- Open Access
Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation
Journal of Translational Medicine volume 21, Article number: 217 (2023)
T cell activation and programming from their naïve/resting state, characterized by widespread modifications in chromatin accessibility triggering extensive changes in transcriptional programs, is orchestrated by several cytokines and transcription regulators. PRDM1 and PRDM2 encode for proteins with PR/SET and zinc finger domains that control several biological processes, including cell differentiation, through epigenetic regulation of gene expression. Different transcripts leading to main protein isoforms with (PR +) or without (PR-) the PR/SET domain have been described. Although many studies have established the critical PRDM1 role in hematopoietic cell differentiation, maintenance and/or function, the single transcript contribution has not been investigated before. Otherwise, very few evidence is currently available on PRDM2. Here, we aimed to analyze the role of PRDM1 and PRDM2 different transcripts as mediators of T lymphocyte activation.
We analyzed the transcription signature of the main variants from PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) genes, in human T lymphocytes and Jurkat cells overexpressing PRDM2 cDNAs following activation through different signals.
T lymphocyte activation induced an early increase of RIZ2 and RIZ1 followed by BLIMP1b increase and finally by BLIMP1a increase. The “first” and the “second” signals shifted the balance towards the PR- forms for both genes. Interestingly, the PI3K signaling pathway modulated the RIZ1/RIZ2 ratio in favor of RIZ1 while the balance versus RIZ2 was promoted by MAPK pathway. Cytokines mediating different Jak/Stat signaling pathways (third signal) early modulated the expression of PRDM1 and PRDM2 and the relationship of their different transcripts confirming the early increase of the PR- transcripts. Different responses of T cell subpopulations were also observed. Jurkat cells showed that the acute transient RIZ2 increase promoted the balancing of PRDM1 forms towards BLIMP1b. The stable forced expression of RIZ1 or RIZ2 induced a significant variation in the expression of key transcription factors involved in T lymphocyte differentiation. The BLIMP1a/b balance shifted in favor of BLIMP1a in RIZ1-overexpressing cells and of BLIMP1b in RIZ2-overexpressing cells.
This study provides the first characterization of PRDM2 in T-lymphocyte activation/differentiation and novel insights on PRDM1 and PRDM2 transcription regulation during initial activation phases.
The activation and programming of T cells from their naïve/resting state is a critical point for the functions of the adaptive immune response. This process, characterized by widespread changes in chromatin accessibility triggering extensive changes in transcriptional programs, is orchestrated by a plethora of signals including cytokines, inflammatory and immune-modulatory products, tissue-specific and transcription regulators that ultimately influence the developmental choices of the T cell .
PRDF1 and RIZ1 homology domain containing proteins (PRDMs) are a family of structurally related transcriptional regulators, controlling several biological processes, including differentiation of a variety of cell types . Their protein structure exhibits several zinc fingers preceded by the PR/SET domain, which is a SET-like domain typical of methyltransferases [3, 4]. PRDMs function in epigenetic regulation of gene expression through their intrinsic histone methyltransferases (HMTase) and/or via interactions with chromatin modifying enzymes. For instance, no methyltransferase activity has been demonstrated for PRDM1, which achieves its repressor activity through other proteins, including histone deacetylases [5,6,7,8,9] whereas the conserved N-terminal PR domain of PRDM2/RIZ1 has an intrinsic H3K9 methyltransferase activity [10, 11]. PRDM genes display a characteristic yin-yang expression pattern where alternative splicing or different promoter use rises to a full-length protein (PR +) and a shorter isoform lacking the PR domain (PR −) [12,13,14]. An imbalance of these isoforms is often observed in several malignancies, including leukemias and lymphomas, with the PR + product commonly lost or downregulated both by genetic inactivation or epigenetic silencing and the PR − isoform always expressed at high concentration levels [12,13,14].
PRDM1 gene, widely known as BLIMP1 (B lymphocyte-induced maturation protein 1), encodes for two PR + and PR − major isoforms designated as BLIMP1a and BLIMP1b respectively, which arise from alternative promoter usage. Indeed, BLIMP1b is transcribed from a promoter and exon positioned upstream of exon 4 of the PRDM1 gene. The BLIMP1b protein lacks the first 101 amino acids of BLIMP1a N-terminal region and instead contains three amino acids fused to residues 102–789 of BLIMP1a (see scheme in Fig. 1) . Currently, a lot of studies support its critical role in differentiation, maintenance and/or function of multiple hematopoietic cells of the myeloid and lymphoid lineages . BLIMP1a orchestrates plasma cell differentiation by repressing genetic programs associated with the germinal center stages, while at the same time activating those programs associated with plasma cell functions. Specifically, PRDM1/BLIMP1 drives plasma cell differentiation by repressing several transcription factors, such as Myc, CIITA (MHC class II trans-activator), PAX5 (paired box 5), which is required for B cell fate specification, and BCL6 (B cell lymphoma 6), which promotes germinal centre B cell identity [16,17,18]. Notably, BCL6 also acts as a transcriptional repressor and forms a negative loop with PRDM1/BLIMP1 . Besides, PRDM1 expression is induced in activated CD4+ and CD8+ T lymphocytes where it regulates many aspects of their differentiation in effector and memory cells . Indeed, PRDM1/BLIMP1 also acts as a repressor in subsets of effector CD4+ and CD8+ T cells [20,21,22,23,24,25,26]. As for B cells, a reciprocal repression of BCL6 and PRDM1 is crucial for T lymphocytes differentiation. In this cell context, PRDM1/BLIMP1 represses IL-2 (interleukin 2). IL2 regulates the initial expansion of naïve T cells then, acting through STAT5 (signal transducer and activator of transcription 5), it induces PRDM1 expression creating a feedback loop that downregulates its own expression during the later stages of T-cell differentiation [27, 28]. PRDM1/BLIMP1 can act also as a transcriptional activator in regulatory T cells by promoting H3K4 methylation at the IL10 (interleukin 10) locus . A BLIMP1a/BLIMP1b imbalance has been detected in several malignancies derived from B, T, and NK cells [reviewed in 12, 29]. Different regulatory mechanisms, both transcriptional and posttranslational, have been reported to control PRDM1/BLIMP1 expression. Numerous transcription factors, including BCL-6, PAX5, and BACH2, as well as TNFR and cytokine signaling pathways, are known to directly affect PRDM1 transcription [30,31,32,33,34].
As PRDM1, also PRDM2 gene expresses two main proteins, PRDM2/RIZ1 (PR +) and PRDM2/RIZ2 (PR −), starting from transcripts initiated by two alternative promoters (see scheme in Fig. 1). PRDM2 is expressed in immature progenitor cells and in mature myeloid cells . A selective PRDM2/RIZ1 expression was correlated with myeloid cell differentiation, suggesting a pivotal role for PRDM2 gene products in the proliferation/differentiation transition . Loss of heterozygosity, promoter hypermethylation or PRDM2 gene mutations have been observed in several human cancer types, including Diffuse Large B-Cell Lymphoma (DLBCL) [10, 37]. For example, Riz1 KO mice, which retain Riz2 expression, show a high incidence of DLBCLs and other rare non-hematopoietic cancers . Furthermore, PRDM2 loss is associated with the chronic myelogenous leukemia (CML) collapse to blast crisis . Noteworthy, several reports indicated that PRDM2/RIZ1 is endowed with tumor suppressor activities, whereas PRDM2/RIZ2 acts as an oncogene with putative intrinsic growth-promoting properties [10, 38].
Although previous findings suggest that PRDM2 proteins could have a role in the regulation of the proliferation/differentiation transition in myeloid cells, more extensive investigations in other models (e.g. lymphocytes, hematopoietic stem cells) are advised to confirm a general role of PRDM2 products in the transition from proliferative activity to a quiescent/differentiate state and vice versa [35, 36].
The aim of this study was to evaluate the mechanisms that regulate the expression of the main molecular variants encoded by PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) genes, both in lymphocytes from healthy donors and in the Jurkat cell line. Specifically, we aimed to investigate the possible action of RIZ1 and RIZ2 on the gene expression regulation of BLIMP1a and BLIMP1b transcripts and their role in lymphocytes maturation as effectors of the immune response.
Isolation, purification, and stimulation of human CD4 + and CD8 + cells
Human peripheral T lymphocytes were obtained from total peripheral blood mononuclear cells (PBMCs) collected from 10 human samples (60–200 ml, depending on the experimental needs) of 4 healthy donors (authors who contributed to the present manuscript). PBMCs were isolated by density gradient centrifugation (400xg for 30’ at room temperature) on FICOLL-Paque PLUS (GE healthcare Amersham) in 50 mL tubes. After centrifugation the layer of PBMCs was carefully removed and transferred to a new conical tube. Cells were diluted with RPMI-1640 and centrifuged at 200xg for 5’ to remove platelets.
