Christopher Maolin Yang

LysRS serves as a key signaling molecule in the immune response by regulating gene expression.

Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.

TRIP-Br: a novel family of PHD zinc finger- and bromodomain-interacting proteins that regulate the transcriptional activity of E2F-1/DP-1

We report the isolation of TRIP‐Br1, a transcriptional regulator that interacts with the PHD‐bromodomain of co‐repressors of Krüppel‐associated box (KRAB)‐mediated repression, KRIP‐1(TIF1β) and TIF1α, as well as the co‐activator/adaptor p300/CBP. TRIP‐Br1 and the related protein TRIP‐Br2 possess transactivation domains. Like MDM2, which has a homologous transactivation domain, TRIP‐Br proteins functionally contact DP‐1, stimulating E2F‐1/DP‐1 transcriptional activity. KRIP‐1 potentiates TRIP‐Br protein co‐activation of E2F‐1/DP‐1. TRIP‐Br1 is a component of a multiprotein complex containing E2F‐1 and DP‐1. Co‐expression of the retinoblastoma gene product (RB) abolishes baseline E2F‐1/DP‐1 transcriptional activity as well as TRIP‐Br/KRIP‐1 co‐activation, both of which are restored by the adenovirus E1A oncoprotein. These features suggest that TRIP‐Br proteins function at E2F‐responsive promoters to integrate signals provided by PHD‐ and/or bromodomain‐ containing transcription factors. TRIP‐Br1 is identical to the cyclin‐dependent kinase 4 (cdk4)‐binding protein p34SEI‐1, which renders the activity of cyclin D/cdk4 resistant to the inhibitory effect of p16INK4a during late G1. TRIP‐Br1(p34SEI‐1) is differentially overexpressed during the G1 and S phases of the cell cycle, consistent with a dual role for TRIP‐Br1(p34SEI‐1) in the regulation of cell cycle progression through sequential effects on the transcriptional activity of E2F‐responsive promoters during G1 and S phases.

TRIP-Br Links E2F to Novel Functions in the Regulation of Cyclin E Expression During Cell Cycle Progression and in the Maintenance of Genomic Stability

The TRIP-Br family of transcriptional regulators (TRIP-Br1 and TRIP-Br2)has been proposed to function at E2F-responsive promoters to integrate regulatorysignals provided by PHD zinc finger- and/or bromodomain-containing transcriptionfactors. To characterize the TRIP-Br “integrator” function(s), we have employeddecoy peptides (*Br1 and *Br2) to antagonize the interaction between TRIP-Br1 orTRIP-Br2 and the PHD zinc finger and/or bromodomain of other transcriptionfactors. Antagonism of the TRIP-Br integrator function elicits anti-proliferativeeffects through the transcriptional downregulation of a subset of E2F-responsive genesin vivo, and induces aberrant cyclin E accumulation, leading to Geminin deregulationand caspase-3-independent cellular sub-diploidization. The observed cyclin Ederegulation is attributed to the downregulation of Fbxw7, which encodes the Fbw7receptor subunit of the SCFFBW7 ubiquitin ligase (E3) responsible for targeting cyclinE for proteolysis. Fbxw7 is identified herein as an E2F-responsive and TRIP-Br coregulatedgene. Our results demonstrate a physiologic role for TRIP-Br in couplingE2F to novel functions in the regulation of cyclin E expression during cell cycleprogression to ensure the proper execution of DNA replication and the maintenance ofgenomic stability.

Human CD8⁺ T cells drive Th1 responses through the differentiation of TNF/iNOS-producing dendritic cells.

TNF/iNOS‐producing dendritic cells (Tip‐DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8+ T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip‐DCs that express high levels of TNF‐α and iNOS. Tip‐DCs exhibited T‐cell priming ability, expressed high levels of MHC class II, upregulated co‐stimulatory molecules CD40, CD80, CD86, toll‐like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip‐DCs was partially dependent on IFN‐γ as blocking the IFN‐γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF‐α production. Importantly, these Tip‐DCs were capable of further driving Th1 responses by priming naive CD4+ T cells for proliferation and IFN‐γ production and this was partially dependent on Tip‐DC production of TNF‐α and NO. Our study hence identifies a role for CD8+ T cells in orchestrating Th1‐mediating signals through the differentiation of monocytes into Th1‐inducing Tip‐DCs.

