Reciprocal regulation of Rag expression in thymocytes by the zinc-finger proteins, Zfp608 and Zfp609

Abstract

Recombination-activating gene 1 (Rag1) and Rag2 enzymes are required for T cell receptor assembly and thymocyte development. The mechanisms underlying the transcriptional activation and repression of Rag1 and Rag2 are incompletely understood. The zinc-finger protein, Zfp608, represses Rag1 and Rag2 expression when expressed in thymocytes blocking T-cell maturation. Here we show that the related zinc-finger protein, Zfp609, is necessary for Rag1 and Rag2 expression in developing thymocytes. Zfp608 represses Rag1 and Rag2 expression indirectly by repressing the expression of Zfp609. Thus, the balance of Zfp608 and Zfp609 plays a critical role in regulating Rag1 and Rag2 expression, which may manifest itself not only during development of immature thymocytes into mature T cells but also in generation of the T-cell arm of the adaptive immune system, which does not fully develop until after birth.

Figure 1

Reciprocal expression of Zfp608, Zfp609, Rag and Aire in wild-type and ZORI thymocytes. Expression levels of the indicated genes (a, b, c, d and g) in wild-type and ZORI thymocytes (a, b, c, and g) and thymic epithelia (d), were determined by PCR using Taqman assays. Results are expressed relative to Gapdh levels: (a) Zfp608, (b) Zfp609, (c) Rag1, (d) Rag2 and (g) Aire. (e) Acetylated histone 4 levels at Rag1 promoter in wild-type and ZORI thymocytes as determined by chromatin immunoprecipitation (ChIP). Results are expressed as fraction of input. (f) Acetylated histone 4 levels at the Rag2 promoter in wild-type and ZORI thymocytes. *P<0.05.

Figure 2

mRNA expression levels of (a) Zfp608, (b) Rag1, (c) Rag2 and (d) Zfp609 in NIH-3T3 fibroblasts and VL3-3M2 thymocytes. Expression levels of the indicated genes were determined as in Figure 1. Results are normalized to expression levels of Gapdh. Error bars represent s.d. *P<0.05.

Figure 3

Zfp608 decreases expression of Rag1, Rag2 and Zfp609 in VL3-3M2 cells. Expression levels of (a) Zfp608, (b) Rag1, (c) Rag2 and (d) Zfp609 (determined as in Figure 1) in VL3-3M2 and in VL3-3M2 cells after stable transfection of a Zfp608 expression plasmid, VL3-3M2-Zfp608 cells. Expression levels were normalized to Gapdh. (e) The levels of Rag1 and Rag2 relative to Zfp608 and Zfp609. Error bars are s.d. *P<0.05.

Figure 4

Zfp609 is a positive regulator of Rag1 and Rag2 in VL3-3M2-Zfp608 cells. VL3-3M2-Zfp608 cells were transiently transfected with a Zfp609 expression plasmid or empty vector control. (a) Zfp609, (b) Rag1 and (c) Rag2 expression levels were determined as in Figure 1. Results are expressed relative to Gapdh. (d) Rag1 and Rag2 relative to Zfp608 and Zfp609. Error bars are s.d. *P<0.05.

Figure 5

siRNA-mediated knockdown of Zfp609 in VL3-3M2 thymocytes and BALB/c thymocytes reduces Rag expression. (a–c) Gene expression levels of Zfp609, Rag1 and Rag2 in VL3-3M2 thymocytes after siRNA-mediated Zfp609 knockdown relative to Gapdh. (d) Rag1 and Rag2 expression levels in VL3-3M2 thymocytes after siRNA-mediated knockdown relative to Zfp609. (e–g) Zfp609, Rag1 and Rag2 expression levels in BALB/c thymocytes after siRNA-mediated Zfp609 knockdown. (h) Rag1 and Rag2 expression levels in BALB/c thymocytes after siRNA knockdown relative to Zfp609. *P<0.05.

Figure 6

Promoter activity of Zfp609 and Rag1 in VL3-3M2 thymocytes. (a) Zfp609 promoter activity in the presence and absence of Zfp608 in VL3-3M2 thymocytes. (b) Rag1 promoter activity in the presence or absence of Zfp609 in VL3-3M2 thymocytes. *P<0.05.