To obtain T naïve cells, PBMCs were resuspended in RPMI-1640 and then transferred to PERCOLL (GE healthcare) 40% and 35%. After centrifugation at 700xg for 25 min at room temperature, cells were washed, suspended in an appropriate volume of RPMI-1640 and counted. T naïve cell concentration was about 6–7 × 106/mL.
To obtain CD4+ and CD8+ T lymphocytes, T naïve cells were incubated for 20’ with specific RosetteSep™ (STEMCELL Technologies), diluted with RPMI-1640 (1:2) and then transferred to FICOLL-Paque PLUS (GE healthcare Amersham) in 50 mL tubes. Isolated cells were analyzed by 6 Color TBNK + Truc assay with BD FACSLyric™ flow cytometer (Becton Dickinson) (Additional file 1: Fig S1).
Isolated cells were maintained in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) at 37 °C with 5% CO2 in a humidified atmosphere. For kinetic analysis, purified CD4+ T cells from PBMC were grown in RPMI-1640 25% homolog serum.
T lymphocyte activation and treatments
To induce T lymphocyte activation, 1 × 106 cells were grown in RPMI-1640 5% FBS at 37 °C with 5% CO2 in a humidified atmosphere supplemented with polyclonal activators 50 ng/mL phorbol 12-myristate 13-acetate (PMA) plus 1 μM Ionomycin A (Ion A) (Sigma Aldrich) for 2 h and 6 h. Activation of T cells receptor (TCR) was carried out with antibodies anti-CD3 and anti-CD28 bound to magnetic beads (Dynabeads™, Thermofisher). The percentage of activated cells was measured at 48 h by BD FACSLyric™ flow cytometer (Becton Dickinson) with BD Multitest™ CD8/CD38/CD3/HLA-DR and anti-CD4 PECy7 and anti-CD45 V500C (Becton Dickinson) (Additional file 1: Fig S1).
The mean percentage of activated cells was 33% (SD = ± 8,2%).
RPMI-1640 was supplemented with IL-2 (30U/mL)(Merck), IL-4 (200 ng/mL), IL-6 (200 ng/mL), IFN-α (180 ng/mL) and IFN-γ (200 ng/mL)(GIBCO™, Thermofisher) where described.
PI3K inhibitor, LY294002 (Ly) or Wortmannin (Calbiochem), were used at final concentration of 10 μM and 1 µM, respectively. The MEK1/2 inhibitor PD98059 (PD) and the Src kinase inhibitor PP1 (Alexis) were used at a final concentration of 5 µM.
Jurkat cell culture and transfection
Jurkat E6.1 (CLS Cell Lines Service GmbH) cells were grown at 37 °C with 5% CO2 in humidified atmosphere in RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin).
The plasmid expressing RIZ1 and RIZ2, were indicated as pSG5_hRIZ1 and pSG5_hRIZ2, respectively. pSG5_hRIZ1 was obtained by cloning in BamH1 site the RIZ1 cDNA from pGEX-RIZ  in the FLAG-pSG5 vector. The RIZ2 insert was obtained by PCR amplification of the RIZ1 cDNA sequence with the enzyme PfuTurbo DNA Polymerase (Stragene) using a forward primer containing the BamH1 site and the RIZ2 codon start site and a reverse primer, RIZ2-R, with the stop coding sequence and the BamH1 site (see primer table). To select stable clones of Jurkat cells transfected with pSG5_hRIZ1 and pSG5_hRIZ2, the plasmid pEGFP-C1 (CLONTECH-TaKaRa) was added to the transfection mixture (1:30 compared to FLAG-pSG5, pSG5_hRIZ1 and pSG5_hRIZ2). The EGFP expression was used to evaluate the transfection efficiency and for the resistance to the antibiotic Geniticin G418 (Sigma-Aldrich).
Plasmids for transfection were prepared with Plasmid Midi Kit (Qiagen), according to manufacturer’s instructions.
Cells were transfected using Lipofectamine™ 2000 Reagent in OptiMem I Reduced Serum Medium (Invitrogen™, Thermofisher) for 6 h, following the manufacturer’s instructions. Reaction mixtures were removed, and cells were cultured for an additional 24 h in RPMI1640 with 15% FBS. For Jurkat E6.1 stable clones, selection was performed by addition of 0.8 mg/ml Geneticin G418 (Sigma-Aldrich). pSG5_hRIZ2 shows an epitope tagged by replacing the terminal stop codon with nucleotides encoding FLAG residues (MDYKDDDDK) by PCR. FLAG-pSG5 vector was used as a control together with pEGFP-C1, used as a transfection marker.
RNA extraction, Semi-Quantitative and Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from cells using Trizol solution (Invitrogen™, Thermofisher), according to the manufacturer's instructions. The quality and quantity of RNA was assessed by denaturing agarose gel electrophoresis and by spectrophotometry analysis (NanoDrop Technologies), after RNAse-free DNAse-I treatments (Invitrogen™, Thermofisher). RNA was reverse transcribed with SuperScript III (Invitrogen™, Thermofisher) using 500 ng of total RNA; 1 μl of cDNA (60 ng) was used as a template in a PCR reaction with 2.5 U AmpliTaq-Gold (Applied Biosystem™ Termofisher) as previously described . The amplification products were also analyzed by agarose gel electrophoresis. Glyceraldehydes-3-phophate dehydrogenase (GAPDH) was used as housekeeping control gene. Densitometry analysis was performed by ImageJ software and differences were assessed by Student’s t-test with a P < 0.05 considered as significant .
Quantitative RT-PCR analysis was performed using the SYBR Green PCR Master Mix (Applied Biosystem™ Termofisher),160 nM of each primer and about 50 ng of cDNA (RNA equivalent) as template in an iCycler thermocycler (Bio-Rad Laboratories). PCR condition were 95 °C for 2 min followed by 45 cycles of 15 s at 95 °C, 60 s at annealing temperature of each primer set and 30 s at 72 °C. All reactions were carried out at least in triplicate for every cDNA template and the melting curves were analyzed to verify the specificity of reaction. The relative gene expression was calculated using the 2−ΔΔCt Pfaffl method . GAPDH or RPS18 were used as housekeeping genes for normalization as indicated .
Primer sets were designed with Primer3 (Table S1). BLAST and BLAT algorithms at NCBI (http://www.ncbi.nlm.nih.gov) and at Genome Browser (Santa Cruz) (http://genome.ucsc.edu/cgi-bin/hgGateway) were used for sequence comparison and analysis of transcripts.
Western Blot assay and Densitometric Analysis
Total cell extracts were separated onto SDS–polyacrylamide gels and blotted onto polyvinylidene difluoride membranes . Western blot analysis was performed as described elsewhere  with mouse monoclonal antibody FLAG™ clone M2 (Sigma-Aldrich) and rabbit polyclonal antibody RIZ1 (Abcam Ltd.).
Results are reported as mean ± SD. Three independent experiments in triplicates were performed. GraphPad Prism 9,5 software was utilized to perform Brown-Forsythe and Welch ANOVA tests. Significances were indicated as: *p < 0.05 and particularly significant at **p < 0.01, ***p < 0.001, ****p < 0.0001. Expression correlation analysis was evaluated on the GEPIA2 database (http://gepia.cancer-pku.cn/) using the Spearman correlation coefficient R.
Polyclonal activation of T Lymphocytes increases PRDM1 and PRDM2 expression levels
To investigate the role of the main molecular variants encoded by PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) (see scheme in Fig. 1) in the lymphocyte activation we firstly used human peripheral blood lymphocytes and activated them with T cell polyclonal activator phorbol 12-myristate 13-acetate and ionomycin (PMA/Ion) or with anti-CD3/CD28 antibodies at 2 h and 6 h.
Activation of T lymphocytes involves the expression changes of several molecules, both on their surface and within the cell, which clearly characterize them from naïve T cells and can be evaluated by several methods, including gene transcription analysis [43,44,45].
In our pilot experiment of RT-PCR analysis, we observed changes of PRDM1 and PRDM2 transcription together with the expression modifications of genes encoding for transcription factors and cytokines involved in lymphocyte activation and differentiation (CD40LG, KLF2, IL2RA, IL2 and MYC) (Additional file 1: Fig. S2).
