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Differential Chromatin Looping Regulates CD4 Expression in Immature Thymocytes

Departments of Medicine, Microbiology, and Immunology, Rosalind Russell Medical Research Center, University of California at San Francisco, San Francisco, California 94143

Received 22 May 2007/ Returned for modification 26 June 2007/ Accepted 7 November 2007

	ABSTRACT

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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.

	INTRODUCTION

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Mammalian transcription starts with the formation of a preinitiation complex at the promoter, where cyclin-dependent kinase 7 (Cdk7) from the general transcription factor TFIIH phosphorylates serines at position 5 of the heptapeptide (YSPTSPS) repeats in the C-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). This phosphorylation allows RNAPII to clear the promoter and to initiate transcription. However, RNAPII then comes under the control of the negative elongation factor (NELF) and the DRB sensitivity-inducing factor (DSIF), which render RNAPII susceptible to pausing and arrest. Positive elongation factor b (P-TEFb) phosphorylates NELF, DSIF, and serines at position 2 of the heptapeptide repeats in the CTD, releasing NELF from the arrested complex and turning DSIF into a positive elongation factor, thus leading to productive transcription elongation. P-TEFb is composed of Cdk9 and one of three C-type regulatory cyclin subunits, CycT1, CycT2, or CycK (reviewed in reference 16). Over the past 2 decades, it has become clear that many mammalian genes are regulated at this step of transcription. To this end, P-TEFb can be recruited to the transcription complex by DNA-bound activators such as NF-B and c-Myc (1, 9), as well as by the RNA-bound transactivator Tat of human immunodeficiency virus (26, 32), the chromatin-bound modifier Brd4 (6, 29), or the coactivator CIITA (8). On the other hand, transcription repressors such as PIE1 and Runx1 can decoy P-TEFb away from the transcription complex, thus inhibiting transcription elongation (7, 30).

Runx1, also called AML1, is a master regulator of hematopoiesis and the most frequent target of translocations and mutations in human leukemias. It belongs to the Runt domain (RD) family of transcription factors, which contain the signature RD that is responsible both for sequence-specific DNA binding and for heterodimerization (reviewed in references 3, 13, and 27). Runx1 is a context-dependent regulator. On certain genes, such as the T-cell antigen receptor (17, 25), it facilitates the assembly of transcription complexes, whereas on others, it acts as a repressor by recruiting mSin3A or Groucho/TLE corepressors (11, 14) and/or by decoying P-TEFb (7).

Newly generated thymocytes do not express CD4 or CD8. These CD4^– CD8^– double-negative (DN) thymocytes will transition through CD4^– CD8^low immature single-positive (ISP) and CD4⁺ CD8⁺ double-positive (DP) stages and eventually develop into two distinct populations: mature CD4⁺ CD8^– single-positive (CD4 SP) or CD4^– CD8⁺ single-positive (CD8 SP) cells. CD4 expression is actively repressed in DN and ISP cells as well as during the transition from DP to CD8 SP cells (2). However, the maintenance of CD4 silencing is achieved by epigenetic silencing in CD8 SP T cells (20). Active repression requires Runx1 and a silencer located in the first intron of the CD4 gene, which contains Runx-binding sites (21, 22, 28). It had been demonstrated previously that the inhibitory domain in Runx1 is required for the repression of CD4 in thymocytes (10, 24) as well as for effects of Runx1 on the CD4 silencer (7). In addition, we demonstrated that Runx1 not only binds P-TEFb but prevents further transcription elongation (7). In this study, we wanted to determine if these interactions are reflected in different chromatin conformations between cis-acting elements in the CD4 gene.

