Analysis of a miR-148a Targetome in B Cell Central Tolerance

A microRNA (miRNA) often regulates the expression of hundreds of target genes. A fundamental question in the field of miRNA research is whether a miRNA exerts its biological function through regulating a small number of key targets or through small changes in the expression of hundreds of target genes. We addressed this issue by performing functional analysis of target genes regulated by miR-148a. We previously identified miR-148a as a critical regulator of B cell central tolerance and found 119 target genes that may mediate its function. We selected 4 of them for validation and demonstrated a regulatory role for Bim, Pten, and Gadd45a in this process. In this study, we performed functional analysis of the other miR-148a target genes in in vitro and in vivo models of B cell central tolerance. Our results show that those additional target genes play a minimal role, if any, in miR-148a-mediated control of B cell central tolerance, suggesting that the function of miRNAs is mediated by a few key target genes. These findings have advanced our understanding of molecular mechanisms underlying miRNA regulation of gene expression and B cell central tolerance.


INTRODUCTION
B cells mount humoral responses against pathogens following their recognition of a vast variety of foreign antigens through their B cell receptors (BCRs). The large diversity of BCR specificities required for this is generated through V(D)J recombination of the heavy and light chains of the immunoglobulin (Ig) M during the early pro-B and pre-B stages of B cell development in the bone marrow. The largely random and combinatorial nature of these rearrangements also generates many autoreactive B cells that recognize self-tissues and, thus, can lead to autoimmunity. To test their autoreactivity, IgM-expressing immature B cells are scrutinized by the process of central tolerance. If a B cell is autoreactive, it continues to rearrange its light chain to eliminate selfreactivity through receptor editing. If this process is not successful, the autoreactive cell is deleted from the repertoire by apoptosis. This is known as clonal deletion. If the cell is non-autoreactive or eliminated its autoreactivity through receptor editing, it exits to the periphery to become a mature B cell (1)(2)(3). B cell central tolerance is a tightly regulated process, but the molecular mechanisms governing this checkpoint remain largely unexplored. Expanding this knowledge is important because defects in B cell tolerance have been shown in many patients with autoimmune diseases such as systemic lupus erythematosus (SLE), type 1 diabetes, rheumatoid arthritis, and multiple sclerosis (4,5).
MiRNAs are small non-coding RNAs of approximately 22 nucleotides in length that regulate the expression of proteincoding genes at the post-transcriptional level, resulting in a fast and robust control of virtually all cellular processes, including development, activation, proliferation, differentiation, and apoptosis (6)(7)(8)(9)(10)(11)(12). MiRNA expression levels are tightly regulated and highly conserved throughout evolution, which supports their functional importance (13)(14)(15). Hundreds of genes can be targeted in any given cell by an individual miRNA through its cognate binding sites on their mRNAs (16,17). MiRNA studies frequently validate a limited number of predicted or experimentally identified target genes, without examining all potential target genes in a systematic manner. Hence, a fundamental question in the field that remains to be answered is whether miRNAs exert their function in a given cellular context through simultaneous regulation of a large number of target genes or, alternatively, through a few critical ones (11,16,18,19).
In a recent study, we identified miR-148a as the first miRNA that functions as a critical regulator of B cell tolerance using the IgM b -macroself mouse model of B cell tolerance (20). The IgM bmacroself mice ubiquitously express an engineered membranebound superantigen reactive to the constant region of IgM heavy chain. In these mice, early B cell development occurs normally, resulting in an immature B cell pool with a large variety of antigen specificities in their bone marrow. However, once immature B cells express IgM molecules on the cell surface, they are recognized by the engineered superantigen. This mimics the recognition of self-antigens by BCR. These "autoreactive" B cells first attempt to eliminate their autoreactivity through receptor editing and then die by apoptosis (clonal deletion), resulting in the absence of B cells in the peripheral lymphoid organs. This murine model provides a robust and clean system to identify novel regulators of B cell tolerance through a hematopoietic and precursor stem cell (HPSC) transduction and reconstitution approach or reconstitution with bone marrow cells from transgenic or knockout mice, with the presence of B cells in the spleens of reconstituted IgM bmacroself mice indicating a break of B cell tolerance. MiR-148a expression is tightly regulated during B cell development and differentiation, and its expression levels frequently increased in lymphocytes of patients with lupus erythematosus and lupusprone mice even before disease onset (13,(20)(21)(22). Increased expression of miR-148a in B cells compromised B cell tolerance in IgM b -macroself mice and facilitated lethal autoimmunity in MRL-lpr mice, a classical mouse model of lupus. Importantly, this effect was recapitulated in WEHI-231 cells, an in vitro model of clonal deletion. Moreover, 119 protein coding genes were identified as potential miR-148a target genes. Four of them were further investigated and 3 of them (Bcl2l11, Pten and Gadd45a) were demonstrated to be bona fide miR-148a target genes and regulate B cell central tolerance in IgM b -macroself mice (20).
However, this study did not investigate the potential contribution of the other 115 target genes to miR-148a regulation of B cell tolerance. This provided the opportunity to perform a systematic and unbiased functional analysis of a miRNA targetome in B cell tolerance to determine whether this miRNA exerts its biological function through a large number or only a few key target genes in this cellular context. No study addressing this question has been previously conducted to our knowledge. By performing in vitro and in vivo functional analysis of miR-148a target genes in B cell tolerance, this work aims to advance our current understanding of miRNA biology in B cell tolerance, with potential implications for the therapy of autoimmune diseases.

