Jangsuk Oh

Analysis of Interleukin-21-Induced Prdm1 Gene Regulation Reveals Functional Cooperation of STAT3 and IRF4 Transcription Factors

Interleukin-21 (IL-21) is a pleiotropic cytokine that induces expression of transcription factor BLIMP1 (encoded by Prdm1), which regulates plasma cell differentiation and T cell homeostasis. We identified an IL-21 response element downstream of Prdm1 that binds the transcription factors STAT3 and IRF4, which are required for optimal Prdm1 expression. Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRF4 in vivo and furthermore revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used for STAT3 binding. Comparing genome-wide expression array data to binding sites revealed that most IL-21-regulated genes were associated with combined STAT3-IRF4 sites rather than pure STAT3 sites. Correspondingly, ChIP-Seq analysis of Irf4(-/-) T cells showed greatly diminished STAT3 binding after IL-21 treatment, and Irf4(-/-) mice showed impaired IL-21-induced Tfh cell differentiation in vivo. These results reveal broad cooperative gene regulation by STAT3 and IRF4.

The Cytokines IL-21 and GM-CSF Have Opposing Regulatory Roles in the Apoptosis of Conventional Dendritic Cells

Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-γ (IFN-γ)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4+ T cells

Significance IL-21 is a type I cytokine important for immune cell differentiation and function. We found that transcription factors STAT1 and STAT3 play partially opposing roles in IL-21 function in CD4+ T cells. Both STAT1 and STAT3 control IL-21–mediated gene regulation, with some genes, including Ifng, Tbx21, and Il21 reciprocally regulated by these STATs. IFN-γ production was also differentially regulated by these STATs in vitro during CD4+ T-cell differentiation and in vivo during chronic lymphocytic choriomeningitis infection. Importantly, IL-21–induced IFNG and TBX21 expression was higher in CD4+ T cells from patients with autosomal dominant hyper-IgE syndrome or with STAT1 gain-of-function mutations, suggesting that dys-regulated IL-21–STAT signaling partially explains the clinical manifestations of these patients. IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4+ T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4+ T cells. RNA-Seq analysis of CD4+ T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21–mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4+ T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21–mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4+ T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.

EGR2 is critical for peripheral naïve T-cell differentiation and the T-cell response to influenza

Significance Early growth response 2 (EGR2) is a transcription factor that can negatively regulate T-cell activation. We unexpectedly found that EGR2 promotes peripheral naïve T-cell proliferation and differentiation, with less T-cell receptor-induced IL-2 production in Egr2-deficient naïve T cells and diminished cytokine production in T-helper differentiated cells. Moreover, EGR2 was required for T-cell responses to influenza, with delayed viral clearance and more severe pathology in lungs of Egr2 conditional knockout mice, as well as decreased effector cytokine production from T cells. Thus, EGR2 can act as a positive regulator essential for a normal T-cell response to viral infection, a finding with potential clinical implications. Early growth response 2 (EGR2) transcription factor negatively regulates T-cell activation, in contrast to the positive regulation of this process by EGR1. Here, we unexpectedly found that EGR2 promotes peripheral naïve T-cell differentiation, with delayed T-cell receptor-induced proliferation in naïve T cells from Egr2 conditional knockout (CKO) mice and decreased production of IFN-γ, IL-4, IL-9, and IL-17A in cells subjected to T-helper differentiation. Moreover, genes that promote T-cell activation, including Tbx21 and Notch1, had decreased expression in Egr2 CKO T cells and are direct EGR2 target genes. Following influenza infection, Egr2 CKO mice had delayed viral clearance, more weight loss, and more severe pathological changes in the lung than did WT and Egr1 KO mice, with decreased production of effector cytokines, increased infiltration of antigen-specific memory-precursor CD8+ T cells, and lower numbers of lung-resident memory CD8+ T cells. Thus, unexpectedly, EGR2 can function as a positive regulator that is essential for naïve T-cell differentiation and in vivo T-cell responses to a viral infection.

