Genome-Wide Analysis Identifies Rag1 and Rag2 as Novel Notch1 Transcriptional Targets in Thymocytes

Abstract

Recombination activating genes 1 (Rag1) and Rag2 are expressed in immature lymphocytes and essential for generating the vast repertoire of antigen receptors. Yet, the mechanisms governing the transcription of Rag1 and Rag2 remain to be fully determined, particularly in thymocytes. Combining cDNA microarray and ChIP-seq analysis, we identify Rag1 and Rag2 as novel Notch1 transcriptional targets in acute T-cell lymphoblastic leukemia (T-ALL) cells. We further demonstrate that Notch1 transcriptional complexes directly bind the Rag1 and Rag2 locus in not only T-ALL but also primary double negative (DN) T-cell progenitors. Specifically, dimeric Notch1 transcriptional complexes activate Rag1 and Rag2 through a novel cis-element bearing a sequence-paired site (SPS). In T-ALL and DN cells, dimerization-defective Notch1 causes compromised Rag1 and Rag2 expression; conversely, dimerization-competent Notch1 achieves optimal upregulation of both. Collectively, these results reveal Notch1 dimerization-mediated transcription as one of the mechanisms for activating Rag1 and Rag2 expression in both primary and transformed thymocytes. Our data suggest a new role of Notch1 dimerization in compelling efficient TCRβ rearrangements in DN progenitors during T-cell development.

Keywords: Double negative thymocyte; Notch1 dimerization; Recombination activating genes; T-cell acute lymphoblastic leukemia; T-cell development.

FIGURE 1

Notch signaling induces Rag1 and Rag2 expression. (A) Identification of Notch1 dimer-dependent target genes. Notch1-dependent genes were determined from a microarray analysis in murine T-ALL T6E cells (GSE97465). Each gene expression is displayed with colored boxes which are scaled in the bottom. ICN1 expression means Notch1 monomer and dimer activation, and R1984A expression denotes only Notch1 monomer activation. Expression data from 20 genes most strictly dependent on the Notch1 dimer and 20 genes equally induced by both Notch1 dimer and monomer are depicted (p < 0.05). (B) T6E cells were treated with JC-18 (1 μM) or DMSO for 24 h. Rag1 and Rag2 mRNA and proteins were detected by real-time PCR (left) and immunoblots (right). (C) T6E cells were transduced with DNMAML1 or empty vector MigR1 surrogated with a GFP marker for 24 h. GFP⁺ cells were sorted and subjected to mRNA analysis by real-time PCR. (D) DN3a and DN3b cells were isolated from C57BL/6 mice. Rag1 and Rag2 mRNA, along with positive control Notch1 mRNA, were evaluated by real-time PCR. (E) T6E cells were infected by retroviruses expressing MigR1, WT ICN1, or designated dimerization mutants, then subjected to JC-18 treatment for 24 h. Rag1 and Rag2 mRNA levels were examined by real-time PCR. Above all, the expression of Rag1 and Rag2 relative to 18S RNA is shown as the mean of values from triplicate wells ± SD. Representative data from three independent experiments are shown. ***p < 0.001, **p < 0.01, and *p < 0.05.

FIGURE 2

Notch1 directly binds the Rag1/2 locus in T-ALL cells and T-cell progenitors. (A) Analysis of the Rag2 locus in the ChIP-seq data (GSE 29600). The binding signals of Notch1 and RBPJ on the -2.8 kb site upstream of the Rag2 gene are highlighted in light gray. (B–D) Chromatin immunoprecipitation (ChIP) was performed on cross-linked fragmented DNAs prepared from T6E cells treated with DMSO or 1 μM JC-18 for 24 h (B), murine primary T-ALL samples (C), or murine DN2 and DN3a cells (D). Eluted DNAs were then analyzed by qPCR using primers flanking the -2.8 kb site. The amount of DNA amplified from immunoprecipitated DNAs was normalized to that amplified from input DNA. Each sample was prepared from at least two independent experiments and run in triplicates. Rag2 NS, non-specific sequence in Rag2 locus; Neg Con, non-specific sequence in the Nanog gene used as the negative control. **p < 0.01 and *p < 0.05.

FIGURE 3

Dimeric Notch1 directly regulates Rag2 transcription. (A) Schematic presentation of the predicted mouse Rag2 SPS. The SPS, within the Rag2 enhancer, consists of a high affinity site (marked by frames) and a low affinity site (marked by dark gray). The high and low affinity sites were mutated as indicated (red), Mut-high and Mut-low, for the following luciferase assays. (B) The Rag2 SPS was constructed to luciferase reporter vector pGL3, then co-transfected with WT ICN1 or indicated mutants. Reporter activities normalized to an empty vector were determined and presented as fold induction. (C) The WT Rag2 SPS or indicated mutants were constructed to luciferase reporter vector pGL3, then co-transfected with WT ICN1. Reporter activities normalized to an empty vector were determined and presented as fold induction. All of the results were shown as the mean of values from triplicate wells ± SD. Data are representative of three independent experiments. ^∗∗p < 0.01.

FIGURE 4

Enforced expression of dimeric Notch1 activates the Rag2 reporter expression in hematopoietic progenitors. (A) Schematic presentation of the experimental procedure. GFP^– LSK (Lin^–Sca⁺c-Kit⁺) cells derived from RAG2-GFP reporter mice (NG-BAC) were transduced with viruses expressing ICN1 or R1984A with NGFR as surrogated marker. The infected cells were co-cultured with stromal cells OP9 for 48 h and then measured the GFP⁺ percentage as an indication of Rag2 expression. (B) Flow cytometry analysis of GFP and NGFR-PE expressions in hematopoietic cells recovered from OP9 culture. (C) A histogram presentation of GFP⁺ cell percentages in NGFR-negative or NGFR-positive populations in panel (B). ***p < 0.001.

FIGURE 5

Dimeric Notch1 activates Rag1 and Rag2 during thymocyte development. (A) Schematic presentation of the experimental procedure. LSK cells derived from C57BL/6 mice were transduced with retroviruses expressing the MigR1 vector, ICN1, or R1984A with GFP as a marker. After transduction, the infected cells were co-cultured with murine OP9 stromal cells for 96 h. GFP⁺ Thy1⁺ T-lineage cells were sorted for mRNA quantification. (B–D) qRT-PCR analysis showing the mRNA expression of Rag1 (B), Rag2 (C), and Ptcra (D) in sorted cells. ***p < 0.001.