PRDM2 gene expression was verified using two sets of primers recognizing sequences exclusive of RIZ1 or a common region to both RIZ1 and RIZ2 (and indicated as RIZex8). Because of the extensive similarity between the two gene products, RIZ2 transcript was measured by subtraction as previously described . Similarly, for PRDM1 two sets of primers, recognizing specific sequences of BLIMP1a and BLIMP1b transcripts, were used (see scheme in Fig. 1).
To further detail the early events and the kinetics of PRDM1 and PRDM2 main transcripts we evaluated their expression by qRT-PCR analysis in human CD4+T lymphocytes (greater than 92,5% of total T cells) activated with anti-CD3/CD28 antibodies at different time points (0, 30’, 1 h, 2 h, 4 h, 6 h and 24 h) (Fig. 1). The expression of CD40LG and IL2RA was also analyzed to monitor lymphocyte activation (Fig. 1).
An expression level increase in RIZ1 and RIZ2 transcripts (at major extend) was observed already at 30’ upon lymphocytes activation. At subsequent time points, the RIZ1 expression is constant over the time, while RIZ2 fluctuates until 6 h where no RIZ2 expression was detected (Fig. 1). BLIMP1a is not expressed in basal conditions and its expression grows progressively to reach the maximum expression level at 4 h. After this time point, the BLIMP1a slows down and maintains a high expression level than the starting point (Fig. 1). An increase in BLIMP1b expression level was detected already 30’ after T cell activation, and gained the maximum expression level at 2 h, which is sustained at 4 h. Thereafter, as BLIMP1a, also BLIMP1b slows down and maintains a higher expression level than the starting point (Fig. 1). An increase in the expression levels of both PRDM1 and PRDM2 has been observed upon 24 h activation (Fig. 1). The CD40LG increases rapidly upon activation reaching its maximum expression level at 2 h after the CD3/CD28 stimulation. Subsequently, its expression level progressively decreases maintaining constant level greater than time 0 (Fig. 1). The IL2RA expression slowly increases during T cell activation, and it peaks upon 4 h CD3/CD28 treatment. At the following time points IL2RA expression remains unchanged and higher than control cells (Fig. 1). In addition, we assessed the ratio among BLIMP1b/BLIMP1a and RIZ2/RIZ1. In basal condition CD4+T lymphocytes express prevalently BLIMP1b; during CD4+T lymphocyte activation BLIMP1b/BLIMP1a decreases progressively in favor of BLIMP1a. In basal conditions, CD4+T lymphocytes express predominantly RIZ2; during CD4+T lymphocyte activation RIZ2/RIZ1increases until 1 h where it begins to slow down reaching the lower level at 6 h.
PRDM2 encodes for several transcripts, which can be distinguished also for the presence of different 3’-ends produced by alternative splicing, although they still need to be experimentally validated (see scheme in Additional file 1: Fig S3) . Thus, we also analyzed these slightly different C- terminal tails during T lymphocyte activation utilizing specific oligonucleotides. Interestingly, the amplicon representing the long tail (RIZ ex10) displays the same lower trend as RIZ1 transcript expression levels during T cell activation whereas the higher expression of short tail amplicon (RIZ ex9a) prevalently correlates with RIZ2 (Additional file 1: Fig S3).
Overall, these data suggested the participation of PRDM2 transcripts other than PRDM1 ones in the early stages of lymphocyte activation.
PI3K and Src modulate PRDM1 and PRDM2 gene expression during T lymphocytes activation
Anti-CD3/CD28 or PMA/Ion treatment prompt the activation of various downstream signaling pathways [47,48,49]. To figure out the cell signaling pathways activated at cell membrane, transduced within the cell, and targeting PRDM1 and PRDM2 gene expression, we employed Ly294002 (Ly) or Wortmannin PI3K inhibitors and the PP1 Src kinase inhibitor to treat T lymphocytes activated with anti-CD3/CD28 antibodies for 2 h and 6 h.
The qRT-PCR analysis showed that Ly and Wortmannin treatment reduced the RIZ1 expression levels and increased the total RIZ products expression levels, probably due to a RIZ2 increase, thus reverting the effect induced by T cell activation on RIZ1 and potentiating the effect on total RIZ (RIZex8) at 2 h. In addition, Ly and Wortmannin increased both RIZ1 and RIZex8 at 6 h. Otherwise, PP1 treatment augmented both RIZ1 and RIZex8 expression levels observed upon T cell activation already at 2 h (Fig. 2).
Wortmannin hindered the BLIMP1a increase induced at 2 h and 6 h without affecting the BLIMP1b expression at 2 h, whereas increased BLIMP1b expression levels at 6 h (Fig. 2). Ly prevented the expression level increase of BLIMP1a at 2 h and 6 h, potentiated the increase of BLIMP1b at 2 h and has not produced any effect on BLIMP1b at 6 h. PP1 prevented and potentiated the BLIMP1a and BLIMP1b increase respectively at 2 h, and has not modified BLIMP1a and BLIMP1b expression at 6 h (Fig. 2). Altogether, these results suggested that the signaling pathways involving PI3K and Src kinase modulates PRDM1 and PRDM2 gene expression during T lymphocyte activation.
IL2, IL4 and IFNA modify the PRDM1 and PRDM2 gene expression levels in activated T lymphocytes
Cytokines trigger intracellular signaling cascades regulating gene expression in target cells, which lead to their activation, proliferation, and differentiation [1, 43]. IL2 provides an additional signal to the antigen recognition that triggers cyclin-dependent kinases and promotes the S phase cell cycle entry through STAT1/STAT3/STAT5 activation . Therefore, IL2 promotes T cell proliferation and differentiation. IL4, by inducing the cell signaling transduction through STAT5/STAT6 and IRS-2 promotes naïve T lymphocyte differentiation in Th2 cells. In addition, IL4 represents the typical growth factor for Th2 cells. IFN-α modulates STAT1/STAT2 activation and prompts Th1 polarization. Thus, we aimed to assess the effect of IL2, IL4 and IFNA on PRDM1 and PRDM2 transcriptional levels in T lymphocytes isolated from healthy donor PBMCs. As shown in Fig. 3, IL2 modified PRDM2 gene expression at 2 h and 6 h, by increasing RIZ1 expression levels without significantly altering PRDM2/RIZex8 amount; however, IL2 induced a RIZ2 downregulation at 2 and 6 h. IL4 increased both RIZ1 and PRDM2/RIZex8 expression levels at 2 h and 6 h; IFNA upregulated RIZ1at 2 and 6 h and particularly influenced PRDM2/RIZex8 expression at 2 h revealing a RIZ2 up-regulation (Fig. 3). As regards to PRDM1, IL2 downregulated both transcripts at 2 h and BLIMP1a also at 6 h. IL4 modulated only BLIMP1b by increasing its expression at 2 h and 6 h. IFN-α down-regulated BLIMP1a and up-regulated BLIMP1b at 2 and 6 h (Fig. 3).
Analysis of PRDM1 and PRDM2 transcripts in different T cell subpopulations
To explore the T cell subset in which polarizing cytokines target PRDM1 and PRDM2 genes expression during differentiation induction, lymphocytes were previously separated into naïve CD4+ and CD8+ T positive cells, activated with anti-CD3/CD28 antibodies and with IL2 supplemented with different further cytokines, as IL4, IL6, IFNA and IFNG for 48 h (Fig. 4A). We also measured the levels of GATA3, which is critical for the development, differentiation and function of CD4+ T cell subsets, as well as CD8+ T cells .
Anti-CD3/CD28 and IL2 treatment of CD4+ T lymphocytes increased PRDM2 expression levels. However, IL4, IL6 or INFG cytokines, but not IFNA, were able to dramatically revert the effect of IL2 on PRDM2 expression level increase (Fig. 4A). Similarly, IL2 treatment of CD4+ T lymphocytes produced a consistent increase of both PRDM1 molecular forms, particularly of BLIMP1a. All the cytokines supplemented to IL2, excluding IFNA, resulted in a reduction of both BLIMP1a and BLIMP1b expression level (Fig. 4A).
In CD8+ T activated lymphocytes, treatment with IL2 reduced RIZ1 expression levels and increased RIZ2 expression level. The other cytokines supplemented to IL2 induced a downregulation of RIZ2 and an upregulation of RIZ1. In CD8+ activated T lymphocytes, IL2 treatment upregulated both PRDM1 transcripts. However, no significant effect was found for BLIMP1b expression with the addition of a second cytokine, except for IL4 that reduced its expression. Besides, BLIMP1a ever decreased in the presence of a second cytokine.
Interestingly, in the same experiment shown in Fig. 4A, our gene expression analysis in CD4+, and CD8+ T subpopulations during T cell activation revealed an upregulation of CD40LG, IL2RA, MYC and KLF2, paralleling previous literature data [51,52,53,54] (Fig. 4B).
