Differential chromatin looping regulates CD4 expression in immature thymocytes - PubMed (original) (raw)

Differential chromatin looping regulates CD4 expression in immature thymocytes

Huimin Jiang et al. Mol Cell Biol. 2008 Feb.

Abstract

Runx1 binds the silencer and represses CD4 transcription in immature thymocytes. In this study, using looping chromatin immunoprecipitation and chromatin conformation capture assays, we demonstrated that interactions between Runx1 and positive elongation factor b (P-TEFb) appose the silencer and enhancer in CD4-negative thymoma cells and double-negative immature thymocytes. This chromatin loop decoys P-TEFb away from the promoter, thus preventing RNA polymerase II from elongating on the CD4 gene. In the absence of Runx1 on the silencer, P-TEFb interacts with the transcription complex, forming a different chromatin loop between the enhancer and the promoter, which leads to the expression of the CD4 gene in CD4-positive hybridoma cells and double-positive thymocytes. Moreover, the knockdown of CycT1 from P-TEFb abolishes both of these chromatin loops. Finally, the selective removal and restoration of Runx1 causes rapid interchanges between these chromatin loops, which reveals the plasticity of this regulatory circuit. Thus, differential looping and decoying of P-TEFb away from the promoter mediate active repression of the CD4 gene during thymocyte development.

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Figures

FIG. 1.

FIG. 1.

Recruitment of RNAPII, P-TEFb (CycT1), and Runx1 to the CD4 locus. (A) Schematic diagram of the CD4 locus and the amplicons used in ChIP assays. The CD4 enhancer (E) is located 12 kb upstream from the promoter (P), and the CD4 silencer (S) is located in the first intron, 2.3 kb downstream from the promoter. I designates the amplicon located between the enhancer and the promoter. (B) FACS analyses of CD4 expression in 1200M (open peak) and 3A9 (solid peak) cells stained with fluorescein isothiocyanate-conjugated anti-mouse CD4 or a rat immunoglobulin G2b(κ) isotype control (thin black line). The intensity of CD4 staining and the relative number of cells are shown on the x and y axes, respectively. (C) ChIP analyses of the CD4 locus in 1200M (CD4−) and 3A9 (CD4+) cells. Anti-RNAPII, anti-CycT1, anti-panRunx, and anti-Runx1 antibodies were used as described in Materials and Methods. Normal rabbit serum served as the negative control for antibody specificity. The presence of the CD4 enhancer, intervening sequence, promoter, and silencer in the immunoprecipitates was examined by PCR with primer sets E, I, P, and S, respectively. PCR analyses with DNA before immunoprecipitation (Input) served as controls for the amplification efficiencies of individual primer sets. PCRs were carried out at various cycle numbers to ensure linear amplification. Representative agarose gels of PCR products within the linear range are presented.

FIG. 2.

FIG. 2.

3C analyses of the CD4 locus. (A) Diagram of 3C analysis. The locations of StuI restriction sites (s) and PCR primers (arrows) used in 3C analyses, relative to the CD4 enhancer (E), promoter (P), and silencer (S), are indicated. Cross-linked and digested chromatin was ligated at very low concentrations so that only intramolecular ligation could occur. Ligation products were detected by quantitative real-time PCR with appropriate primer sets in the presence of Sybr green. The relative cross-linking frequency of the CD4 enhancer with the silencer and that of the enhancer with the promoter were evaluated by the PCR signals obtained from the ES and EP amplicons, both normalized to Hprt1 and the control template as described in Materials and Methods. (B) Negative controls of 3C analysis. Real-time PCRs were performed from StuI-digested, non-cross-linked 1200M genomic DNA and cross-linked 1200M chromatin before and after ligation. Sybr green signals from the EP and ES amplicons were normalized to signals from a fragment that was not cut by StuI. Since no signal was obtained from non-cross-linked or nonligated samples after 40 cycles (lanes 1 to 3 and 5 to 7), the signal from the EP amplicon from cross-linked and ligated 1200M chromatin (lane 8) was set to 1. (C) 3C analyses of the CD4 locus in 1200M and 3A9 cells. The y axis shows the relative cross-linking frequency of the enhancer with the promoter (EP) and of the enhancer with the silencer (ES) compared to the Hprt1 locus (set to 1; not shown on the graph). At least two independent experiments were performed for each cell population, and more than three measurements were carried out for each experiment. Results of one typical experiment are presented. Error bars, standard deviations. (D) 3C analyses of the CD4 locus in primary DN and DP thymocytes were carried out as described for panel C.

FIG. 3.

FIG. 3.

Runx1 and CycT1 knockdowns disrupt the looping between the CD4 enhancer and silencer in 1200M cells. (A) FACS analyses of CD4 expression in 1200M cells transduced either with the empty vector, with a retroviral vector containing a shRNAmir against Runx1 followed by IRES-green fluorescent protein (GFP) (Runx1-KD1), or with Runx1-KD1 plus MSCV vectors containing Runx1-IRES-human CD2 (Runx1-hCD2). The intensity of CD4 staining and the levels of enhanced GFP (EGFP) or hCD2 are presented on the y and x axes, respectively. (B) Western blotting of Runx1 and CycT1 levels in 1200M cells transduced either with the empty vector, with a retroviral vector containing a different shRNAmir against Runx1 followed by IRES-GFP (Runx1-KD2), with Runx1-KD2 plus Runx1-hCD2, or with a shRNAmir against CycT1 (CycT1-KD). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were used as controls for equal loading. (C) FACS analyses of CD4 expression in 1200M (white peak to the left), 3A9 (dotted line to the right), Runx1-KD2 (black peak), and Runx1-KD2/Runx1-hCD2 (solid gray peak) cells stained with phycoerythrin-conjugated anti-mouse CD4. The intensity of CD4 staining and the relative numbers of cells are shown on the x and y axes, respectively. (D) 3C analyses of the CD4 locus. The relative cross-linking frequencies of the enhancer with the promoter (EP) and of the enhancer with the silencer (ES) are presented as described for Fig. 2C.

FIG. 4.

FIG. 4.

A model for active repression by Runx1. In DN and ISP thymocytes, the interaction between Runx1 and P-TEFb brings the enhancer (E) into the close proximity of the silencer (S) and prevents P-TEFb from activating RNAPII, which is arrested at the promoter (P), thus actively repressing transcription elongation. In DP and CD4 SP thymocytes, the lack of Runx1 binding to the CD4 silencer frees P-TEFb to interact with RNAPII and to activate transcription elongation. Only RNAPII on the CD4 promoter is depicted in the model.

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