Sequential chromatin immunoprecipitation assay and analysis.

Sequential chromatin immunoprecipitation (SeqChIP) assays have been developed for the study of interactions of two or more proteins (or simultaneous histone modifications) at genomic sites. It is based on the principle that chromatin and associated proteins can be first immunoprecipitated with a first antibody and the obtained immunoprecipitate can be subjected to a second antibody. At the end of the assay the immunoprecipitated material contains only chromatin that concomitantly carries both DNA-associated proteins (or both histone modifications). The SeqChIP protocol described here combines speed (minimum of 3-4 h to perform the complete assay), sensitivity (known targets can be detected with only about 20,000 cell equivalents), and avoidance of antibody-antigen disruption after the first ChIP step. In addition, specific SeqChIP controls and potential shortcomings are discussed, the main characteristics of different SeqChIP protocols are described and several examples of protein complexes and protein-protein interactions at genomic sites that have been solved by SeqChIP in the recent years are presented.

Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells.

BACKGROUND: The investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. High-throughput approaches based on chromatin immunoprecipitation (ChIP) have been widely used for this purpose. However, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and require complex statistical analysis. Advances in this field will greatly benefit from the development and use of simple, fast, sensitive and straightforward ChIP assay and analysis methodologies. RESULTS: We initially developed a simplified, basic ChIP protocol that combines simplicity, speed and sensitivity. ChIP analysis by real-time PCR was compared to analysis by densitometry with the ImageJ software. This protocol allowed the rapid identification of known target genes for SOX2, NANOG, OCT3/4, SOX17, KLF4, RUNX2, OLIG2, SMAD2/3, BMI-1, and c-MYC in a human embryonic stem cell line. We then developed a novel Sequential ChIP protocol to investigate in vivo promoter co-occupancy, which is basically characterized by the absence of antibody-antigen disruption during the assay. It combines centrifugation of agarose beads and magnetic separation. Using this Sequential ChIP protocol we found that c-MYC associates with the SOX2/NANOG/OCT3/4 complex and identified a novel RUNX2/BMI-1/SMAD2/3 complex in BG01V cells. These two TF complexes associate with two distinct sets of target genes. The RUNX2/BMI-1/SMAD2/3 complex is associated predominantly with genes not expressed in undifferentiated BG01V cells, consistent with the reported role of those TFs as transcriptional repressors. CONCLUSION: These simplified basic ChIP and novel Sequential ChIP protocols were successfully tested with a variety of antibodies with human embryonic stem cells, generated a number of novel observations for future studies and might be useful for high-throughput ChIP-based assays.

The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of Rap1.

WAVE2 regulates T cell receptor (TCR)-stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

Distinct regulation of integrin-dependent T cell conjugate formation and NF-kappa B activation by the adapter protein ADAP.

Following TCR stimulation, T cells utilize the hematopoietic specific adhesion and degranulation-promoting adapter protein (ADAP) to control both integrin adhesive function and NF-kappaB transcription factor activation. We have investigated the molecular basis by which ADAP controls these events in primary murine ADAP(-/-) T cells. Naive DO11.10/ADAP(-/-) T cells show impaired adhesion to OVAp (OVA aa 323-339)-bearing APCs that is restored following reconstitution with wild-type ADAP. Mutational analysis demonstrates that the central proline-rich domain and the C-terminal domain of ADAP are required for rescue of T:APC conjugate formation. The ADAP proline-rich domain is sufficient to bind and stabilize the expression of SKAP55 (Src kinase-associated phosphoprotein of 55 kDa), which is otherwise absent from ADAP(-/-) T cells. Interestingly, forced expression of SKAP55 in the absence of ADAP is insufficient to drive T:APC conjugate formation, demonstrating that both ADAP and SKAP55 are required for optimal LFA-1 function. Additionally, the ADAP proline-rich domain is required for optimal Ag-induced activation of CD69, CD25, and Bcl-x(L), but is not required for assembly of the CARMA1/Bcl10/Malt1 (caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1/B-cell CLL-lymphoma 10/mucosa-associated lymphoid tissue lymphoma translocation protein 1) signaling complex and subsequent TCR-dependent NF-kappaB activity. Our results indicate that ADAP is used downstream of TCR engagement to delineate two distinct molecular programs in which the ADAP/SKAP55 module is required for control of T:APC conjugate formation and functions independently of ADAP/CARMA1-mediated NF-kappaB activation.

T-cell receptor signaling to integrins.