Collectively, T cell subpopulations revealed a different response to the “third signal” provided by the several cytokines supplemented to the activated T cells treated with IL2 when we observed the PRDM gene expression levels. Indeed, while BLIMP1a and BLIMP1b variants exhibited a substantial similarity in their trend, PRDM2 products showed a great variation.
PRDM1 and PRDM2 expression level analysis during Jurkat cell activation
The Jurkat cell line is an immortalized T lymphocyte cell line originally obtained from the peripheral blood sample of a young patient with T cell leukemia . The Jurkat cell line has often been used as a prototypical T cell line to study multiple events in T cell biology, including signal transduction pathways involved in lymphocyte activation . Moreover, considering the high difficulties to transfect normal lymphocytes, this cell line is suitable for evaluating the overexpression of genes by transfection. Firstly, we assessed the PRDM1 and PRDM2 expression levels in Jurkat cell line by qRT-PCR analysis. In standard culture conditions (see Methods), Jurkat cells expressed prevalently PRDM2; besides, they showed similar expression levels of PRDM1 transcripts whereas RIZ2 was expressed at higher levels than RIZ1 (about six folds) (Fig. 5). Then, we assessed the expression levels of PRDM1 and PRDM2 transcripts at early time points after activation with PMA/Ion (45’, 80’, 2 h, 4 h and 8 h) in Jurkat cells (Fig. 5). As observed in T cell lymphocytes, both PRDM1 and PRDM2 transcripts showed a cyclic expression variation even though with different magnitudes and kinetics. Specifically, in basal conditions and at 45’ the predominant PRDM2 isoform is RIZ2 that is downregulated at 2 h and 4 h. At 4 h a decrease of both isoforms was observed. Upon 8 h activation, RIZ1 and RIZ2 expression level is equal to the level observed at 45’ (Fig. 5).
A cyclic variation in BLIMP1a and BLIMP1b expression level has been observed during lymphocyte activation: both BLIMP1a and BLIMP1b are upregulated at 45'. BLIMP1a increased at 2 h and 8 h while at 80’ and 4 h BLIMP1a showed an expression level similar to that observed in basal conditions. An opposite trend has been revealed for BLIMP1b at the same time (Fig. 5).
PRDM2 transcript overexpression in Jurkat cells
Hypothesizing a role of master regulator gene for PRDM2 in T lymphocyte activation and a hierarchical organization in the expression level regulation of PRDM genes, Jurkat cell line was transiently transfected with plasmids encoding for RIZ1 (pSG5_rRIZ1), RIZ2 (pSG5_hRIZ2) and with the empty vector (pSG5) to investigate the ability of RIZ1 and RIZ2 to modulate the gene expression of pivotal factors involved in lymphocyte activation and differentiation. The RIZ1 and RIZ2 overexpression was evaluated after transfection by qRT-PCR of reverse-transcribed total cellular RNA and Western blot analysis. qRT-PCR analysis revealed a significant increase of RIZ1 and RIZ2 transcripts compared to control cells (pSG5) (Additional file 1: Fig S4). RIZ1 protein level was evaluated by Western blot analysis with the monoclonal antibody recognizing the PR domain exclusive of RIZ1. RIZ2 protein overexpression was revealed using a monoclonal antibody recognizing the Flag epitope. Altogether, both qRT-PCR and Western blot analyses confirmed RIZ1 and RIZ2 overexpression (Additional file 1: Fig S4).
Then, we analyzed if the overexpression of RIZ1 and RIZ2 was able to modulate PRDM1 expression level and modify the ratio among the BLIMP1a and BLIMP1b forms.
As depicted in Fig. 6, RIZ1 transient overexpression in Jurkat cell line has not significantly modified BLIMP1a and BLIMP1b gene expression levels, while RIZ2 overexpression increased BLIMP1b without affecting BLIMP1a expression levels. When we co-transfected both RIZ1 and RIZ2, we observed a reduction of both BLIMP1a and BLIMP1b transcripts. Besides, RIZ1 overexpression increased the BLIMP1a/BLIMP1b ratio also in the presence of RIZ2 (> 2 folds). RIZ2 alone, instead, reduced the ratio in favor of BLIMP1b (Fig. 6).
In standard culture conditions, Jurkat cells exhibited different expression levels of transcription factors involved in lymphocyte differentiation (Fig. 6). Specifically, they expressed high levels of GATA3, alike CD40LG, moderate levels of FOXP3 and RORC (about 10 folds lower than CD40LG) and very low levels of TBX21 (about 100 folds lower than CD40LG). Interestingly, the overexpression of RIZ1 or RIZ2 in Jurkat cells altered the expression of these factors; RIZ2 overexpressing cells showed a significant reduction in GATA3 expression. In addition, RIZ2 overexpression reduced RORC expression level while RIZ1 overexpression has not significantly affected its transcription. In both RIZ1 and RIZ2 overexpressing cells we observed a reduction in CD40LG expression, while FOXP3 was upregulated only in RIZ1 overexpressing cells (Fig. 6). Collectively, RIZ2 forced expression through an imbalance of RIZ1/RIZ2 ratio in favor of RIZ2, induced a BLIMP1b increase. Consequently, the balance among BLIMP1a and BLIMP1b has shifted in disfavor of the first form. Otherwise, a balance in favor of BLIMP1a was promoted by RIZ1 overexpression through BLIMP1b repression. In conclusion, we speculate that PRDM2 may act upstream PRDM1 by regulating its gene expression. In addition, upon PRDM2 overexpression the main transcription factors involved in lymphocyte differentiation showed a down-regulation except for FOXP3 that is up-regulated by RIZ1 suggesting a putative PRDM2 involvement in the regulation of lymphocyte differentiation. Meanwhile, RIZ1 and/or RIZ2 could act on CD40LG in T lymphocytes activation with a negative feedback mechanism of regulation. TBX21 was expressed at very low levels; thus, we were not able to appreciate statistically significant variations.
Polyclonal activation of Jurkat cells stably transfected with RIZ1 and RIZ2
To investigate the possible effect of stable overexpression of PRDM2 molecular variants on target genes, qRT-PCR analysis was performed in Jurkat cells stably transfected with the plasmid encoding for RIZ1 (pSG5_rRIZ1), RIZ2 (pSG5_hRIZ2) or with pSG5 control vector (see Additional file 1: Fig S4 for details). In non-activated cells, only RIZ2 stable overexpression increased BLIMP1a level, as shown in Fig. 7. Instead, both RIZ1 and RIZ2 increased BLIMP1b expression levels. In addition, RIZ2 significantly increased the expression level of the main transcription factor of lymphocyte differentiation, GATA3, a similar effect as observed for BLIMP1b, while RIZ1 increased it to a lesser extent. Besides, a reduction of BCL6 expression was detected in RIZ2 overexpressing cells. No significant changes were observed for FOXP3, RORC and TBX21. CD40LG was noticeably increased by RIZ1, and to a less extent by RIZ2.
Activation of Jurkat cells stably transfected with pSG5_rRIZ1 or pSG5_hRIZ2 increased the level of both PRDM1 transcripts compared to control cells. RIZ1 overexpression reduced the BLIMP1a and BLIMP1b expression increase induced by cell activation, particularly for BLIMP1b. RIZ2 overexpression did not affect the BLIMP1a expression level whereas significantly amplified BLIMP1b expression increase observed after activation (of about 2.5 folds) (Fig. 7).
The RIZ1 overexpression prevented the FOXP3 expression level increase observed upon activation. No significant variations were detected for TBX21. Only the RIZ1 overexpression augmented GATA3 expression level increase observed in Jurkat cell activation. Despite its elevated basal level, CD40LG showed a conserved inducibility with a markedly higher increase of total levels in RIZ1 overexpressing cells. By observing the expression of genes encoding chemokine receptors related to the effector functions of different lymphocyte subsets, the CCR4 gene was significantly increased in activated RIZ1 and RIZ2 overexpressing cells. Instead, CCR6 was repressed by both RIZ1 and RIZ2 while CXCR3 was repressed only by RIZ2 (Fig. 7). Moreover, RIZ1 and RIZ2 overexpression did not modify the IL2 levels in basal conditions but halved the IL2 upregulation observed in activated lymphocytes. RIZ1 overexpression increased IL2RA levels in basal conditions. Both RIZ1 and RIZ2 overexpression did not significantly modify IL2RA upon lymphocyte activation. RIZ1 and RIZ2 overexpression increased the basal level of CTLA4 that was not modulated during lymphocyte activation (Fig. 7).
