CTCF mediates long-range interaction between silencer Sis and enhancer Ei and inhibits VJ rearrangement in pre-B cells

Introduction

It is becoming increasingly evident that precise regulation of eukaryotic gene expression requires communication between cis-acting elements through alterations in higher order chromatin structure [1-4]. The mouse Igk light chain gene locus represents an ideal system to elucidate a paradigm for nuclear reorganization during cellular differentiation [5, 6]. The Igk locus contains 95 potentially functional Vk genes, four functional and one nonfunctional Jk regions, a single Ck region. The locus also possesses a silencer Sis and three transcriptional enhancers: an intronic enhancer (Ei), a 3’ enhancer (E3’), and a further downstream enhancer (Ed) [7]. In pre-B cells, one Vk gene carrying its own promoter is semi-randomly rearranged to one Jk region. This VJ rearrangement results in transcriptional activation because it puts the Vk gene promoter under the control of three powerful enhancers. Sis and Ei are reported to regulate VJ rearrangement. Sis inhibits VJ rearrangement through targeting the Igk locus to pericentromeric heterochromatin whereas Ei has a positive role in VJ rearrangement through activation of germline transcription [8, 9].

CCCTC-binding factor (CTCF) is a highly conserved zinc finger protein implicated in diverse regulatory functions, including transcriptional activation/repression, insulation, imprinting [10-13]. Moreover, CTCF mediates intra- and interchromosomal contacts at several developmentally regulated genomic loci [14-17]. It is reported that CTCF associates with Sis in pre-B cells. However, the role of CTCF is not clear. Here we demonstrate that CTCF associates with Sis and Ei, mediates long-range chromatin interaction between Sis and Ei, inhibits germline transcription and downregulates VJ rearrangements in pre-B cells.

Material and methods

Cell culture



70Z/3 and P815 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 50 µM 2-ME, 100 U of penicillin/ml, and 100 µg of streptomycin/ml at 37°C with 5% CO2.



Chromosome conformation capture 3C assays were performed as described previously [18]. Cells (106) were

cross-linked, lysed, and nuclei were digested with BglII. After ligation and subsequent DNA purification, the cross-linking frequencies between the anchor and test fragments, as measured by the amount of the corresponding ligation product, were estimated by PCR reactions relative to standards. DNA including Sis, Ei, E3’ and Ed was purified from E.coli cells and was ligated at high concentration following BglII digestion to generate equamolar mixtures of all possible ligation products, and was used to generate standards. The cross-linking and ligation efficiencies between different samples and different experiments were normalized by setting the highest cross-linking frequency for each experimental series to 1.0. Error bars represent the SDs from three to five independent experiments as indicated in the figure legends. Primers used in this study are listed in Table 1.



Chromatin immunoprecipitation assay



Chromatin immunoprecipitation (ChIP) assays were performed as described previously [19]. Antibody against CTCF was purchased from Millipore. Results were quantified by real-time PCR with SYBR Green dye using the ABI Prism 7500 system (Applied Biosystems). All PCR signals from immunoprecipitation samples were referenced to their respective inputs to normalize for differences in primer efficiencies. -actin was treated as a negative control and its enrichment fold was considered as 1.0. The enrichment of every test fragment was referenced to -actin. Primers used in this study were listed in Table 1.



Short hairpin RNA (shRNA) knockdown of CTCF



shRNA sequence for RNA interference of CTCF

were described previously [20]. The effective sequence proved to be 5'-GATCCCCGCAGAGAAAGTAGTTGG TAATTTCAAGAGAATTACCAACTACTTTCTCTGCTTTTTA-3'. The lentiviral transfer construct pCCL. PPT.hPGK.GFP.Wpre, as well as helper viruses pMD2. VSVG, pMDLg/pRRE, and pRSV-REV were transfected into Fx293T cells. Lentiviruses were harvested and infected into target cells 70Z/3 as previously described [21].



Immunoblots



The following antibodies were used: CTCF (Millipore, Billerica, MA, USA) and Actin (Millipore, Billerica, MA, USA).



Real-time PCR for germline transcription



Total RNA, extracted from 10 × 105 cells using TRIzol reagent (Invitrogen, Carlsbad, CA), was reverse transcribed into cDNA with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCR was performed with the following primers: sense 5'-GCACACTTAGCTCTCATTTCCCACCCCAG-3' and antisense 5'-ATGAACTA GAAATTAATATTGTTTGTCTG-3'.



PCR amplification assays for Vk-Jk joining



Genomic DNA was purified from cell populations as described previously [22]. For the assay of Vkhf24 rearrangement to Jk1 and Jk2 only, the reverse primer used was 5'-TCATACAAAGGACACAATGGGAC-3'. The family-specific forward Vkhf24 primers used were 5'- TGGGTCAGGAACTGCTTTCACACT-3'. PCR products were separated in 1% agarose gels.

