65 ± 0.47 and 1.97 ± 0.35, p = 0.2 and 0.05, for the pGL2-Zeta-2 construct 1 ± 0 vs 2.47 ± 0.73 and 3.79 ± 1.1, p = 0.01 and 0.04, for the pGL2-Zeta-3 construct 1 ± 0 vs 2.05 ± 0.55 and 4.45 ± 1.27, p = 0.01 and 0.03, and for the pGL2-Zeta-4 construct 1 ± 0 vs 2.64 ± 0.39 and 3.57 ± 0.5, p = 0.02 and 0.01 for the 2:1 and 3:1 conditions respectively. To confirm these findings in primary T cells, we performed promoter-reporter assays in peripheral blood T cells. We co-transfected the -307/+58 construct (Fig 4A) with increasing amounts of SRSF1 (pSRSF1) or empty plasmid (pcDNA) as control (Fig 4B). We found significant dose dependent increase in CD3ζ promoter activity in the SRSF1 transfected cells compared with control-transfected cells (Fig 4B, 1 ± 0 vs 1.82 ± 0.23, 3 ± 0.48, 4.43 ± 0.83 and p = 0.03, 0.02 and 0.02 respectively for the 3:1, 4:1 and 5:1 conditions). Data show average values from three independent experiments, error bars represent SEM. These results indicate that SRSF1 positively regulates transcriptional activity of the CD3ζ promoter.


Serine Arginine-Rich Splicing Factor 1 (SRSF1) Contributes to the Transcriptional Activation of CD3ζ in Human T Cells.

Moulton VR, Gillooly AR, Perl MA, Markopoulou A, Tsokos GC - PLoS ONE (2015)

SRSF1 regulates transcriptional activity of the CD3ζ promoter in 293T cells.(A) Schematic showing luciferase constructs with different lengths of the CD3ζ promoter. (B) 293T cells were co-transfected with an empty pGL2-basic plasmid or the indicated CD3ζ promoter-luciferase constructs and empty vector (pcDNA) or SRSF1 expression vector (pSRSF1) in increasing concentrations such that the ratio of the pcDNA or pSRSF1 (i.e., effector) plasmid to the luciferase (i.e., reporter) plasmid was 2:1 or 3:1. 24hours post transfection, cells were lysed, and luciferase activity measured using the dual-luciferase assay. Graphs show average values from at least three independent experiments and error bars represent SEM. Asterisks indicate p <0.05; NS = not significant.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4489909&req=5

pone.0131073.g003: SRSF1 regulates transcriptional activity of the CD3ζ promoter in 293T cells.(A) Schematic showing luciferase constructs with different lengths of the CD3ζ promoter. (B) 293T cells were co-transfected with an empty pGL2-basic plasmid or the indicated CD3ζ promoter-luciferase constructs and empty vector (pcDNA) or SRSF1 expression vector (pSRSF1) in increasing concentrations such that the ratio of the pcDNA or pSRSF1 (i.e., effector) plasmid to the luciferase (i.e., reporter) plasmid was 2:1 or 3:1. 24hours post transfection, cells were lysed, and luciferase activity measured using the dual-luciferase assay. Graphs show average values from at least three independent experiments and error bars represent SEM. Asterisks indicate p <0.05; NS = not significant.
Mentions: In order to examine if SRSF1 can control transcriptional activity of CD3ζ, we performed promoter-luciferase reporter assays. We used three promoter-luciferase constructs (zeta-2, zeta-3 and zeta-4) with varying lengths (-160/+58, -307/+58 and -485/+58) of the CD3ζ promoter as shown in schematic in Fig 3A, according to the numbering published previously [26]. 293T cells were transfected with the CD3ζ promoter-luciferase ‘reporter’ construct and co-transfected with increasing amounts of the SRSF1 or empty ‘effector’ constructs such that effector: reporter plasmid ratios were 2:1, or 3:1, and promoter activity measured using dual-luciferase assays. As seen in Fig 3B, SRSF1 induced a dose dependent increase in CD3ζ promoter activity with all three lengths of the CD3ζ promoter (Fig 3B). As control, an empty luciferase construct (pGL2-basic) was co-transfected with increasing amounts of SRSF1. Data show average values from at least three independent experiments, error bars represent SEM Data for control vs SRSF1-transfected cells for the pGL2-basic construct were 1 ± 0 vs 1.65 ± 0.47 and 1.97 ± 0.35, p = 0.2 and 0.05, for the pGL2-Zeta-2 construct 1 ± 0 vs 2.47 ± 0.73 and 3.79 ± 1.1, p = 0.01 and 0.04, for the pGL2-Zeta-3 construct 1 ± 0 vs 2.05 ± 0.55 and 4.45 ± 1.27, p = 0.01 and 0.03, and for the pGL2-Zeta-4 construct 1 ± 0 vs 2.64 ± 0.39 and 3.57 ± 0.5, p = 0.02 and 0.01 for the 2:1 and 3:1 conditions respectively. To confirm these findings in primary T cells, we performed promoter-reporter assays in peripheral blood T cells. We co-transfected the -307/+58 construct (Fig 4A) with increasing amounts of SRSF1 (pSRSF1) or empty plasmid (pcDNA) as control (Fig 4B). We found significant dose dependent increase in CD3ζ promoter activity in the SRSF1 transfected cells compared with control-transfected cells (Fig 4B, 1 ± 0 vs 1.82 ± 0.23, 3 ± 0.48, 4.43 ± 0.83 and p = 0.03, 0.02 and 0.02 respectively for the 3:1, 4:1 and 5:1 conditions). Data show average values from three independent experiments, error bars represent SEM. These results indicate that SRSF1 positively regulates transcriptional activity of the CD3ζ promoter.

Bottom Line: T lymphocytes from many patients with systemic lupus erythematosus (SLE) express decreased levels of the T cell receptor (TCR)-associated CD3 zeta (ζ) signaling chain, a feature directly linked to their abnormal phenotype and function.Chromatin immunoprecipitation assays show that SRSF1 is recruited to the CD3ζ promoter.Thus our study identifies a novel mechanism whereby SRSF1 regulates CD3ζ expression in human T cells and may contribute to the T cell defect in SLE.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston MA, United States of America.

ABSTRACT
T lymphocytes from many patients with systemic lupus erythematosus (SLE) express decreased levels of the T cell receptor (TCR)-associated CD3 zeta (ζ) signaling chain, a feature directly linked to their abnormal phenotype and function. Reduced mRNA expression partly due to defective alternative splicing, contributes to the reduced expression of CD3ζ chain. We previously identified by oligonucleotide pulldown and mass spectrometry approaches, the serine arginine-rich splicing factor 1 (SRSF1) binding to the 3' untranslated region (UTR) of CD3ζ mRNA. We showed that SRSF1 regulates alternative splicing of the 3'UTR of CD3ζ to promote expression of the normal full length 3`UTR over an unstable splice variant in human T cells. In this study we show that SRSF1 regulates transcriptional activation of CD3ζ. Specifically, overexpression and silencing of SRSF1 respectively increases and decreases CD3ζ total mRNA and protein expression in Jurkat and primary T cells. Using promoter-luciferase assays, we show that SRSF1 enhances transcriptional activity of the CD3ζ promoter in a dose dependent manner. Chromatin immunoprecipitation assays show that SRSF1 is recruited to the CD3ζ promoter. These results indicate that SRSF1 contributes to transcriptional activation of CD3ζ. Thus our study identifies a novel mechanism whereby SRSF1 regulates CD3ζ expression in human T cells and may contribute to the T cell defect in SLE.

No MeSH data available.


Related in: MedlinePlus