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RFX transcription factors control a miR-150/PDAP1 axis that restrains the proliferation of human T cells

['Michele Chirichella', 'Institute For Research In Biomedicine', 'Irb', 'Università Della Svizzera Italiana', 'Usi', 'Bellinzona', 'Niccolò Bianchi', 'Graduate School For Cellular', 'Biomedical Sciences', 'University Of Bern']

Date: 2022-02 

Within the immune system, microRNAs (miRNAs) exert key regulatory functions. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9–mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes.

Funding: This work was supported by the Swiss National Science Foundation grant 31003A_175569 ( www.snf.ch ), the NCCR “RNA & Disease", the Novartis Foundation for medical-biological Research and the Ceresio Foundation (all to SM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Specifically, we focused on the single miRNA accounting for almost 50% of miRNAs constitutively expressed in human T cells, miR-150-5p (hereafter miR-150). This miRNA is abundantly expressed in both T and B lymphocytes [ 11 ], and its deletion in mouse models revealed that it modulates B lymphocyte and CD8 + T-cell differentiation [ 12 – 15 ]. To identify the mechanisms controlling constitutive miR-150 expression and its activation-induced down-regulation, we used an unbiased genomic approach to map the cis-regulatory elements in the MIR150 locus that controlled its expression, leading to the identification of regulatory factor X (RFX) transcription factors as crucial regulators of constitutive miR-150 expression in resting cells and stimulus-induced down-regulation. Finally, we used miRNA pull-down and sequencing to identify the mRNAs specifically targeted by miR-150 in human T lymphocytes. MiR-150 targeted modulators of T-cell proliferation, including the transcription factor MYB and a previously unidentified target, PDGFA-associated protein 1 (PDAP1), which we characterized as an RNA-binding protein (RBP). Deletion of MYB, PDAP1, or MIR150 itself by CRISPR/Cas-9–mediated gene editing in primary human T lymphocytes revealed the contribution of each of these factors to the regulation of T-cell proliferation in response to activating signals. Overall, our data identified a miRNA-regulated network involved in restraining proliferative responses of circulating resting T lymphocytes.

Through their ability to target a variety of mRNAs and regulate their translation and stability, microRNAs (miRNAs) modulate all aspects of the biology of T lymphocytes, including cell differentiation, activation, and proliferation [ 1 , 2 ]. The effect of any given miRNA is dependent on its expression level relative to that of its targets [ 3 , 4 ] and also on the specific context and cell-specific usage of target sites in the 3′ untranslated region (UTR) of mRNAs [ 5 ], resembling the cell type–specific regulation of gene expression mediated by transcription factors. The quantitative analysis of miRNA expression in different T-cell subsets and in response to T cell receptor (TCR) triggering may thus provide clues on the functional impact of individual miRNAs on T-cell responses. Abundant miRNAs that are down-regulated after stimulation may be involved in restraining T-cell activation, as shown in the case of miR-125b, which is required to maintain the naive state of human T cells [ 6 ]. By contrast, miRNAs that are expressed at very low levels are highly unlikely to reach the concentrations required to exert biological functions [ 4 , 7 ]. Finally, modestly expressed but inducible miRNAs may dynamically reach intracellular concentrations relevant in the modulation of T-cell activation. Examples in this group include miR-155 [ 8 , 9 ] and miR-146a [ 10 ], which are responsible for enhancing and attenuating T-cell responses, respectively.

