Amplification of poly(I:C)-induced interleukin-6 production in human bronchial epithelial cells by priming with interferon-γ.

Proinflammatory cytokine interleukin (IL)-6 was associated with disease severity in patients with COVID-19. The mechanism underlying the excessive IL-6 production by SARS-Cov-2 infection remains unclear. Respiratory viruses initially infect nasal or bronchial epithelial cells that produce various inflammatory mediators. Here, we show that pretreatment of human bronchial epithelial cells (NCl-H292) with interferon (IFN)-γ (10 ng/mL) markedly increased IL-6 production induced by the toll-like receptor (TLR) 3 agonist poly(I:C) (1 µg/mL) from 0.4 ± 0.1 to 4.1 ± 0.4 ng/mL (n = 3, P < 0.01). A similar effect was observed in human alveolar A549 and primary bronchial epithelial cells. TLR3 knockdown using siRNA in NCl-H292 cells diminished the priming effects of IFN-γ on poly(I:C)-induced IL-6 production. Furthermore, the Janus kinase (JAK) inhibitor tofacitinib (1 µM) inhibited IFN-γ-induced upregulation of TLR3, and suppressed poly(I:C)-induced IL-6 production. Quantitative chromatin immunoprecipitation revealed that IFN-γ stimulated histone modifications at the IL-6 gene locus. Finally, IFN-γ priming significantly increased lung IL-6 mRNA and protein levels in poly(I:C)-administrated mice. Thus, priming bronchial epithelial cells with IFN-γ increases poly(I:C)-induced IL-6 production via JAK-dependent TLR3 upregulation and chromatin remodeling at the IL-6 gene locus. These mechanisms may be involved in severe respiratory inflammation following infection with RNA viruses.

The Il6 -39 kb enhancer containing clustered GATA2- and PU.1-binding sites is essential for Il6 expression in murine mast cells.

Mast cells serve as a first-line defense of innate immunity. Interleukin-6 (IL-6) induced by bacterial lipopolysaccharide (LPS) in mast cells plays a crucial role in antibacterial protection. The zinc finger transcription factor GATA2 cooperatively functions with the ETS family transcription factor PU.1 in multiple mast cell activities. However, the regulatory landscape directed by GATA2 and PU.1 under inflammation remains elusive. We herein showed that a large proportion of GATA2-binding peaks were closely located with PU.1-binding peaks in distal cis-regulatory regions of inflammatory cytokine genes in mast cells. Notably, GATA2 and PU.1 played crucial roles in promoting LPS-mediated inflammatory cytokine production. Genetic ablation of GATA2-PU.1-clustered binding sites at the Il6 -39 kb region revealed its central role in LPS-induced Il6 expression in mast cells. We demonstrate a novel collaborative activity of GATA2 and PU.1 in cytokine induction upon inflammatory stimuli via the GATA2-PU.1 overlapping sites in the distal cis-regulatory regions.

Mouse Tryptase Gene Expression is Coordinately Regulated by GATA1 and GATA2 in Bone Marrow-Derived Mast Cells.

Mast cell tryptases have crucial roles in allergic and inflammatory diseases. The mouse tryptase genes represent a cluster of loci on chromosome 16p3.3. While their functional studies have been extensively performed, transcriptional regulation of tryptase genes is poorly understood. In this study, we examined the molecular basis of the tryptase gene expression in bone marrow-derived mast cells (BMMCs) of C57BL/6 mice and in MEDMC-BRC6 mast cells. The expression of the Tpsb2 and Tpsg1 genes, which reside at the 3'-end of the tryptase locus, is significantly decreased by the reduction of the GATA transcription factors GATA1 or GATA2. Chromatin immunoprecipitation assays have shown that the GATA factors bind at multiple regions within the locus, including 1.0 and 72.8 kb upstream of the Tpsb2 gene, and that GATA1 and GATA2 facilitate each other's DNA binding activity to these regions. Deletion of the -72.8 kb region by genome editing significantly reduced the Tpsb2 and Tpsg1 mRNA levels in MEDMC-BRC6 cells. Furthermore, binding of CTCF and the cohesin subunit Rad21 was found upstream of the -72.8 kb region and was significantly reduced in the absence of GATA1. These results suggest that mouse tryptase gene expression is coordinately regulated by GATA1 and GATA2 in BMMCs.

GATA2 and PU.1 Collaborate To Activate the Expression of the Mouse Ms4a2 Gene, Encoding FcεRIß, through Distinct Mechanisms.