Results and discussion

Recombination silencer, Sis, exhibits interaction with an intronic enhancer, Ei, in 70Z/3 cells



Sis (silencer in the intervening sequence) was discovered as a cluster of four DNase I hypersensitive sites within Igk-locus-chromatin that reside in the intervening sequence between the closest Vk gene and the Jk region [23]. In subsequent studies we showed that Sis acted as a recombination silencer and could target germline Igk transgenes to pericentromeric heterochromatin in pre-B cells [8]. The intronic enhancer Ei resides within the Jk and Ck region and it exhibits a significant function in activating VJ rearrangement. Subsequent studies showed that Ei activates germline transcription to open Igk chromatin to facilitate the association of recombination complexes. To explore how Sis and Ei cooperate to regulate VJ recombination, we studied the proximity of these two cis-regulatory elements using chromosome conformation capture (3C) technology in pre-B cells 70Z/3. In brief, cross-linked chromatin was digested at a commonly-occurring site with BglII, diluted, re-ligated, and long-range association frequencies were assessed with PCR. When the Ei fragment was used as a PCR anchor, a very strong association was observed between Ei and Sis in 70Z/3 cells. In P815 cells, a non-B cell-negative control with inactive Igk loci, however, the interaction between Ei and Sis was not detected (Fig. 1A, B). Therefore, silencer Sis and intronic enhancer, Ei, physically interact with each other in pre-B cells.



CTCF binds to Sis and Ei elements and mediates Sis-Ei physical interaction



We next asked what protein mediates this Sis-Ei physical interaction. Feeney and co-workers have found that Sis possesses bound CTCF in pre-B cells, a protein known to mediate silencing and DNA looping in other systems [24]. Consistently with this report, our ChIP analysis detected the association of CTCF in Sis in 70Z/3 cells and besides that, CTCF was also found to bind to Ei (Fig. 2A). It should be pointed out that Ei does not contain CTCF binding site, the detection of association of CTCF with Ei might be indirect. For example, CTCF associates with Sis. Sis physically interacts with Ei. The whole looping complex was pulled down by CTCF antibody. This result suggests that CTCF may be present in the Sis-Ei looping complexes.

To explore the role of CTCF in Sis-Ei loop formation, we knocked down CTCF in 70Z/3 cells. As shown by the western blot in Fig. 2B, about 90% of CTCF was knocked down.

We then employed 3C to examine the change of Ei-Sis physical interaction in the presence or absence of CTCF. When the Sis fragment is used as a PCR anchor, a very strong association was detected between Sis and Ei in 70Z/3-iluc cells. But this interaction was lost in 70Z/3-iCTCF cells (Fig. 2C). The same result was observed with Ei as an anchor fragment (Fig. 2D). These results indicate that CTCF plays an essential role in mediating the interaction between Sis and Ei.



CTCF inhibits VJ rearrangement through repressing germline transcription



It is the first report showing that the silencer and enhancer can physically interact with each other. To address the role of Sis-Ei physical interaction in VJ recombination, we disrupted the colocalization of Sis and Ei by knocking down CTCF. We then used PCR assay to analyze the VJ rearrangement. The forward primer resides within Vk gene hf24 that is the most upstream Vk gene. The reverse primer locates downstream of Jk2 region. PCR products generated from these two primers represent the rearrangement of Vkhf24 to Jk1 or Jk2. To quantitate results, we performed PCR assays with one, two, and three times template DNA concentrations. Fig. 3A showed that a two-fold increase of hf24-J1 and hf24-J2 rearrangement in CTCF knock-down 70Z/3 cells, indicating that CTCF mediated Sis-Ei looping interaction inhibits VJ rearrangement. This result implies that the interaction between silencer and enhancer may interrupt the function of enhancer.

Since VJ rearrangement requires germline transcription in both Vk gene and Jk region and Ei plays an important role in activation of germline transcription. We next examined the change of germline transcription. Two germline transcription starting sites were identified in the upstream of Jk region. The transcription generated from these two starting sites are termed 5’germline transcription and 3’germline transcription. As shown in Fig. 3B, the level of 5’germline transcripts increased 3-fold in 70Z/3-iCTCF cells compared with those in 70Z/3-iluc cells, indicating that CTCF mediated Sis-Ei interaction represses germline transcription.

70Z/3 is a late pre-B cell line. It contains a rearranged Vk4-J1 gene. The VJ rearrangement in one allele inhibits activation of the other allele and this is termed allelic exclusion. Allelic exclusion plays an essential role in ensuring that a single B cell expresses only one kind of antigen receptor (BCR). This is important not only for optimizing antibody-antigen specificities by single cells but also for preventing autoimmune reaction because autoimmunity could result if a single cell expressed at least two different BCRs, one directed at an infectious agent and the other against a self-antigen. Previous studies have shown that allelic exclusion of the mouse Igk locus occurs by the monoallelic silencing and a low level of monoallelic activation for rearrangement combined with a negative feedback loop blocking additional functional rearrangements due to downregulation of RAG expression [25, 26]. Our results lead us to suggest that CTCF-mediated physical interaction between Sis and Ei blocks the function of Ei in stimulating germline transcription and promoting VJ joining. Thus, we revealed a novel mechanism that participates in the monoallelic silencing aspect of allelic exclusion regulation.



The authors declare no conflict of interests.



This work was supported by grants (91019012, 31071128 to Z.L., 81071730 to Z.M., 81101546 to X.L.) from the National Natural Science Foundation of China and grants (11JCZDJC19000 to Z.L. and 11JCZDJC18700 to Z.M.) from Tianjin Municipal Science and Technology Commission, grant 20110490792 to X.L. from China Postdoctoral Science Foundation, grant 2009CB918903 to Z.Y. from the Ministry of Science and Technology of China.

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