PRDM1 and PRDM2 transcripts positively correlate with expression of T cell activation genes
To further support the relationship between PRDM1 and PRDM2 and lymphocyte activation we explored the expression of their transcripts by in silico analysis of GEPIA2 database . Specifically, we measured the Spearman correlation coefficient R to evaluate the expression correlation of PRDM1, PRDM2 and their main transcripts on three datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx): whole blood, thymoma normal and EBV-transformed lymphocytes. The mRNA levels of the analyzed genes were normalized by RPS18 expression. As depicted in Fig. 8A, a high positive correlation was found between PRDM1 and PRDM2 (R = 0.76) when their whole transcription was considered. When we analyzed the PR + (RIZ1 and BLIMP1a) and PR- (RIZ2 and BLIMP1b) transcripts, we found a stronger correlation in both cases (R = 0.93 and R = 0.88 respectively). Interestingly, high correlations were also observed between PRDM1 and PRDM2 transcripts with GATA3 (R > 0.7) (Fig. 8B). Again, when we matched the whole PRDM1 or PRDM2 transcripts with gene signatures of immune response, a moderate correlation was found for PRDM1 (R ≥ 0.57) whereas a high correlation resulted for PRDM2 (R ≥ 0.85) (Fig. 8C).
PRDM family genes play a pivotal role in the control of several aspects of cell behavior, such as cell cycle progression, cell development and differentiation as well as the homeostasis maintenance of immune system cells [2, 3, 13]. The lymphocyte differentiation is mainly due to histone modifications caused by methyltransferase proteins [58, 59]. A very close relationship between PRDM gene expression and lymphocyte function regulation could exist . To date, several studies have discussed the PRDM1 pivotal role in lymphocyte activation and differentiation. Indeed, BLIMP1 protein is considered an essential regulator of terminal B cell differentiation into antibody-secreting plasma cells and controls the differentiation of Th1/Th2 cells [15, 61]. PRDM2 products are expressed in T lymphocytes where they interact with GATA-3, an essential transcription factor for T cell development and the Th2 lymphocyte differentiation, thereby suggesting that PRDM2 regulates GATA-3 function . The role of PRDM2/RIZ in lymphocytes has not been elucidated so far. Several findings highlighted the relationship between PRDM2 and proliferation control, represented by the Yin-Yang paradigmatic model. Here, we have investigated the role of PRDM2 gene and its molecular variants using the human peripheral blood lymphocytes. Indeed, normal lymphocytes represent a good model to evaluate events induced by mitogenic signals, understand their control mechanisms than tumor cell lines or cells transformed with oncogenes. We have analyzed the transcription of PRDM1 and PRDM2 variants through qRT-PCR because of the high specificity and sensitivity of this method. Besides, it represents a proper tool to study the early events of T cell activation. Indeed, it is well established that distinct transcriptional profiles and chromatin modifications are observed during T cell response .
Our findings represent the first evidence on the putative role of PRDM2 proteins (RIZ1 and RIZ2) in the commitment of T lymphocytes to the functions subsequent to their activation. Moreover, as regard to PRDM1, we have also characterized the expression of the different PRDM1/BLIMP1 (a and b) transcripts during activation. Specifically, we have analyzed the transcription signature of these genes in both human peripheral blood T lymphocytes and Jurkat cells after activation operating through the “first” signal mediated by TCR, and the “second” signal triggered by CD28, or through the cell signaling transduction pathways downstream the receptors. Noteworthy, our findings are also supported by in silico data on GEPIA2; indeed, gene expression correlation analyses clearly indicated a high correlation between PRDM1 and PRDM2, and between their transcripts and other recognized genes involved in immune response (Fig. 8).
We observed that the activation of PBMC or naïve CD4+ T lymphocytes induced an increase in the expression levels of the PRDM2/RIZ and PRDM1/BLIMP1 genes, with a correlated increase of MYC gene expression, a target of both PRDM1 and PRDM2 proteins [64, 65] (Fig. 1). The expression profile of PRDM1 and PRDM2 variants over the time showed the early increase of RIZ2 and RIZ1 followed by BLIMP1b increase and finally by BLIMP1a increase. The “first” and the “second” signals shifted the balance between the PR + and the PR- forms in favor of the PR- ones, for both studied genes. Furthermore, we have also evidenced that the short tail amplicon (RIZ ex9a) prevalently correlates with RIZ2 transcription (Additional file 1: Fig S3); however, further studies are still needed to fully elucidate the functional role of these PRDM2 transcription variants .
These genes showed a trend similar to that of the Immediate Early Growth Response Genes, which determine important changes in the cellular state of Jurkat T cells . Specifically, they regulate the switch of resting lymphocytes from quiescent to activated state, followed by cell proliferation essential for clonal expansion and subsequent differentiation. Furthermore, to determine the role of the first and second signal transduction pathways, TCR and CD3/CD28 respectively, in the gene expression regulation of PRDMs, the effects of several inhibitors of key enzymes of transduction pathways were evaluated, such as PI3 and MEK1/2 kinases. The obtained results suggested that the signaling pathway involving PI3K modulates the RIZ1/RIZ2 ratio in favor of RIZ1 gene during T lymphocyte activation while the balance versus RIZ2 was promoted by the MAPK pathway. Similarly, in the CML model, RIZ1 was induced from myeloid cells, where PI3K/AKT upregulates RIZ1 .
The presence of cytokines that mediate different Jak/Stat signaling pathways are observed as agents capable of modulating the expression of PRDM1/PRDM2 genes and the relationship of their forms (Fig. 2). IL-2/STAT5 promotes the increase in the RIZ1 amount compared to RIZ2; instead, IL-4/STAT6 induces an increase of PRDM2/RIZ, most likely of the RIZ2 form. Besides, IFN-∝/STAT1/2/IRF9 produces the most marked increase in RIZ2. All these effects seem to be very quick, already at 2 hrs whereas at 6 hrs we observed non-significant variation in expression. In the same T cells, stimulated as above, we observed an early increase in the BLIMP1b form with IFN-∝ and late as with IL-2 or IL-4, while the increase in BLIMP1a is only late. The relationship between the PRDM1 forms towards the form a is evident in the presence of IL-2 and IL-4 while with IFN-∝ the increase in the form a balances the form b. Overall, this analysis would confirm what we observed with the activation of the 1st and 2nd signals, i.e. the early increase of the PR-minus transcripts compared to the PR-plus ones. The physiological activation by the antigen presenting cell (APC) generates clonal expansion by means of 1st and 2nd signals and the 3rd signal, which is prompted by the presence of “inducing” cytokines produced by different innate cells present in the microenvironment or by the APCs themselves, promotes the differentiation of T cell subtypes. The presence of all the three signals, simulated in the CD4+ T cells and CD8+ T cells populations, involves a highly different expression trend between PRDM2 and PRDM1 for many of the cytokines added, such as IL-4, IFN-γ, IFN-∝ and IL-6, to the main stimulus CD3/CD28 + IL-2 (Fig. 4). In summary, it is likely that in CD4+ cells IL-4 produces a reduction in the induction of PRDM1 and a repression of PRDM2. The latter effect is also observed with other pro-differentiating cytokines, such as IFN-γ and IL-6. In CD8+ T cells, the RIZ2 and BLIMP1b increase occurs through the activation with IL-2 and CD3/CD28 alone, while the presence of cytokines pushes the prevalence of RIZ1 (IL-4) or the repression of RIZ2 (IFN-∝ and IL-6). On the other hand, BLIMP1b increases, and in the presence of IL-4, a balance is produced towards a while b form is repressed. In the presence of the other cytokines, IFN-γ, IFN-∝ and IL-6, the negative regulation of a form promotes a greater balance towards the b form. From these findings, we deduce that the response of the CD4+ or CD8+ effector T cells is different regarding the expression of the PRDM1 and PRDM2 genes and their variants, if we compare the trend of T cell activation markers (KLF2, CD40LG, IL2RA, MYC). The used model of enriched polyclonal human T cells, which represent the sum of different T subsets (Th1, Th2, TH17, TFh etc.), does not allow a precise assessment of the associations between PRDM1 and PRDM2 transcripts and the different cytokine stimuli, but these data solicit further correlation and functional studies to better understand the cause/effect role of PRDM1 and PRDM2 and their variants in the mechanism of action of the cytokines themselves.