	MATERIALS AND METHODS

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Cells. The mouse 3A9 T-cell hybridoma, a gift from Nigel Killeen (University of California at San Francisco [UCSF], San Francisco, CA), and 1200M cells, a gift from Dan Littman (New York University, New York, NY), were maintained at 37°C under 5% CO₂ in RPMI medium supplemented with 10% fetal bovine serum, 100 mM L-glutamine, 50 µM β-mercaptoethanol, and 50 µg each of penicillin and streptomycin per ml. To isolate primary wild-type DP cells, thymocytes from C57BL/6 mice were stained with anti-CD4 (RM4-5) and anti-CD8a (53-6.7), and DP cells were sorted. Total thymocytes from Rag2^–/– mice were used as wild-type DN thymocytes.

ChIP assays. Chromatin immunoprecipitation (ChIP) was carried out essentially as described previously (7). Cross-linking was achieved by incubating 60 million cells in medium containing 1% formaldehyde for 10 min at room temperature. Cells were then pelleted and washed once with cold phosphate-buffered saline with freshly added protease inhibitors (Sigma-Aldrich, St. Louis, MO). The cell pellets were then resuspended in 6 ml of homogenization buffer (10 mM HEPES at pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.1% NP-40) with freshly added protease inhibitors, incubated for 10 min on ice, and homogenized with 10 strokes of a B pestle in a Dounce homogenizer. After spinning, the isolated nuclei were lysed in 600 µl of lysis buffer (1% sodium dodecyl sulfate [SDS], 10 mM EDTA, 50 mM Tris-HCl at pH 8.0) with freshly added protease inhibitors for 10 min on ice and were sonicated to obtain DNA fragments averaging approximately 200 to 500 bp. Chromatin-protein complexes from 10 million cells were used in each ChIP. Chromatin solutions were diluted 10-fold in TES-150 buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl at pH 8.0, 150 mM NaCl) with freshly added protease inhibitors, precleared with protein A-Sepharose beads, and then incubated with appropriate antibodies at 4°C overnight. Antibodies used included normal rabbit serum (as a negative control) (Sigma-Aldrich), rabbit polyclonal anti-RNAPII (sc-899; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-cyclin T1 (sc-10750; Santa Cruz Biotechnology), rabbit polyclonal anti-panRunx (a gift from Masanobu Satake, Tohoku University, Japan), and anti-Runx1 (pc285; EMD Biosciences, San Diego, CA) antibodies. Protein A-Sepharose beads were added the next day. After another 2 h of incubation, the beads were washed once in TSE-150, once in TSE-500 (like TSE-150 but with 500 mM NaCl), once in buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl at pH 8.0), and twice in TE (10 mM Tris, I mM EDTA, pH 8.0) buffer, all with freshly added protease inhibitors. Immunocomplexes were eluted from the beads with elution buffer (1% SDS and 0.5% NaHCO₃) for 15 min at room temperature. Reverse cross-linking was performed at 65°C for 4 h and followed by treatment with proteinase K. DNA was extracted with phenol-chloroform, precipitated with ethanol, and dissolved in 30 µl of TE buffer. Two microliters of DNA was used in PCR with appropriate primer sets to amplify specific DNA fragments. Primer sequences are available upon request. PCR products, taken at various cycle numbers to ensure linear amplification, were separated on agarose gels and visualized with Sybr green. Each ChIP was repeated at least twice with similar results.