STAT5-mediated chromatin interactions in superenhancers activate IL-2 highly inducible genes: Functional dissection of the Il2ra gene locus

Significance Superenhancers regulate the expression of genes that specify cell type-specific development, but little is known regarding their function and regulation in vivo. Here, we study the cytokines IL-2 and IL-21, which critically control the immune response. These cytokines induce the binding of transcription factors STAT5 and STAT3, respectively, at superenhancers in a cytokine- and gene-specific manner. STAT5-bound superenhancers regulate genes highly induced by IL-2, with STAT5 mediating chromatin looping within such genes, including Il2ra, which mediates responsiveness to IL-2. By deleting three STAT5 binding sites that mediate IL-2–induced chromatin looping at the Il2ra locus, we demonstrate that superenhancer elements cooperatively control gene expression. Overall, we elucidate cytokine-dependent superenhancer function in general and provide detailed analysis of the Il2ra superenhancer. Cytokines critically control immune responses, but how regulatory programs are altered to allow T cells to differentially respond to distinct cytokine stimuli remains poorly understood. Here, we have globally analyzed enhancer elements bound by IL-2–activated STAT5 and IL-21–activated STAT3 in T cells and identified Il2ra as the top-ranked gene regulated by an IL-2–activated STAT5-bound superenhancer and one of the top genes regulated by STAT3-bound superenhancers. Moreover, we found that STAT5 binding was rapidly superenriched at genes highly induced by IL-2 and that IL-2–activated STAT5 binding induces new and augmented chromatin interactions within superenhancer-containing genes. Based on chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing data, we used CRISPR-Cas9 gene editing to target three of the STAT5 binding sites within the Il2ra superenhancer in mice. Each mutation decreased STAT5 binding and altered IL-2–induced Il2ra gene expression, revealing that individual elements within the superenhancer were not functionally redundant and that all were required for normal gene expression. Thus, we demonstrate cooperative utilization of superenhancer elements to optimize gene expression and show that STAT5 mediates IL-2–induced chromatin looping at superenhancers to preferentially regulate highly inducible genes, thereby providing new insights into the mechanisms underlying cytokine-dependent superenhancer function.

IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells.

The canonical pathway for IL-1β production requires TLR-mediated NF-κB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1β. Here we show that IL-21 unexpectedly induces IL-1β production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-κB-independent pathway. IL-21 does not induce Il1b expression in CD4+ T cells, with differential histone marks present in these cells versus cDCs. IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1β expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with pneumonia virus of mice. These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo.

Genetic background-dependent role of Egr1 for eyelid development.

Significance Eyelid formation begins at approximately day E15.5 in mice. Over the next 24 h, the epidermis of both upper and lower eyelids rapidly grows and merges to cover the cornea. Here, we demonstrate that Egr1−/− mice on the C57BL/6 background have normal eyelid development, but back-crossing to BALB/c background for four or five generations resulted in defective eyelid development by embryonic day E15.5. This defective eyelid formation was then further associated with profound ocular anomalies evident by postnatal days 1-4. The BALB/c albino phenotype associated with the Tyrc tyrosinase mutation also appeared to contribute to the phenotype. Thus EGR1 in a genetic background-dependent manner plays a critical role in mammalian eyelid development, with subsequent impact on ocular integrity. EGR1 is an early growth response zinc finger transcription factor with broad actions, including in differentiation, mitogenesis, tumor suppression, and neuronal plasticity. Here we demonstrate that Egr1−/− mice on the C57BL/6 background have normal eyelid development, but back-crossing to BALB/c background for four or five generations resulted in defective eyelid development by day E15.5, at which time EGR1 was expressed in eyelids of WT mice. Defective eyelid formation correlated with profound ocular anomalies evident by postnatal days 1–4, including severe cryptophthalmos, microphthalmia or anophthalmia, retinal dysplasia, keratitis, corneal neovascularization, cataracts, and calcification. The BALB/c albino phenotype-associated Tyrc tyrosinase mutation appeared to contribute to the phenotype, because crossing the independent Tyrc-2J allele to Egr1−/− C57BL/6 mice also produced ocular abnormalities, albeit less severe than those in Egr1−/− BALB/c mice. Thus EGR1, in a genetic background-dependent manner, plays a critical role in mammalian eyelid development and closure, with subsequent impact on ocular integrity.