Retroviral TransOMIC-shRNA Library Generation
The TransOMIC-shRNA sublibrary was obtained from the RNAi Core at La Jolla Institute for Allergy and Immunology (La Jolla, CA), amplified from a glycerol stock, and DNA prepared using an endotoxin-free DNA purification kit (K210001, ThermoFisher). Retrovirus preparations were obtained as previously described (20). Briefly, packaging vectors GAG-POL, VSV-G and individual shRNA-encoding or empty plasmids were co-transfected into HEK293T cells using TransIT-LT1 (Mirus) following the manufacturer´s instructions. Supernatants containing retrovirus were collected after 48 and 72 hours, filtered through a 0.45mm low protein retention filter, aliquoted and stored at −80°C until use.

In Vitro Screen of Stable shRNA WEHI-231 Cell Lines
To generate stable WEHI-231 cell lines, 0.5x10 6 cells were transduced with retroviruses encoding individual shRNAs in 24-well plates overnight. Media was replaced with fresh one the next morning. After 24 hours of culture, 10µg/ml puromycin was added to medium to select transduced cells for 5 days. After selection, stable cell lines were maintained in the presence of 2µg/ ml puromycin in culture medium. Stable WEHI-231 cells were stimulated with 2µg/ml anti-IgM (115-006-020, µ chain specific, Jackson) for 72 hours, stained for intracellular active caspase-3, and analyzed by flow cytometry as described above.

In Vivo Validation of Positive Hits in IgM b -Macroself Mice
For in vivo validation, plasmids encoding positive shRNA hits were modified to delete the puromycin resistance gene, amplified using an endotoxin-free DNA purification kit (12362, Qiagen), and packaged to generate retroviral supernatants as described above. For HPSC enrichment, wild type C57BL/6J mice were treated with 4 mg/mouse 5-fluorouracil (5-FU) (Sigma) by intravenous (iv) injection, followed by bone marrow cell collection 5 days later. HPSCs were stimulated with SCF (100ng/ ml, Novoprotein), IL-3 (20ng/ml, Novoprotein) and IL-6 (25ng/ ml, Novoprotein) for 36 hours before transduction. Retrovirus transduction of HPSCs was performed by spinoculation (2000rpm, 2 hours without brake) and media was replaced 4 hours later. Transduced cells were harvested after 20 hours for intravenous transfer into IgM b -macroself mice. Alternatively, freshly isolated BM cells from mutant mice were depleted of red blood cells using ACK lysis buffer, counted, and resuspended in PBS. 1-10x10 6 retrovirally transduced HPSCs or freshly isolated BM cells were iv injected into recipient mice. IgM b -macroself mice were irradiated with 6 Gy by X-Ray before reconstitution. Recipient mice were euthanized and their splenocytes analyzed by flow cytometry 8 weeks after reconstitution.

Statistical Analysis
Data were analyzed with an unpaired two-tailed Student's t test or one-way ANOVA using the Prism software Version9 as indicated in the figure legends. Statistical significance was set at P<0.05.