Results

MiR-150 is the most highly expressed miRNA in human T cells and is down-regulated by activation To identify and accurately quantify miRNAs that are expressed by ex vivo isolated primary human T cells, we performed NanoString digital profiling of CD4+ naive, central memory (T CM ), and effector memory (T EM ) T-cell subsets isolated from 4 independent donors. Among the 827 miRNAs quantified, only 48 were detectable in these subsets (S1 Table). The levels of expression of these miRNAs differed widely, with the combined expression of only 2 of them (miR-150 and miR-142) representing >70% of the overall miRNA content in all the T-cell subsets analyzed (Fig 1A). MiR-150 was the most highly expressed miRNA, with an average number of approximately 110,000 molecules per 100 ng of total RNA (S1A Fig). While miR-150 expression was substantially similar among subsets, a few moderately expressed miRNAs (such as miR-222) were preferentially expressed in memory T cells (both T CM and T EM ) compared to naive cells, while miR-181a was instead preferentially expressed in naive compared to memory T lymphocytes (S1B Fig). No significant differences were observed between T CM and T EM cells (S1B Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 1. MiRNA expression in human CD4+ T-cell subsets. (A) Total RNA was extracted from freshly isolated CD4+ naive, T CM , and T EM T-cell subsets, and miRNA expression was measured by NanoString SPRINT profiling. The most highly expressed miRNAs are shown, and data are expressed as percentage of normalized counts over the total. N = 3 independent donors. (B) Total RNA was extracted from the indicated T-cell subsets freshly isolated from peripheral blood. MiRNA expression was measured by qRT-PCR, and data are expressed as 2−ΔCt. N = 3 independent donors. (C) Freshly isolated memory T lymphocytes were loaded with CFSE, transfected with either a miR-150 mimic or a control oligonucleotide, and activated with anti-CD3 and anti-CD28 antibodies. The extent of cell proliferation was measured 3 days after activation. Data in the bar graph were normalized to the overall baseline signal on day 0, prior to stimulation, to compensate from experimental differences in basal CFSE loading. N = 6 independent experiments. Mean ± SD. Student t test, 2 tailed, paired. Underlying data can be found in S1 Data. CFSE, carboxyfluorescein succinimidyl ester; miRNA, microRNA; qRT-PCR, quantitative RT-PCR. https://doi.org/10.1371/journal.pbio.3001538.g001 Next, we selected some of the highly expressed or differentially expressed miRNAs to assess their regulation in response to T-cell activation. T cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies, and miRNA expression was measured by reverse transcription quantitative PCR (RT-qPCR) over time (Fig 1B). Some of the miRNAs expressed at moderate levels in resting lymphocytes (miR-155, miR-222, and miR-146a) were substantially induced upon TCR stimulation, especially in naive cells. Abundant miRNAs such as miR-150 and miR-342 were instead markedly reduced after 2 days of activation, while miR-181a had a more variable pattern of expression across the different subsets and time points. We further measured the expression of these highly abundant or inducible miRNAs in different ex vivo isolated effector subsets, namely T H 1, T H 2, T H 17, and T H 22 cells. We observed quantitatively modest and nonsignificant differences, concordant with differential miRNA expression being limited primarily to naive versus memory cells (S1C Fig). The dynamic regulation of miR-150 upon activation together with its high levels of expression in resting cells pointed toward its possible role in the regulation of T-cell responses upon TCR triggering. To determine the functional role of miR-150 in human T cells, we transfected freshly isolated memory T lymphocytes with either a miR-150 mimic or a control oligonucleotide, and we measured cell proliferation over time by carboxyfluorescein succinimidyl ester (CFSE) dilution. We found that in the presence of miR-150, T-cell proliferation was significantly affected after 3 days of anti-CD3 and anti-CD28 stimulation, as shown by the reduced dilution of CFSE, leading to higher mean fluorescence intensity (MFI) (Fig 1C). Proliferation (measured by BrdU incorporation) was similarly reduced in Jurkat T cells stably transduced with a miR-150–expressing lentivirus (S1D Fig). Overall, miR-150 was the most highly expressed miRNA in human T lymphocytes, in which it controlled proliferation in response to stimuli.