TRIP-Br2 Promotes Oncogenesis in Nude Mice and is Frequently Overexpressed in Multiple Human Tumors

BackgroundMembers of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2).MethodsOncogenic potential of TRIP-Br2 was demonstrated by (1) inoculation of NIH3T3 fibroblasts, which were engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2) comprehensive immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and human tumor tissue microarrays (TMAs). Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2 as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target.ResultsOverexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier survival analysis. Small interfering RNA-mediated (siRNA) knockdown of TRIP-Br2 was sufficient to inhibit cell-autonomous growth of HCT-116 cells in vitro.ConclusionThis study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a novel prognostic marker and a chemotherapeutic drug target in human cancer.

Importin Beta Plays an Essential Role in the Regulation of the LysRS-Ap 4 A Pathway in Immunologically Activated Mast Cells

ABSTRACT We recently reported that diadenosine tetraphosphate hydrolase (Ap4A hydrolase) plays a critical role in gene expression via regulation of intracellular Ap4A levels. This enzyme serves as a component of our newly described lysyl tRNA synthetase (LysRS)-Ap4A biochemical pathway that is triggered upon immunological challenge. Here we explored the mechanism of this enzymes translocation into the nucleus and found its immunologically dependent association with importin beta. Silencing of importin beta prevented Ap4A hydrolase nuclear translocation and affected the local concentration of Ap4A, which led to an increase in microphthalmia transcription factor (MITF) transcriptional activity. Furthermore, immunological activation of mast cells resulted in dephosphorylation of Ap4A hydrolase, which changed the hydrolytic activity of the enzyme.

Exploiting the TRIP-Br family of cell cycle regulatory proteins as chemotherapeutic drug targets in human cancer.

TRIP-Br1 and TRIP-Br2 are potent cell growth promoting factors that function as components of the E2F1/DP1 transcription complex to integrate positive growth signals provided by PHD zinc finger- and/or bromodomain-containing transcription factors. TRIP-Br1 has been demonstrated to be an oncogene. We recently reported that antagonism of the TRIP-Br integrator function by synthetic decoy peptides that compete with TRIP-Br for binding to PHD zinc finger- and/or bromodomain-containing proteins elicit an anti-proliferative effect and induces caspase-3-independent sub-diploidization in cancer cells in vitro. We now demonstrate the chemotherapeutic potential of TRIP-Br decoy peptides for the treatment of cutaneous and intracavitary lesions in vitro as well as in vivo in representative human nasopharyngeal cancer (CNE2), cervical cancer (Ca Ski) and melanoma (MeWo) cancer cell lines. In vitro, BrdU incorporation, colony formation assays and cell cycle analysis confirmed that TRIP-Br decoy peptides possess strong anti-proliferative effects and induce nuclear sub-diploidization in cancer cells. In vivo, NPC2, Ca Ski and MeWo-derived chick embryo chorioallantoic membrane (CAM) tumor xenografts were used to evaluate the effect of topically applied TRIP-Br peptides. Confocal microscopy and flow cytometric analysis demonstrated that cells comprising the tumor xenografts efficiently internalized topically applied FITC-labeled peptides. Fifty µM of TRIP-Br1 decoy peptide significantly suppressed the growth of NPC2-derived human nasopharyngeal tumors, while 50 µM of TRIP-Br2 decoy peptide significantly inhibited tumor growth in all three CAM tumor xenograft models. Two hundred µM of TRIP-Br1 decoy peptide significantly inhibited MeWo-derived tumors. These results suggest that the TRIP-Br integrator function may represent a novel chemotherapeutic target for the treatment of human cutaneous and intracavitary proliferative lesions.