In Vitro Functional Screen Platform Development
WEHI-231 is a commonly used immature B cell line that undergoes cell cycle arrest at G0/G1 phase and apoptosis upon anti-IgM-induced BCR crosslinking (25). This mimics the recognition of self-antigens by and subsequent clonal deletion (apoptosis) of autoreactive B cells that occur in vivo under homeostatic conditions. We previously found that miR-148a protects WEHI-231 cells from BCR engagement-induced apoptosis triggered by anti-IgM stimulation. This recapitulated the role of miR-148a in B cell central tolerance, where it prevented clonal deletion of autoreactive B cells following selfantigen encounter (20). We further identified 119 protein-coding genes that contained predicted miR-148a binding sites in their mRNAs and showed at least 1.5-fold reduction in their mRNA levels upon miR-148a overexpression. Four of them were chosen for experimental validation. Among them, Bcl2l11 (encoding Bim), Pten and Gadd45a were validated as regulators of B cell tolerance, while Tnfrsf1b did not play any significant role in this process (20). In this study, we investigated the function of the other miR-148a target genes in B cell tolerance, first in the WEHI-231 system, followed by validation in animal models ( Figure 1A). To this end, a retroviral TransOMIC shRNA library containing 288 shRNAs (Supplementary Table 1) covering 106 miR-148a target genes, with one to nine shRNAs for each gene, was generated. An empty vector (EV) and a vector encoding an shRNA against Bcl2l11 (shRNA-Bcl2l11) were included as negative and positive controls. The backbone vector used to generate this library contains a shRNA cassette followed by a puromycin resistance gene that enables puromycin selection of transduced cells ( Figure 1B). Retroviruses were produced from individual constructs to generate the shRNA retroviral library as previously described (20).
The strategy for our in vitro functional screen is outlined in Figure 1A. WEHI-231 cells were transduced with retroviruses expressing individual shRNAs. After 48 hours, transduced cells were selected with puromycin for 5 days. Half of the cells were then stimulated with anti-IgM (2mg/ml) for 72 hours to induce BCR-engagement induced apoptosis, while the other half were left without stimulation and served as control. After 72 hours of stimulation, cells were harvested, stained for active Caspase 3, and analyzed by flow cytometry to quantify the percentage of apoptotic cells as a readout for clonal deletion. WEHI-231 cells transduced with empty retroviruses (WEHI-EV) and with retroviruses encoding shRNA-Bcl2l11 (WEHI-shRNA-Bcl2l11) were included in every independent experiment as controls.
The feasibility of this approach was first validated with WEHI-EV and WEHI-shRNA-Bcl2l11 controls. As expected, a significant fraction of non-transduced and WEHI-EV-transduced WEHI-231 cells died by apoptosis when stimulated with anti-IgM, and cell death was largely abrogated in shRNA-Bcl2l11-transduced cells (Figures 1C, D). This protection from clonal deletion by decreasing the expression levels of Bim recapitulated our previous results obtained in the IgM bmacroself B cell tolerance mouse model (20).

Functional Screen of the miR-148a Targetome shRNA Library
Once the in vitro screen platform had been validated, 288 WEHI-231 stable cell lines expressing individual shRNAs for miR-148a target genes were generated. WEHI-231 cells were transduced with retroviruses encoding individual shRNAs, selected with puromycin, stimulated with anti-IgM, and analyzed for apoptosis as described above ( Figure 1A). The effect of shRNAs that significantly impaired BCR engagement-induced apoptosis were confirmed in three independent experiments.
Among the 106 miR-148a target genes tested, nearly half of them showed an effect in protecting WEHI-231 cells from BCRinduced apoptosis when knocked down by shRNAs, with protection ratios ranging from 5% to 65% (Figure 2A). Among those, 22 shRNAs, which correspond to 13 genes, showed an average protection effect greater than 30% ( Figure 2B and Figure 3). Of note, knockdown of B4galt5, Phip and Nsd1 showed the most robust protection against BCR engagement-induced apoptosis, with all shRNAs showing consistent effect ( Figures 2C, D).

In Vivo Analysis of Positive Hits in IgM b -Macroself Mouse Model
We next investigated the function of those positive hits in regulating B cell tolerance in vivo, by retroviral transduction of HSPCs and reconstitution of IgM b -macroself mice. Briefly, C57BL/6J mice were treated with 5-fluorouracil (5-FU) for 5 days. HSPCs from treated mice were enriched, transduced with shRNA-encoding retroviruses, and transferred to sublethally irradiated IgM b -macroself mice. After 8 weeks, the presence of peripheral B cells was analyzed in the spleen of these reconstituted mice by flow cytometry ( Figure 4A). We set the presence of 5% splenic B cells as the threshold to consider an effect of breaking B cell tolerance because irradiated IgM bmacroself mice reconstituted with wild-type C57BL/6J bone marrow cells show some background of splenic B cells after 8 weeks, up to this percentage in some experiments.
We observed a significant number of splenic B cells in 6 out of 16 (37%) and 6 out of 19 (32%) IgM b -macroself mice reconstituted with HPSCs transduced with shRNA-B4galt5 and shRNA-Nsd1, respectively. Of note, a combination of two different shRNAs for B4galt5 was required for this effect, as transduction of HSPCs with individual shRNAs failed to break B  Figure 1). The remaining mice did not show compromised B cell tolerance ( Figures 4B, C). Although shRNA-Phip showed a significant protection against BCR engagement-induced apoptosis in WEHI-231 cells, those shRNAs did not show much effect in IgM b -macroself mice, suggesting that Phip does not play any significant role in regulating B cell tolerance ( Figures 4B, C). All other hits from the screen failed to break B cell tolerance in vivo.