GATA factors GATA1 and GATA2 and ETS factor PU.1 are known to function antagonistically during hematopoietic development. In mouse mast cells, however, these factors are coexpressed and activate the expression of the Ms4a2 gene encoding the ß chain of the high-affinity IgE receptor (FcεRI). The present study showed that these factors cooperatively regulate Ms4a2 gene expression through distinct mechanisms. Although GATA2 and PU.1 contributed almost equally to Ms4a2 gene expression, gene ablation experiments revealed that simultaneous knockdown of both factors showed neither a synergistic nor an additive effect. A chromatin immunoprecipitation analysis showed that they shared DNA binding to the +10.4-kbp region downstream of the Ms4a2 gene with chromatin looping factor LDB1, whereas the proximal -60-bp region was exclusively bound by GATA2 in a mast cell-specific manner. Ablation of PU.1 significantly reduced the level of GATA2 binding to both the +10.4-kbp and -60-bp regions. Surprisingly, the deletion of the +10.4-kbp region by genome editing completely abolished the Ms4a2 gene expression as well as the cell surface expression of FcεRI. These results suggest that PU.1 and LDB1 play central roles in the formation of active chromatin structure whereas GATA2 directly activates the Ms4a2 promoter.

The Gata2 repression during 3T3-L1 preadipocyte differentiation is dependent on a rapid decrease in histone acetylation in response to glucocorticoid receptor activation.

The transcription factor GATA2 is an anti-adipogenic factor whose expression is downregulated during adipocyte differentiation. The present study attempted to clarify the molecular mechanism underlying the GATA2 repression and found that the repression is dependent on the activation of the glucocorticoid receptor (GR) during 3T3-L1 preadipocyte differentiation. Although several recognition sequences for GR were found in both the proximal and distal regions of the Gata2 locus, the promoter activity was not affected by the GR activation in the reporter assays, and the CRISPR-Cas9-mediated deletion of the two distal regions of the Gata2 locus was not involved in the GR-mediated Gata2 repression. Notably, the level of histone acetylation was markedly reduced at the Gata2 locus during 3T3-L1 differentiation, and the GR-mediated Gata2 repression was significantly relieved by histone deacetylase inhibition. These results suggest that GR regulates the Gata2 gene by reducing histone acetylation in the early phase of adipogenesis.

GATA2 is critical for the maintenance of cellular identity in differentiated mast cells derived from mouse bone marrow.

GATA2 plays a crucial role for the mast cell fate decision. We herein demonstrate that GATA2 is also required for the maintenance of the cellular identity in committed mast cells derived from mouse bone marrow (BMMCs). The deletion of the GATA2 DNA binding domain (GATA2ΔCF) in BMMCs resulted in a loss of the mast cell phenotype and an increase in the number of CD11b- and/or Ly6G/C-positive cells. These cells showed the ability to differentiate into macrophage- and neutrophil-like cells but not into eosinophils. Although the mRNA levels of basophil-specific genes were elevated, CD49b, a representative basophil marker, never appeared on these cells. GATA2 ablation led to a significant upregulation of C/EBPα, and forced expression of C/EBPα in wild-type BMMCs phenocopied the GATA2ΔCF cells. Interestingly, simultaneous deletion of the Gata2 and Cebpa genes in BMMCs restored the aberrant increases of CD11b and Ly6G/C while retaining the reduced c-Kit expression. Chromatin immunoprecipitation assays indicated that GATA2 directly binds to the +37-kb region of the Cebpa gene and thereby inhibits the RUNX1 and PU.1 binding to the neighboring region. Upregulation of C/EBPα following the loss of GATA2 was not observed in cultured mast cells derived from peritoneal fluid, whereas the repression of c-Kit and other mast cell-specific genes were observed in these cells. Collectively, these results indicate that GATA2 maintains cellular identity by preventing Cebpa gene activation in a subpopulation of mast cells, whereas it plays a fundamental role as a positive regulator of mast cell-specific genes throughout development of this cell lineage.

Transcription factor GATA1 is dispensable for mast cell differentiation in adult mice.

Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1(-/y)), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1(-/y) bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5' end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit(+) FcεRIα(+) mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.

Mast cell deficiency results in the accumulation of preadipocytes in adipose tissue in both obese and non-obese mice.

Mast cells have been suggested to play key roles in adipogenesis. We herein show that the expression of preadipocyte, but not adipocyte, marker genes increases in the white adipose tissue of mast cell-deficient (Kit(W-sh/W-sh) ) mice under both obese and non-obese conditions. In vitro culturing with adipogenic factors revealed increased adipocytes differentiated from the Kit(W-sh/W-sh) stromal vascular fraction, suggesting the accumulation of preadipocytes. Moreover, the increased expression of preadipocyte genes was restored by mast cell reconstitution in the Kit(W-sh/W-sh) mice. These results suggest positive effects of mast cells on the preadipocyte to adipocyte transition under both physiological and pathological conditions.

GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation.

Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the 'GATA factor switching'. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1-knockdown embryos that concomitantly express Gata2-GFP reporter, high-level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1-binding sites widely overlap with the sites pre-occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient-phase genes.