3C assays. Chromatin conformation capture (3C) assays were carried out as previously described with modifications (18). Cross-linking was achieved by incubating 8 million cells in 10 ml medium containing 2% formaldehyde for 5 min at room temperature and was stopped by adding glycine to 0.125 M and incubating for another 5 min at room temperature. Cells were then pelleted in a conical tube and lysed in 10 ml of lysis buffer (10 mM Tris-HCl at pH 8.0, 10 mM NaCl, 0.2% NP-40) with freshly added protease inhibitors for 90 min at 4°C with rotation. The nuclei were collected and incubated in 400 µl of 1.2x restriction buffer 2 (New England Biolabs [NEB], Beverly, MA) containing 0.3% SDS at 37°C for 1 h with shaking. The SDS was then sequestered by adding Triton X-100 to 1.8% and incubating at 37°C for another hour with shaking. Three batches of StuI (NEB), a total of 900 U, were added in a 22-h period to achieve >80% digestion. The reaction was stopped by adding SDS to 1.6% and incubating at 65°C for 20 min. The extent of digestion was verified by quantitative real-time PCR using Sybr green detection (Sigma) across the StuI sites. Two micrograms of digested chromatin was diluted in 800 µl ligation buffer (NEB) (final concentration, 2.5 ng/µl, to ensure that only intramolecular ligation would occur). Residual SDS was sequestered by adding Triton X-100 to 2% and incubating at 37°C for 1 h with shaking. The reaction mixture was then cooled to 16°C, and 2,000 U of T4 DNA ligase (NEB) was added. After 4 h of ligation, the chromatin mixture was incubated with 100 µg/ml proteinase K at 65°C overnight to reverse cross-links. RNA was removed by RNase A (0.5 µg/ml) treatment for 30 min at 37°C, and DNA was purified by phenol extraction. Quantitative real-time PCRs were performed, in the presence of Sybr green (Sigma), with appropriate primers (sequences available upon request) from purified DNA as well as the control template. The control template was generated by PCR amplifying sequences around individual StuI sites to be analyzed and mixing them in equal molar amounts, followed by restriction digestion and ligation to include all possible ligation products. The relative cross-linking frequency between two fragments of the CD4 locus (X_{CD4 locus}) is calculated as [(S_{CD4 locus}/S_{Hprt1 locus}) cell]/[(S_{CD4 locus}/S_{Hprt1 locus}) control], where S_{CD4 locus} is the signal obtained with primer pairs for two restriction fragments of the CD4 locus from cells or the control template and S_{Hprt1 locus} is the signal obtained with primer pairs for two restriction fragments of the Hprt1 locus from cells or the control template. This calculation corrects for differences in cross-linking and ligation efficiencies, PCR amplification efficiency, the amount of the initial template used, and the sizes of the PCR products. For negative controls, we performed real-time PCR from StuI-digested, non-cross-linked genomic DNA and cross-linked chromatin, before and after ligation. In these control reactions, the Sybr green signals obtained from the EP and ES amplicons were normalized to signals obtained from a fragment that was not cut by StuI.

Retrovirus production and transduction. Retroviruses were produced by transfecting Phoenix cells with pSM2 containing a microRNA-adapted short hairpin RNA (shRNAmir) cassette targeting the 3' untranslated region (3' UTR) of Runx1 mRNA (OpenBiosystems, Huntsville, AL) or with pMSCV-Runx1-IRES-hCD2 plus the pCL-10A1 packaging plasmid by using Fugene reagents (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Polybrene (4 µg/ml) was added to the viral supernatant, which was then added to 1200M cells in 6-well plates. The cells were spun at 2,500 rpm for 2 h at room temperature. Fresh medium was then added, and cells were incubated at 37°C under 5% CO₂ overnight. For RNA interference experiments, transduced cells were selected in 1 µg/ml of puromycin.

Fluorescence-activated cell sorting (FACS). Half a million cells were stained with fluorescein isothiocyanate- or phycoerythrin-conjugated anti-mouse CD4 (GK1.5; BD Biosciences-Pharmingen, San Diego, CA) or a rat immunoglobulin G2b() isotype control (BD Biosciences-Pharmingen), or with biotin-conjugated anti-human CD2 (B-E2; Diaclone Research, Besancon, France) antibodies in conjunction with allophycocyanin-conjugated streptavidin (Caltag Laboratories, Burlingame, CA), and were analyzed using FACSCalibur flow cytometers and CellQuest software (BD Biosciences-Pharmingen).

Western blotting. Runx1 and CycT1 protein levels were determined by Western blotting with rabbit polyclonal anti-panRunx and rabbit polyclonal anti-Cyc T1 (sc-10750; Santa Cruz Biotechnology) antibodies, respectively.