In Vivo Validation by a Genetic Approach
To confirm that B4galt5 and Nsd1 indeed regulate B cell tolerance, we obtained conditional knockout (KO) mice with specific deletion of these genes in B cells.

DISCUSSION
B cell tolerance mechanisms are essential for the generation of a vast pool of B cells that enable the defense against a large variety of pathogens without attacking self-tissues. When B cell tolerance checkpoints are compromised, the development of autoimmune diseases ensue (1,6,20,(26)(27)(28)(29)(30)(31)(32)(33). Aiming to advance our knowledge on B cell tolerance, we investigated the function of more than one hundred miR-148a target genes in this process.
Our previous study showed that elevated expression of miR-148a impaired BCR engagement-induced apoptosis in WEHI-231 cells, and that this was recapitulated in IgM b -macroself mice, suggesting that WEHI-231 cells are a good in vitro model for studying B cell central tolerance. In this study, we performed initial screen of miR-148a target genes in WEHI-231 cells and identified 13 genes whose knockdown protected BCR Further validation of the screen hits by retroviral transduction and bone marrow reconstitution of IgM b -macroself mice showed that shRNA-mediated knockdown of two of those 13 genes, B4galt5 or Nsd1, led to break of B cell tolerance in a fraction of IgM b -macroself mice. When irradiated IgM b -macroself mice were reconstituted with bone marrow cells from mutant mice with B cell-specific deletion of B4galt5 or Nsd1, very few B cells escaped B cell central tolerance. Therefore, none of those 13 genes whose knockdown protected BCR engagement-induced apoptosis in WEHI-231 cells plays important roles in regulating B cell central tolerance. We speculate that the escape of B cell tolerance in some of the IgM b -macroself mice reconstituted with HPSCs transduced with retroviruses encoding shRNAs for B4galt5 or Nsd1 was due to off-target effect of these shRNAs. MiRNAs bind target gene mRNAs mainly through pairing with their seed regions, allowing for the regulation of hundreds of target genes by one miRNA. Exactly how many targets mediate the function of a miRNA has been hotly debated. Cellular context-dependence of miRNA functions adds another layer of complexity to this issue (16,18,34,35). For example, miR-148a regulates antigen presentation of TLR-triggered dendritic cells (36), osteoclastogenesis of CD14 + peripheral blood mononuclear cells (37), LDL receptor activity in human hepatic cells (38), and functions as a tumor suppressor miRNA in various types of cancers including gastric, colorectal, hepatocellular and pancreatic cancers (39)(40)(41)(42). How the same miRNA exerts such diverse functions in different cell types remains elusive. It is possible that these differences rely on the differential gene expression programs of each individual cell type and that miRNA-mediated regulation of target genes is only efficient when target genes are expressed at appropriate levels in the cell type of interest. Another possibility is that only a small number of the hundreds of target genes regulated by a given miRNA are both functionally important and sensitive to changes in their cellular concentrations in any given cell type. Those genes would be the key target genes mediating the function of this miRNA in this particular cellular context. In the context of B cell central tolerance, three key target genes (i.e. Bcl2l11, Pten and Gadd45a) drive the function of miR-148a upon engagement of their BCRs with anti-IgM, with a minor contribution, if any, from the other one hundred or so target genes. Further exploring if miR-148a functions similarly or not in B cells receiving different stimuli such as TLR agonist or anti-CD40 and anti-IgM would be of interest (21,(43)(44)(45).
Our results support the concept that miRNAs exert their regulatory functions through a limited number of key target genes. In future studies, it would be interesting to perform functional analysis of miR-148a target genes in other cellular contexts to find out whether the same or different sets of key target genes mediate the function of this miRNA in different types of cells.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Care and Use Committees of Xiamen University and Autonoma University of Madrid.  (20). Statistical analysis was performed with an unpaired two-tailed Student's T-test with ***P < 0.001 and ****P < 0.0001. No statistically significant differences were found in (D, F).

AUTHOR CONTRIBUTIONS
the manuscript. All authors contributed to the article and approved the submitted version.