Establishment of erythroleukemic GAK14 cells and characterization of GATA1 N-terminal domain.

GATA1 is a transcription factor essential for erythropoiesis and megakaryopoiesis. It has been found that Gata1 gene knockdown heterozygous female (Gata1(G1.05/+)) mice spontaneously develop erythroblastic leukemias. In this study, we have generated a novel Gata1 knockdown erythroblastic cell line, designated GAK14, from the leukemia cells in the Gata1(G1.05/+) mice. Although GAK14 cells maintain immature phenotype on OP9 stromal cells in the presence of erythropoietin and stem cell factor, the cells produce Gr-1-, Mac1-, B220-, CD3e- or CD49b-positive hematopoietic cells when co-cultured with DAS104-8 feeder cells. However, GAK14 cells did not produce erythroid and megakaryocytic lineages, perhaps due to the absence of GATA1. Indeed, GAK14 cells became capable of differentiating into mature erythroid cells when complemented with full-length GATA1 and co-cultured with fetal liver-derived FLS5 stromal cells. This differentiation potential was impaired when GATA1 lacking the N-terminal domain was complemented. The N-terminal domain is known to contribute to the pathogenesis of transient abnormal myelopoiesis and acute megakaryoblastic leukemia related to Down syndrome. These results thus showed that GAK14 cells will serve as a powerful tool for dissecting domain function of GATA1 and that the GATA1 N-terminal domain is essential for the erythroid differentiation of GAK14 cells.

Regulation of GATA factor expression is distinct between erythroid and mast cell lineages.

The zinc finger transcription factors GATA1 and GATA2 participate in mast cell development. Although the expression of these factors is regulated in a cell lineage-specific and differentiation stage-specific manner, their regulation during mast cell development has not been clarified. Here, we show that the GATA2 mRNA level was significantly increased while GATA1 was maintained at low levels during the differentiation of mast cells derived from mouse bone marrow (BMMCs). Unlike in erythroid cells, forced expression or small interfering RNA (siRNA)-mediated knockdown of GATA1 rarely affected GATA2 expression, and vice versa, in mast cells, indicating the absence of cross-regulation between Gata1 and Gata2 genes. Chromatin immunoprecipitation assays revealed that both GATA factors bound to most of the conserved GATA sites of Gata1 and Gata2 loci in BMMCs. However, the GATA1 hematopoietic enhancer (G1HE) of the Gata1 gene, which is essential for GATA1 expression in erythroid and megakaryocytic lineages, was bound only weakly by both GATA factors in BMMCs. Furthermore, transgenic-mouse reporter assays revealed that the G1HE is not essential for reporter expression in BMMCs and peritoneal mast cells. Collectively, these results demonstrate that the expression of GATA factors in mast cells is regulated in a manner quite distinct from that in erythroid cells.

UG4 enhancer-driven GATA-2 and bone morphogenetic protein 4 complementation remedies the CAKUT phenotype in Gata2 hypomorphic mutant mice.

During renal development, the proper emergence of the ureteric bud (UB) from the Wolffian duct is essential for formation of the urinary system. Previously, we showed that expression of transcription factor GATA-2 in the urogenital primordium was demarcated anteroposteriorly into two domains that were regulated by separate enhancers. While GATA-2 expression in the caudal urogenital mesenchyme is controlled by the UG4 enhancer, its more-rostral expression is regulated by UG2. We found that anteriorly displaced budding led to obstructed megaureters in Gata2 hypomorphic mutant mice, possibly due to reduced expression of the downstream effector bone morphogenetic protein 4 (BMP4). Here, we report that UG4-driven, but not UG2-driven, GATA-2 expression in the urogenital mesenchyme significantly reverts the uropathy observed in the Gata2 hypomorphic mutant mice. Furthermore, the data show that transgenic rescue by GATA-2 reverses the rostral outgrowth of the UB. We also provide evidence for a GATA-2-BMP4 epistatic relationship by demonstrating that reporter gene expression from a Bmp4 bacterial artificial chromosome (BAC) transgene is altered in Gata2 hypomorphs; furthermore, UG4-directed BMP4 expression in the mutants leads to reduced incidence of megaureters. These results demonstrate that GATA-2 expression in the caudal urogenital mesenchyme as directed by the UG4 enhancer is crucial for proper development of the urinary tract and that its regulation of BMP4 expression is a critical aspect of this function.

GATA transcription factors are involved in IgE-dependent mast cell degranulation by enhancing the expression of phospholipase C-γ1.