	RESULTS AND DISCUSSION

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Looping ChIP reveals differential interactions between the enhancer, silencer, and promoter in CD4^– and CD4⁺ cells. To investigate the role of chromatin structure in the expression of the CD4 gene in thymic development, we first performed ChIP assays and examined the recruitment of RNAPII, P-TEFb, and Runx1 to the CD4 locus. The expression of the mouse CD4 gene in immature T cells is controlled by a proximal promoter (P), an enhancer (E) located about 12 kb upstream of the transcription start site (TSS), and a silencer (S) within the first intron, 2.3 kb downstream from the TSS (Fig. 1A) (reviewed in reference 4). 3A9 is a T-cell hybridoma that represents CD4 SP thymocytes (Fig. 1B) (24). Previously, we demonstrated that RNAPII is present on the CD4 promoter as well as in the coding region (about 1 kb from the TSS) in these cells (7). In Fig. 1C, we demonstrate that RNAPII is also present on the CD4 enhancer in 3A9 cells (lane 2). Although it was reported that the locus control region of the human -globin locus can deliver RNAPII to the cis-linked promoter via facilitated tracking (31), we did not detect any transcription complexes in the intervening region (I) between the CD4 enhancer and promoter (Fig. 1C, panels I). Thus, this situation does not pertain to the CD4 gene. On the other hand, 1200M is a thymoma that represents ISP thymocytes, which do not express CD4 (Fig. 1B). It has been used extensively to characterize mechanisms of CD4 repression during thymocyte development. In these cells, RNAPII is detected on the enhancer and promoter but not on the silencer (Fig. 1C, lane 8), consistent with our previous finding that RNAPII is arrested at the promoter and cannot elongate in CD4^– cells (7). That RNAPII is found on the CD4 enhancer is consistent with reports of sterile transcription from immunoglobulin (19) and major histocompatibility complex (MHC) class II (15) enhancers. The presence of CycT1 on the CD4 enhancer, promoter, and silencer in 3A9 cells (Fig. 1C, lane 3) is consistent with P-TEFb being recruited to proximal and distal sites and traveling with RNAPII during elongation (23). However, in 1200M cells, CycT1 is present at the CD4 enhancer but not at the promoter (Fig. 1C, lane 9), indicating that P-TEFb is recruited only to the distal enhancer in these cells. More surprisingly, the CD4 silencer was immunoprecipitated by anti-CycT1 antibodies in these cells (Fig. 1C, lane 9). This observation suggested that chromatin looping brought the CD4 enhancer close to the silencer in 1200M cells, allowing Runx1 to exert its inhibitory effects on P-TEFb. This hypothesis was confirmed by the presence of both enhancer and silencer fragments in anti-Runx1 immunoprecipitates from 1200M cells (Fig. 1C, lanes 10 and 11). Importantly, no Runx1 binding to the CD4 locus was observed in 3A9 cells (Fig. 1C, lanes 4 and 5).

View larger version (28K): [in this window] [in a new window]   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() 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.

View larger version (26K): [in this window] [in a new window]   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.

View larger version (29K): [in this window] [in a new window]   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.

View larger version (23K): [in this window] [in a new window]   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.

	ACKNOWLEDGMENTS

 
We thank Dan Littman and Takeshi Egawa for fruitful discussions and helping us to isolate primary thymocytes.

This work was supported by grants from the NIH and the Nora Eccles Treadwell Foundation.

We declare no conflict of interest with any aspects of this work.

	FOOTNOTES

 
* Corresponding author. Mailing address: Box 0703, UCSF, 3rd and Parnassus Avenues, San Francisco, CA 94143-0703. Phone: (415) 502-1905. Fax: (415) 502-1901. E-mail: matija.peterlin{at}ucsf.edu

Published ahead of print on 26 November 2007.

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