These observations highlight the relationship between stimulus and response downstream of the PRDM1 and PRDM2 genes and of the different expressivity of their variants. Thus, the question is how and in what way the different pathways are concatenated and if there could be a cause-effect hierarchy between the early activation of RIZ1 and RIZ2 and the late activation of BLIMP1a and BLIMP1b. To this purpose, we performed overexpression experiments of PRDM2/RIZ isoforms in Jurkat cell line (although they already overexpress RIZ2 at baseline condition) to reproduce significant variations of RIZ1 and RIZ2 as those induced by the 1st/2nd signals over time and observed in T cells from PBMCs and assess the effects on BLIMP1a and BLIMP1b expression levels. This approach allowed us observing that the acute (transiently) increase of RIZ2 promotes the balancing of the PRDM1 forms towards the b form (Fig. 5). Noteworthy, the generation of stable Jurkat clones overexpressing RIZ1 or RIZ2 allowed us to evidence a different equilibrium of the BLIMP1a/b molecules in basal conditions beyond the modification of the response to the polyclonal activators (Fig. 6). The RIZ2-mediated adaptation of the cells led to a basal increase in BLIMP1b and preserved its induction by the 1st/2nd signals. Obviously, through an autocrine mechanism, cells can promote the expression of IL2, which is not expressed in basal conditions in the different stable RIZ1 or RIZ2 overexpressing cell lines. Stable RIZ1 cell line promotes a lower increase in the baseline BLIMP1b level and, on the other hand, a strong repression effect of the 1st/2nd signal induction, thus highlighting the importance of RIZ1 and RIZ2. GATA3 gene appears strongly modulated under basal conditions in RIZ2 overexpressing cells in a similar manner as BLIMP1b. Otherwise, in RIZ1 overexpressing cells, GATA3 increases little in basal conditions but responds to activation stimuli. This evidence confirms the hypothesis of a close relationship between PRDM2 and GATA3, and it indicates significant differences between the action of the PRDM2 forms on the GATA3 expression regulation. Different changes of other analyzed genes (FOXP3, RORC, BCL6, etc.), compared to BLIMP1 and GATA3, seem to confirm the different activity of the PRDM2 forms, RIZ1 and RIZ2, both in the increase or decrease of the basal amount and in the induction capacity by the activation signals. This finding is in line with the basal GATA3 increase, which predisposes to the cell function increase in the T helper 2 direction. Likely, RIZ2 could work in concert with GATA3 to strengthen its functions, including the modulation of CCR4 expression. Instead, comparing the effects on other key genes, such as those coding for chemokine receptors, CCR6 and CXCR3, we observed a general reduction in the induction of their expression following activation. Therefore, we can assume that RIZ2 strengthens the polarization of T lymphocytes in Th2. RIZ1, as RIZ2, is capable of repressing CCR6 and CXCR3 but not of increasing CCR4 levels. Probably the effect could be due to the concomitant repression of BLIMP1b (Fig. 7).
Interestingly, the CD40LG high expression level in basal condition, which was particularly observed in RIZ1 overexpressing cells than RIZ2 ones, and the CD40LG up-regulation, observed during cell activation, could be related to the variations in the expression levels of positive and negative regulators induced by the adaptation of the cell line to the overexpression of PRDM2 forms that mimic key steps of cell activation or memory cell differentiation.
In summary, the stable forced expression of RIZ1 or RIZ2 in clones of Jurkat T cell line induced a significant variation in the expression level of key transcription factors involved in T lymphocyte differentiation observed already in steady state. Accordingly, these clones showed a different ability to modulate the expression level of these transcription factors upon cell activation. The PRDM1a/b balance has shifted in favor of BLIMP1a in RIZ1-overexpressing cells and in favor of BLIMP1b in RIZ2-overexpressing cells. In addition, GATA3 expression level is noticeably modified by both RIZ1 and RIZ2.
Overall, our observations represent the first step for the characterization of PRDM2 in lymphocyte activation/differentiation. In the future, we point out to clarify deeply the mechanism of action and functions of PRDM2 in lymphocyte activation/differentiation. In our opinion, it could be interesting evaluating the gene expression regulation by performing time-course Next Generation Sequencing (NGS) analysis in single cell. Furthermore, it could be remarkable to study the chromatin architecture of genes regulated by PRDM2 proteins, identifying any PRDM2 responsive element sequence. Considering that literature data shows the involvement of other genes of the family, including PRDM15 in lymphoproliferative diseases , the preliminary evidence of our study allows us to hypothesize the use of drugs directed against RIZ or BLIMP1 to prevent or modify the response of cells to activation and cell differentiation and as a useful tool also for cancer therapy.
Consistent with its established role as an essential regulator of immune cell function, numerous PRDM1-associated risk alleles have been linked with autoimmune pathologies [15, 68]. Noteworthy, a recent meta-analysis of multi-trait genome-wide association studies identified PRDM2 as a locus associated with six autoimmune and allergic diseases thus suggesting the implication of this gene . Abnormal gene expression level or expression of genes containing deleterious variants can be at the basis of these genetic diseases. Currently, several molecular tools have been developed and are in clinical trials for therapeutic purposes acting to restore the physiological gene expression . Thus, the whole knowledge of the transcription regulation mechanisms involving PRDM1 and PRDM2 genes in the immune biology can provide the molecular bases for the application of these new strategies to the autoimmune diseases.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Protein kinase B
Antigen presenting cell
BTB Domain and CNC Homolog 2
B cell lymphoma 6
B lymphocyte-induced maturation protein 1
- CCR C–C:
Motif Chemokine Receptor
- CIITA MHC:
Class II trans-activator
Chronic myelogenous leukemia
Cytotoxic T-Lymphocyte Antigen 4
C-X-C Motif Chemokine Receptor
Diffuse Large B-Cell Lymphoma
Fetal bovine serum
Forkhead box P3
Interleukin 2 receptor subunit alpha
Interferon regulatory factor
Insulin receptor substrate
KLF Transcription Factor 2
Mitogen-activated protein kinase
Mitogen-activated protein kinase kinase)
Paired box 5
SPeripheral blood mononuclear cells
Phorbol 12-myristate 13-acetate
PRDF1 and RIZ1 homology domain containing proteins
Retinoblastoma protein interacting zinc finger gene
RAR Related Orphan Receptor C
Ribosomal protein S18
Signal transducer and activator of transcription
T-Box Transcription Factor 21
T cell receptor
Tumor necrosis factor receptor
Pennock ND, White JT, Cross EW, Cheney EE, Tamburini BA, Kedl RM. T cell responses: naive to memory and everything in between. Adv Physiol Educ. 2013;37(4):273–83. https://doi.org/10.1152/advan.00066.2013.
Di Zazzo E, De Rosa C, Abbondanza C, Moncharmont B. PRDM proteins: molecular mechanisms in signal transduction and transcriptional regulation. Biology. 2013;2(1):107–41.
Fog CK, Galli GG, Lund AH. PRDM proteins: important players in differentiation and disease. BioEssays. 2012;34(1):50–60. https://doi.org/10.1002/bies.201100107.
Hohenauer T, Moore AW. The Prdm family: expanding roles in stem cells and development. Development. 2012;139(13):2267–82.
Yu J, Angelin-Duclos C, Greenwood J, Liao J, Calame K. Transcriptional repression by blimp-1 (PRDI-BF1) involves recruitment of histone deacetylase. Mol Cell Biol. 2000;20(7):2592–603. https://doi.org/10.1128/MCB.20.7.2592-2603.2000.
Gyory I, Wu J, Fejér G, Seto E, Wright KL. PRDI-BF1 recruits the histone H3 methyltransferase G9a in transcriptional silencing. Nat Immunol. 2004;5(3):299–308. https://doi.org/10.1038/ni1046.
Ancelin K, Lange UC, Hajkova P, Schneider R, Bannister AJ, Kouzarides T, et al. Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nat Cell Biol. 2006;8(6):623–30. https://doi.org/10.1038/ncb1413.
Su ST, Ying HY, Chiu YK, Lin FR, Chen MY, Lin KI. Involvement of histone demethylase LSD1 in Blimp-1-mediated gene repression during plasma cell differentiation. Mol Cell Biol. 2009;29(6):1421–31. https://doi.org/10.1128/MCB.01158-08.
Ren B, Chee KJ, Kim TH, Maniatis T. PRDI-BF1/Blimp-1 repression is mediated by corepressors of the Groucho family of proteins. Genes Dev. 1999;13(1):125–37. https://doi.org/10.1101/gad.13.1.125.
Sorrentino A, Rienzo M, Ciccodicola A, Casamassimi A, Abbondanza C. Human PRDM2: structure, function and pathophysiology. Biochim Biophys Acta Gene Regul Mech. 2018;1861(7):657–71. https://doi.org/10.1016/j.bbagrm.2018.06.002.