Mast cell degranulation is a dynamic, highly organized process involving numerous signaling molecules and enzymes. Although the molecular mechanisms underlying antigen-mediated mast cell degranulation have been studied intensively, little is known about the transcriptional control of this process. Here, we show that the hematopoietic transcription factors GATA1 and GATA2 are involved in mast cell degranulation through the control of phospholipase C-γ1 (PLC-γ1) expression. Knockdown of GATA1 and/or GATA2 by specific siRNA significantly reduced antigen-induced degranulation and Ca(2+) mobilization in the rat basophilic leukemia cell line RBL-2H3. RT-PCR analyses showed that PLC-γ1 expression was significantly decreased by this GATA factor repression. Other GATA factor targets, such as the previously reported α and ß subunits of the high-affinity IgE receptor (FcεRI), were unaffected. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that GATA factors directly activate PLC-γ1 gene transcription through a conserved GATA-binding motif that resides in the 5'-upstream sequence. Furthermore, we show evidence that the PLC-γ1 expression is regulated by GATA2 in mast cells derived from mouse bone marrow. These data indicate that PLC-γ1 is a target gene of GATA factors in mast cells and provide evidence that GATA1 and GATA2 control antigen-mediated mast cell degranulation by regulating the expression of PLC-γ1.

Characterization of a functional ZBP-89 binding site that mediates Gata1 gene expression during hematopoietic development.

GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5' region of the enhancer. However, the importance of the GC-rich region adjacent to the 3'-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G(5) string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G(5) string (G(4) mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G(5) string sequence. Finally, transgenic mouse studies reveal that the G(4) mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G(5) string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region.

Systematic screening for genes specifically expressed in the anterior neuroectoderm during early Xenopus development.

A cDNA library derived from the anterior neuroectoderm (ANE) of Xenopus late-gastrula embryos was systematically screened to isolate novel developmental regulatory genes involved in early brain development. We isolated 1,706 5 expressed sequence tags (ESTs), which were subdivided into 1,383 clusters and categorized into 19 classes based on predicted functions according to their similarities to other known genes. Of these, 757 clusters that were considered possible novel regulatory genes or unknown genes were subjected to expression pattern analysis using whole-mount in situ hybridization. Genes from 69 clusters (9%) were expressed in the ANE region. Based on their expression patterns and predicted amino acid sequences, 25 genes were selected for further analysis as novel Xenopus genes expressed broadly or region-specifically in the ANE. Eighteen genes were expressed in postulated patterning centers in the neuroectoderm, including the anterior (four genes) and lateral (nine genes) neural ridges, the midbrain-hindbrain boundary region (one gene) and the midline region of the neural plate (two genes), whereas 13 genes were expressed in the eye anlagen. Therefore, early regionalization of the neuroectoderm appears to occur mainly in those neural patterning centers and the eye anlagen. We determined the entire coding regions of p54nrb, Semaphorin 6D and a novel gene designated scribble-related protein 1 (SCRP1). Interestingly, Semaphorin 6D is expressed in the mesoderm with a dorsoventral gradient, as well as in the ectoderm at the gastrula stage, implying a new role for this protein in development other than in axon guidance.

Selective degradation of excess Ldb1 by Rnf12/RLIM confers proper Ldb1 expression levels and Xlim-1/Ldb1 stoichiometry in Xenopus organizer functions.

The Xenopus LIM homeodomain (LIM-HD) protein, Xlim-1, is expressed in the Spemann organizer and cooperates with its positive regulator, Ldb1, to activate organizer gene expression. While this activation is presumably mediated through Xlim-1/Ldb1 tetramer formation, the mechanisms regulating proper Xlim-1/Ldb1 stoichiometry remains largely unknown. We isolated the Xenopus ortholog (XRnf12) of the RING finger protein Rnf12/RLIM and explored its functional interactions with Xlim-1 and Ldb1. Although XRnf12 functions as a E3 ubiquitin ligase for Ldb1 and causes proteasome-dependent degradation of Ldb1, we found that co-expression of a high level of Xlim-1 suppresses Ldb1 degradation by XRnf12. This suppression requires both the LIM domains of Xlim-1 and the LIM interaction domain of Ldb1, suggesting that Ldb1, when bound to Xlim-1, escapes degradation by XRnf12. We further show that a high level of Ldb1 suppresses the organizer activity of Xlim-1/Ldb1, suggesting that excess Ldb1 molecules disturb Xlim-1/Ldb1 stoichiometry. Consistent with this, Ldb1 overexpression in the dorsal marginal zone suppresses expression of several organizer genes including postulated Xlim-1 targets, and importantly, this suppression is rescued by co-expression of XRnf12. These data suggest that XRnf12 confers proper Ldb1 protein levels and Xlim-1/Ldb1 stoichiometry for their functions in the organizer. Together with the similarity in the expression pattern of Ldb1 and XRnf12 throughout early embryogenesis, we propose Rnf12/RLIM as a specific regulator of Ldb1 to ensure its proper interactions with LIM-HD proteins and possibly other Ldb1-interacting proteins in the organizer as well as in other tissues.

XMAN1, an inner nuclear membrane protein, antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos.

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.