Identification of miR-150 targets in human T cells Cellular context–dependent regulation is a crucial aspect of miRNA-mediated regulation that is mainly based on the relative abundance of a miRNA and its targets within a specific cell type or activation state [5,16]. Such context-dependent regulation mediated by miRNAs cannot be predicted by the available databases and can only be experimentally explored. To identify the mRNAs that are directly and specifically regulated by miR-150 in T lymphocytes, we transfected activated memory T cells from 3 independent donors with either a biotinylated version of a miR-150 mimic or a control oligonucleotide, followed by streptavidin agarose pull-down and sequencing [17–20]. As a control of target specificity, we performed the same experiment using biotinylated miR-146a. We found that the pull-down of both miR-150 and miR-146a recovered established targets for these miRNAs, namely MYB for miR-150 and IRAK1 and TRAF6 for miR-146a, thus confirming target specificity (Fig 2A, S2 and S3 Tables). Further analysis of the recovered targets showed that 31 out of the 33 miR-150 putative targets contained at least one 6-mer seed within either the 3′ UTR, 5′ UTR, or the coding sequence (CDS), and approximately 50% (17 out of 33) of these were predicted miR-150 targets by the miRWalk 2.0 database [21] (Fig 2B). Our results are in line with previous observations, reported by other groups, showing that about half of bound miRNA sites are noncanonical, and that most noncanonical sites are bound and functional in a cell type–specific manner [5,22,23]. To which extent these noncanonical sites (that are efficiently bound in vivo) mediate effective target repression remains to be fully understood [24]. Similar results were obtained for miR-146a (Fig 2B). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Identification of miR-150 targets. (A) Volcano plot of differentially expressed genes between the indicated miRNA mimic and control oligonucleotides. Genes in red were considered significantly differentially expressed when log 2 FC ≥ 0.6, −log 10 p-value ≥ 2. (B) The list of genes obtained from (A) was intersected with several prediction databases using miRWalk 2.0. Both the 3′ UTRs and the CDS were manually searched for the presence of at least a 6-mer miR-150 or miR-146a binding site. (C) Activated memory cells were transfected with miR-150 mimic or control oligonucleotide. Twenty-four hours or 48 hours after transfection, the expression of the indicated genes was measured by qRT-PCR. N = 3 to 5 independent donors. Mean ± SD. Student t test, 2 tailed, paired. (D) The 3′ UTR of the indicated genes was cloned in a dual-luciferase reporter vector and transfected into HEK cells together with either a miR-150 mimic or a control oligonucleotide. Luciferase reads were normalized to the renilla ones. N = 3 to 4 independent experiments. Mean ± SEM. Student t test, 2 tailed, paired. (E) Same as in (D), except that the 4 putative miR-150 binding sites identified in the PDAP1 3′ UTR were mutated by site-directed mutagenesis. N = 3 independent experiments. Mean ± SEM. Student t test, 2 tailed, paired. Underlying data can be found in S1 Data. A.U., arbitrary unit; CDS, coding sequence; miRNA, microRNA; qRT-PCR, quantitative RT-PCR; UTR, untranslated region; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001538.g002 Next, as validation of these pull-down data, we selected 10 putative miR-150 targets and tested the effects of a miR-150 mimic on their expression in T cells from an independent set of donors. Memory T cells were transfected with either the miR-150 mimic or control oligonucleotide, and mRNA expression was analyzed 24 hours or 48 hours later (Fig 2C). For some of the targets (HNRNPAB, MYB, PDAP1, PIK3R1, and RMND1), a suppressive effect of miR-150 was already observed after 24 hours, while for others (SMAD7 and VPS36), a significant reduction was observed only after 48 hours, most likely due to varying mRNA stability and turnover. To determine whether the observed down-regulation of these putative miR-150 targets was mediated by a direct activity of miR-150 on their 3′ UTRs, we cloned either the entire 3′ UTR or the regions containing the predicted miR-150 binding site(s) in a reporter vector. Cotransfection of these plasmids with a miR-150 mimic oligonucleotide led to significantly reduced luciferase expression for 3 out of 4 targets tested, namely MYB, PDAP1, and HNRNPAB, which were therefore the highest confidence targets, while the effect on PIK3R1 appeared to be more variable (Fig 2D). The PDAP1 3′ UTR contains 5 putative miR-150 binding sites predicted by TargetScan 7.2 [25], 4 of which are clustered in the distal region of the 3′ UTR. We cloned the region containing the 4 clustered sites, and we evaluated the impact of mutating these sites on miR-150–mediated repression. We found that mutation of only one site was sufficient to abrogate repression by miR-150, suggesting that all 4 clustered sites are required for full miR-150 activity on the PDAP1 3′ UTR (Fig 2E), although this effect may be different in vivo. To further investigate the relationship between PDAP1 and miR-150 in a more physiological setting, we deleted 1 or 3 clustered miR-150 binding sites from the 3′ UTR of the PDAP1 gene in primary human T lymphocytes, using CRISPR/Cas-9 editing. We found that deletion of one single site was insufficient to completely abrogate miR-150 activity, while the partial deletion of 3 sites reduced miR-150 responsiveness (S2 Fig). Overall, our target analysis in primary human T lymphocytes recovered established targets of miR-150, such as MYB, and identified additional ones, such as PDAP1, as direct miR-150 targets in human T cells.

[END]

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001538

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