Derunes C, Briknarová K, Geng L, Li S, Gessner CR, Hewitt K, et al. Characterization of the PR domain of RIZ1 histone methyltransferase. Biochem Biophys Res Commun. 2005;333(3):925–34. https://doi.org/10.1016/j.bbrc.2005.05.190.
Casamassimi A, Rienzo M, Di Zazzo E, Sorrentino A, Fiore D, Proto MC, et al. Multifaceted role of PRDM proteins in human cancer. Int J Mol Sci. 2020;21(7):2648. https://doi.org/10.3390/ijms21072648.
Mzoughi S, Tan YX, Low D, Guccione E. The role of PRDMs in cancer: one family, two sides. Curr Opin Genet Dev. 2016;36:83–91. https://doi.org/10.1016/j.gde.2016.03.009.
Di Tullio F, Schwarz M, Zorgati H, Mzoughi S, Guccione E. The duality of PRDM proteins: epigenetic and structural perspectives. FEBS J. 2022;289(5):1256–75. https://doi.org/10.1111/febs.15844.
Nadeau S, Martins GA. Conserved and Unique Functions of Blimp1 in Immune Cells. Front Immunol. 2022;12:805260. https://doi.org/10.3389/fimmu.2021.805260.
Belz GT, Kallies A. Effector and memory CD8+ T cell differentiation: toward a molecular understanding of fate determination. Curr Opin Immunol. 2010;22(3):279–85. https://doi.org/10.1016/j.coi.2010.03.008.
Crotty S, Johnston RJ, Schoenberger SP. Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation. Nat Immunol. 2010;11(2):114–20. https://doi.org/10.1038/ni.1837.
Martins G, Calame K. Regulation and functions of Blimp-1 in T and B lymphocytes. Annu Rev Immunol. 2008;26:133–69. https://doi.org/10.1146/annurev.immunol.26.021607.090241.
Cretney E, Leung PS, Trezise S, Newman DM, Rankin LC, Teh CE, et al. Characterization of Blimp-1 function in effector regulatory T cells. J Autoimmun. 2018;91:73–82. https://doi.org/10.1016/j.jaut.2018.04.003.
Cretney E, Xin A, Shi W, Minnich M, Masson F, Miasari M, et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nat Immunol. 2011;12(4):304–11. https://doi.org/10.1038/ni.2006.
Kallies A, Hawkins ED, Belz GT, Metcalf D, Hommel M, Corcoran LM, et al. Transcriptional repressor Blimp-1 is essential for T cell homeostasis and self-tolerance. Nat Immunol. 2006;7(5):466–74. https://doi.org/10.1038/ni1321.
Kallies A, Xin A, Belz GT, Nutt SL. Blimp-1 transcription factor is required for the differentiation of effector CD8(+) T cells and memory responses. Immunity. 2009;31(2):283–95. https://doi.org/10.1016/j.immuni.2009.06.021.
Martins GA, Cimmino L, Shapiro-Shelef M, Szabolcs M, Herron A, Magnusdottir E, et al. Transcriptional repressor Blimp-1 regulates T cell homeostasis and function. Nat Immunol. 2006;7(5):457–65. https://doi.org/10.1038/ni1320.
Nutt SL, Kallies A, Belz GT. Blimp-1 connects the intrinsic and extrinsic regulation of T cell homeostasis. J Clin Immunol. 2008;28(2):97–106. https://doi.org/10.1007/s10875-007-9151-6.
Rutishauser RL, Martins GA, Kalachikov S, Chandele A, Parish IA, Meffre E, et al. Transcriptional repressor Blimp-1 promotes CD8(+) T cell terminal differentiation and represses the acquisition of central memory T cell properties. Immunity. 2009;31(2):296–308. https://doi.org/10.1016/j.immuni.2009.05.014.
Fu SH, Yeh LT, Chu CC, Yen BL, Sytwu HK. New insights into Blimp-1 in T lymphocytes: a divergent regulator of cell destiny and effector function. J Biomed Sci. 2017;24(1):49. https://doi.org/10.1186/s12929-017-0354-8.
Martins GA, Cimmino L, Liao J, Magnusdottir E, Calame K. Blimp-1 directly represses Il2 and the Il2 activator Fos, attenuating T cell proliferation and survival. J Exp Med. 2008;205(9):1959–65. https://doi.org/10.1084/jem.20080526.
Wu H, Deng Y, Zhao M, Zhang J, Zheng M, Chen G, et al. Molecular control of follicular helper T cell development and differentiation. Front Immunol. 2018;9:2470. https://doi.org/10.3389/fimmu.2018.02470.
Boi M, Zucca E, Inghirami G, Bertoni F. PRDM1/BLIMP1: a tumor suppressor gene in B and T cell lymphomas. Leuk Lymphoma. 2015;56(5):1223–8. https://doi.org/10.3109/10428194.2014.953155.
Mora-López F, Reales E, Brieva JA, Campos-Caro A. Human BSAP and BLIMP1 conform an autoregulatory feedback loop. Blood. 2007;110(9):3150–7. https://doi.org/10.1182/blood-2007-05-092262.
Ochiai K, Katoh Y, Ikura T, Hoshikawa Y, Noda T, Karasuyama H, et al. Plasmacytic transcription factor Blimp-1 is repressed by Bach2 in B cells. J Biol Chem. 2006;281(50):38226–34. https://doi.org/10.1074/jbc.M607592200.
Tunyaplin C, Shaffer AL, Angelin-Duclos CD, Yu X, Staudt LM, Calame KL. Direct repression of prdm1 by Bcl-6 inhibits plasmacytic differentiation. J Immunol. 2004;173(2):1158–65. https://doi.org/10.4049/jimmunol.173.2.1158.
Yasuda T, Hayakawa F, Kurahashi S, Sugimoto K, Minami Y, Tomita A, et al. B cell receptor-ERK1/2 signal cancels PAX5-dependent repression of BLIMP1 through PAX5 phosphorylation: a mechanism of antigen-triggering plasma cell differentiation. J Immunol. 2012;188(12):6127–34. https://doi.org/10.4049/jimmunol.1103039.
Calame K. Activation-dependent induction of Blimp-1. Curr Opin Immunol. 2008;20(3):259–64. https://doi.org/10.1016/j.coi.2008.04.010.
Lakshmikuttyamma A, Takahashi N, Pastural E, Torlakovic E, Amin HM, Garcia-Manero G, et al. RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression. J Hematol Oncol. 2009;2:28. https://doi.org/10.1186/1756-8722-2-28.
Gazzerro P, Bontempo P, Schiavone EM, Abbondanza C, Moncharmont B, Armetta I, et al. Differentiation of myeloid cell lines correlates with a selective expression of RIZ protein. Mol Med. 2001;7(8):552–60.
Steele-Perkins G, Fang W, Yang XH, Van Gele M, Carling T, Gu J, et al. Tumor formation and inactivation of RIZ1, an Rb-binding member of a nuclear protein-methyltransferase superfamily. Genes Dev. 2001;15(17):2250–62. https://doi.org/10.1101/gad.870101.
Rienzo M, Sorrentino A, Di Zazzo E, Di Donato M, Carafa V, Marino MM, et al. Searching for a putative mechanism of RIZ2 tumor-promoting function in cancer models. Front Oncol. 2021;10:583533. https://doi.org/10.3389/fonc.2020.583533.
Medici N, Abbondanza C, Nigro V, Rossi V, Piluso G, Belsito A, et al. Identification of a DNA binding protein cooperating with estrogen receptor as RIZ (retinoblastoma interacting zinc finger protein). Biochem Biophys Res Commun. 1999;264(3):983–9. https://doi.org/10.1006/bbrc.1999.1604.
Rienzo M, Schiano C, Casamassimi A, Grimaldi V, Infante T, Napoli C. Identification of valid reference housekeeping genes for gene expression analysis in tumor neovascularization studies. Clin Transl Oncol. 2013;15(3):211–8. https://doi.org/10.1007/s12094-012-0904-1.
Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29(9):e45. https://doi.org/10.1093/nar/29.9.e45.
Abbondanza C, Medici N, Nigro V, Rossi V, Gallo L, Piluso G, et al. The retinoblastoma-interacting zinc-finger protein RIZ is a downstream effector of estrogen action. Proc Natl Acad Sci USA. 2000;97(7):3130–5. https://doi.org/10.1073/pnas.97.7.3130.
Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009;27:591–619. https://doi.org/10.1146/annurev.immunol.021908.132706.
Shipkova M, Wieland E. Surface markers of lymphocyte activation and markers of cell proliferation. Clin Chim Acta. 2012;413(17–18):1338–49. https://doi.org/10.1016/j.cca.2011.11.006.
