Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in ...respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators
and
as well as
mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.
Specification of the T helper 17 (Th17) cell lineage requires a well-defined set of transcription factors, but how these integrate with posttranscriptional and epigenetic programs to regulate gene ...expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient Th17 and T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9, and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2.
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•miR-155 is highly induced during mouse and human Th17 cell differentiation•Jarid2 and miR-155 are epistatic in Th17 and Treg cells•Jarid2 is required to recruit PRC2 to genomic sites in Th17 cells•Direct targets of PRC2 in Th17 cells include Il22, Il10, Il9, and Atf3
miR-155 is known to promote inflammatory Th17 cell responses, but the mechanism has been unclear. Escobar et al. find that miR-155 promotes cytokine expression in Th17 cells by repressing Jarid2 to relieve Polycomb-mediated gene silencing.
Abstract
MicroRNA miR-155 is an important regulatory molecule in the immune system and is highly expressed and functional in Th17 cells, a subset of CD4
+
T helper cells which are key players in ...autoimmune diseases. Small molecules that can modulate miR-155 may potentially provide new therapeutic avenues to inhibit Th17 cell-mediated autoimmune diseases. Here, we present a novel high-throughput screening assay using primary T cells from genetically engineered
Mir155
reporter mice, and its use to screen libraries of small molecules to identify novel modulators of Th17 cell function. We have discovered a chemical series of (
E
)-1-(phenylsulfonyl)-2-styryl-1
H
-benzo
d
imidazoles as novel down-regulators of
Mir155
reporter and cytokine expression in Th17 cells. In addition, we found that FDA approved antiparasitic agents belonging to the ‘azole’ family also down-regulate
Mir155
reporter and cytokine expression in Th17 cells, and thus could potentially be repurposed to treat Th17-driven immunopathologies.
After peripheral nerve axotomy, vasoactive intestinal peptide (VIP) gene expression is upregulated in neurons, whereas ciliary neurotrophic factor (CNTF) accumulates extracellularly at the lesion ...site. Although CNTF-induced VIP gene expression has been reported in cultured sympathetic neurons and neuroblastoma cells, it still remains to be determined if CNTF and VIP play interrelated roles in nerve injury. The corneal endothelium, like sympathetic neurons, derives from the neural crest. Previously, we demonstrated that a sublethal-level of oxidative stress induces CNTF release from corneal endothelial (CE) cells
in situ. Here, we show that human CE cells express the 53
kDa ligand-binding α subunit of the CNTF receptor (CNTFRα). We further demonstrate that CNTF induces VIP immunoreactivity in human donor corneas. To determine if the increase in VIP immunoreactivity was reflected by an increase in gene expression, donor human corneas were bisected and treated with CNTF or vehicle, and analyzed by real-time RT-qPCR. Two experiments using different sets of bisected corneas indicated that CNTF induced increases in VIP mRNA levels of 6.5
±
2.2-fold (
N
=
7 corneas) and 2.3
±
0.6-fold (
N
=
10 corneas) (mean
±
S.E.M.), respectively. Whereas VIP is produced as a CE autocrine factor against oxidative stress, the present study suggested that oxidative stress-released CNTF plays a role in protecting CE cells against oxidative stress injury by upregulating VIP expression.
Vasoactive intestinal polypeptide (VIP) is a pleiotropic neurotransmitter with roles in development, circadian function, immune regulation, and pain sensation. VIP gene expression is synergistically ...activated by elevation of cAMP and Ca2+ (Hamelink et al., J. Neurosci. 22:, 2002). The effects of these two signaling pathways on VIP gene regulation were examined in NBFL human neuroblastoma cells, which endogenously express VIP. Treatment with forskolin or the Ca2+ionophore A23187, alone modestly (10‐fold) increased VIP mRNA, but together caused a synergistic (75‐fold) increase in NBFL cells. Although stimulation of neuroendocrine genes by both cAMP and Ca2+ via convergence on a common cis‐active element, the cyclic AMP‐response element (CRE) is common, we found, unexpectedly, that the CRE of a full‐length VIP luciferase reporter gene, while essential for cAMP‐stimulated transcription and synergistic stimulation with cAMP and Ca2+, was dispensable for activation by Ca2+ alone. Instead, Ca2+ activation required a single element of 280 bases located 4 kb upstream from the VIP proximal promoter that was previously identified as a tissue specifier element (TSE; Hahm et al., JBC 273:,1998). The TSE of the VIP gene is a novel calcium response element in a neuron‐specific gene that is independent of the CRE, but acts together with it to enable synergistic cAMP and Ca2+signaling.
Pituitary adenylate cyclase activating polypeptide (PACAP) is the slow transmitter that mediates sustained catecholamine secretion and stimulus‐secretion‐synthesis coupling for repletion of ...catecholamine and neuropeptide stores in the adrenal medulla during prolonged metabolic stress (Hamelink et al., PNAS 99: , 2002). PACAP acts through the PAC1 receptor to effect these calcium‐dependent actions, and the hop cassette of this receptor is specifically required for sustained calcium elevation stimulated by PACAP (Mustafa et al., JBC 2007, PMID 17213203). Here we demonstrate that expression of the bovine PAC1hop receptor in PC12 cells, at levels found in bovine chromaffin cells, significantly enhances both short‐term (0–5 min) and sustained (5–60 min) catecholamine secretion, and enhances transcription from the VIP promoter specifically dependent on the calcium‐response element of this gene. The endogenous PAC1 receptor of PC12 cells supports PACAP‐induced short‐term secretion and stimulation of the VIP gene from its cAMP response element, but not prolonged PACAP‐stimulated secretion and activation of transcription of a PACAP target gene (the VIP promoter) via its calcium response element. These data implicate both PAC1 receptor density, and the specific isoform of the receptor that is expressed, in physiological slow transmitter function of PACAP at the adrenomedullary synapse.
Mutations of c-Ha-ras at codon 12 were detected from 11 out of 27 fresh tissues and cell-lines of human gastric cancer patients using PCR-restriction analysis. Further statistical investigation ...showed that the presence of point mutations was related to the distal metastases and the survival time of gastric cancer patients after surgical operations.
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