Wieland E, Shipkova M. Lymphocyte surface molecules as immune activation biomarkers. Clin Biochem. 2016;49(4–5):347–54. https://doi.org/10.1016/j.clinbiochem.2015.07.099.
Abbondanza C, De Rosa C, D’Arcangelo A, Pacifico M, Spizuoco C, Piluso G, et al. Identification of a functional estrogen-responsive enhancer element in the promoter 2 of PRDM2 gene in breast cancer cell lines. J Cell Physiol. 2012;227(3):964–75. https://doi.org/10.1002/jcp.22803.
Hwang JR, Byeon Y, Kim D, Park SG. Recent insights of T cell receptor-mediated signaling pathways for T cell activation and development. Exp Mol Med. 2020;52(5):750–61. https://doi.org/10.1038/s12276-020-0435-8.
Agrahari G, Sah SK, Bang CH, Kim YH, Kim TY. Superoxide dismutase 3 controls the activation and differentiation of CD4+T Cells. Front Immunol. 2021;12:628117. https://doi.org/10.3389/fimmu.2021.628117.
Baldanzi G. Immune Checkpoint Receptors Signaling in T Cells. Int J Mol Sci. 2022;23(7):3529. https://doi.org/10.3390/ijms23073529.
Wan YY. GATA3: a master of many trades in immune regulation. Trends Immunol. 2014;35(6):233–42. https://doi.org/10.1016/j.it.2014.04.002.
Marchingo JM, Sinclair LV, Howden AJ, Cantrell DA. Quantitative analysis of how Myc controls T cell proteomes and metabolic pathways during T cell activation. Elife. 2020;9:e53725. https://doi.org/10.7554/eLife.53725.
Preston GC, Sinclair LV, Kaskar A, Hukelmann JL, Navarro MN, Ferrero I, et al. Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes. EMBO J. 2015;34(15):2008–24. https://doi.org/10.15252/embj.201490252.
Stout RD, Suttles J. The many roles of CD40 in cell-mediated inflammatory responses. Immunol Today. 1996;17(10):487–92. https://doi.org/10.1016/0167-5699(96)10060-i.
Hart GT, Hogquist KA, Jameson SC. Krüppel-like factors in lymphocyte biology. J Immunol. 2012;188(2):521–6. https://doi.org/10.4049/jimmunol.1101530.
Abraham RT, Jurkat Weiss A. T cells and development of the T-cell receptor signalling paradigm. Nat Rev Immunol. 2004;4(4):301–8. https://doi.org/10.1038/nri1330.
Kim JE, White FM. Quantitative analysis of phosphotyrosine signaling networks triggered by CD3 and CD28 costimulation in Jurkat cells. J Immunol. 2006;176(5):2833–43. https://doi.org/10.4049/jimmunol.176.5.2833.
Tang Z, Kang B, Li C, Chen T, Zhang Z. GEPIA2: an enhanced web server for large-scale expression profiling and interactive analysis. Nucleic Acids Res. 2019;47(W1):W556–60. https://doi.org/10.1093/nar/gkz430.
Wei G, Wei L, Zhu J, Zang C, Hu-Li J, Yao Z, et al. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. Immunity. 2009;30(1):155–67. https://doi.org/10.1016/j.immuni.2008.12.009.
Wilson CB, Rowell E, Sekimata M. Epigenetic control of T-helper-cell differentiation. Nat Rev Immunol. 2009;9(2):91–105. https://doi.org/10.1038/nri2487.
Mzoughi S, Fong JY, Papadopoli D, Koh CM, Hulea L, Pigini P, et al. PRDM15 is a key regulator of metabolism critical to sustain B-cell lymphomagenesis. Nat Commun. 2020;11(1):3520. https://doi.org/10.1038/s41467-020-17064-0.
Turner CA Jr, Mack DH, Davis MM. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell. 1994;77(2):297–306.
Shapiro VS, Lee P, Winoto A. Identification and cloning of the G3B cDNA encoding a 3’ segment of a protein binding to GATA-3. Gene. 1995;163(2):329–30. https://doi.org/10.1016/0378-1119(95)00420-b.
Weng NP, Araki Y, Subedi K. The molecular basis of the memory T cell response: differential gene expression and its epigenetic regulation. Nat Rev Immunol. 2012;12(4):306–15. https://doi.org/10.1038/nri3173.
Lin Y, Wong K, Calame K. Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation. Science. 1997;276(5312):596–9. https://doi.org/10.1126/science.276.5312.596.
Gazzerro P, Abbondanza C, D’Arcangelo A, Rossi M, Medici N, Moncharmont B, et al. Modulation of RIZ gene expression is associated to estradiol control of MCF-7 breast cancer cell proliferation. Exp Cell Res. 2006;312(3):340–9. https://doi.org/10.1016/j.yexcr.2005.11.002.
Skerka C, Decker EL, Zipfel PF. Coordinate expression and distinct DNA-binding characteristics of the four EGR-zinc finger proteins in Jurkat T lymphocytes. Immunobiology. 1997;198(1–3):179–91. https://doi.org/10.1016/S0171-2985(97)80039-3.
Pastural E, Takahashi N, Dong WF, Bainbridge M, Hull A, Pearson D, et al. RIZ1 repression is associated with insulin-like growth factor-1 signaling activation in chronic myeloid leukemia cell lines. Oncogene. 2007;26(11):1586–94. https://doi.org/10.1038/sj.onc.1209959.
Abugessaisa I, Gomez-Cabrero D, Snir O, Lindblad S, Klareskog L, Malmström V, Tegnér J. Implementation of the CDC translational informatics platform–from genetic variants to the national Swedish Rheumatology Quality Register. J Transl Med. 2013;11:85. https://doi.org/10.1186/1479-5876-11-85.
Shirai Y, Nakanishi Y, Suzuki A, Konaka H, Nishikawa R, Sonehara K, et al. Multi-trait and cross-population genome-wide association studies across autoimmune and allergic diseases identify shared and distinct genetic component. Ann Rheum Dis. 2022;81(9):1301–12. https://doi.org/10.1136/annrheumdis-2022-222460.
Tsai HC, Pietrobon V, Peng M, Wang S, Zhao L, Marincola FM, Cai Q. Current strategies employed in the manipulation of gene expression for clinical purposes. J Transl Med. 2022;20(1):535. https://doi.org/10.1186/s12967-022-03747-3.
We thank our colleagues Dr Luciano Pezone, Dr Grazia Maglio and Dr Marina D’Agostino for their technical assistance.
This work was supported by ordinary funds from University of Campania “Luigi Vanvitelli.
Ethics approval and consent to participate
Human peripheral T lymphocytes utilized in this study were obtained from total peripheral blood mononuclear cells (PBMCs) collected from authors who consented to participate.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1
: Figure S1: Analysis of T cell subpopulations and activated cells at 48h by flow cytometer. CD4+, CD8+ T lymphocytes, and T naïve cells were analyzed by 6 Color TBNK + Truc assay with BD FACSLyricTM flow cytometer. The percentage of activated cells (scatter plots in blue) was measured at 48h with BD MultitestTM CD8/CD38/CD3/HLA-DR and anti-CD4 PECy7 and anti-CD45 V500C. Figure S2: Lymphocyte activation increases PRDM1 and PRDM2 expression levels. RT-PCR analysis of PRDM1 and PRDM2 transcripts upon activation in naïve T cells after stimulation with anti-CD3/CD28 and PMA/Ion for 2h and 6h. Semiquantitative analysis of genes related to lymphocyte activation is also reported. Bar graphs represent quantitative data obtained by ImageJ analysis of gel electrophoresis images. Figure S3: A. Schematic presentation of primer localizations for selectively measuring the expression levels of transcript variants with different 3' ends generated by alternative splicing . The use of specific reverse oligonucleotides allowed to distinguish two amplicons with different tails. B. Bar graphs represent data from qRT-PCR analysis of RIZ ex10 amplicon ad RIZ ex9a amplicon at different time points. Expression levels were calculated using the DDCt method with the indicated control gene. The ratio between the RIZ ex10 and RIZ ex9a amplicons was also calculated. Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *P<0.05 vs control cells. *p<0.05, **p<0.01, ***p<0.001. Figure S4: PRDM2 transcript levels in Jurkat cell line. Expression levels of RIZ1 and RIZ2 and their proteins in Jurkat cells after transfection with the indicated plasmids through qRT-PCR and Western blot analyses respectively.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Di Zazzo, E., Rienzo, M., Casamassimi, A. et al. Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation. J Transl Med 21, 217 (2023). https://doi.org/10.1186/s12967-023-04066-x
- T lymphocyte activation
- T lymphocyte commitment
- Transcription factors
- Transcription regulation