Integrin adhesion receptors are critical for antigen recognition by T cells and for regulated recirculation and trafficking into and through various tissues in the body. T-cell receptor (TCR) signaling induces rapid increases in integrin function that facilitate T-cell activation by promoting stable contact with antigen-presenting cells and extracellular proteins in the environment. In this review, we outline the molecular mechanisms by which the TCR signals to integrins and present a model that highlights four key events: (i) initiation of proximal TCR signals nucleated by the linker for activated T cells (LAT) adapter protein and involving Itk, phospholipase C-gamma1, Vav1, and Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa; (ii) transmission of integrin activation signals from the LAT signalosome to integrins by protein kinase (PK) C and the adapter protein, adhesion and degranulation-promoting adapter protein; (iii) assembly of integrin-associated signaling complexes that include PKD, the guanosine triphosphatase Rap1 and its effectors, and talin; and (iv) reorganization of the actin cytoskeleton by WAVE2 and other actin-remodeling proteins. These events coordinate changes in integrin conformation and clustering that result in enhanced integrin functional activity following TCR stimulation.

WAVE2 regulates high-affinity integrin binding by recruiting vinculin and talin to the immunological synapse.

T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.

Regulation of NF-kappaB activation in T cells via association of the adapter proteins ADAP and CARMA1.

The adapter protein ADAP regulates T lymphocyte adhesion and activation. We present evidence for a previously unrecognized function for ADAP in regulating T cell receptor (TCR)-mediated activation of the transcription factor NF-kappaB. Stimulation of ADAP-deficient mouse T cells with antibodies to CD3 and CD28 resulted in impaired nuclear translocation of NF-kappaB, a reduced DNA binding, and delayed degradation and decreased phosphorylation of IkappaB (inhibitor of NF-kappaB). TCR-stimulated assembly of the CARMA1-BCL-10-MALT1 complex was substantially impaired in the absence of ADAP. We further identified a region of ADAP that is required for association with the CARMA1 adapter and NF-kappaB activation but is not required for ADAP-dependent regulation of adhesion. These findings provide new insights into ADAP function and the mechanism by which CARMA1 regulates NF-kappaB activation in T cells.

MHC class II deprivation impairs CD4 T cell motility and responsiveness to antigen-bearing dendritic cells in vivo.

The role continuous contact with self-peptide/MHC molecules (self ligands) in the periphery plays in the function of mature T cells remains unclear. Here, we elucidate a role for MHC class II molecules in T cell trafficking and antigen responsiveness in vivo. We find that naïve CD4 T cells deprived of MHC class II molecules demonstrate a progressive and profound defect in motility (measured by real-time two-photon imaging) and that these cells have a decreased ability to interact with limiting numbers of cognate antigen-bearing dendritic cells, but they do not demonstrate a defect in their responsiveness to direct stimulation with anti-CD3 monoclonal antibody. Using GST fusion proteins, we show that MHC class II availability promotes basal activation of Rap1 and Rac1 but does not alter the basal activity of Ras. We propose that tonic T cell receptor signaling from self-ligand stimulation is required to maintain a basal state of activation of small guanosine triphosphatases critical for normal T cell motility and that T cell motility is critical for the antigen receptivity of naïve CD4 T cells. These studies suggest a role for continuous self-ligand stimulation in the periphery for the maintenance and function of mature naïve CD4 T cells.

Formins regulate the actin-related protein 2/3 complex-independent polarization of the centrosome to the immunological synapse.

T cell receptor (TCR)-mediated cytoskeletal reorganization is considered to be actin-related protein (Arp) 2/3 complex dependent. We therefore examined the requirement for Arp2/3- and formin-dependent F-actin nucleation during T cell activation. We demonstrated that without Arp2/3-mediated actin nucleation, stimulated T cells could not form an F-actin-rich lamellipod, but instead produced polarized filopodia-like structures. Moreover, the microtubule-organizing center (MTOC, or centrosome), which rapidly reorients to the immunological synapse through an unknown mechanism, polarized in the absence of Arp2/3. Conversely, the actin-nucleating formins, Diaphanous-1 (DIA1) and Formin-like-1 (FMNL1), did not affect TCR-stimulated F-actin-rich structures, but instead displayed unique patterns of centrosome colocalization and controlled TCR-mediated centrosome polarization. Depletion of FMNL1 or DIA1 in cytotoxic lymphocytes abrogated cell-mediated killing. Altogether, our results have identified Arp2/3 complex-independent cytoskeletal reorganization events in T lymphocytes and indicate that formins are essential cytoskeletal regulators of centrosome polarity in T cells.

Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.

Nuclear translocation of NF-kappaB initiates transcription of numerous genes, many of which are critical to host defense. Fluorescent image-based methods that quantify this event have historically utilized adherent cells with large cytoplasm-to-nuclear area ratios. However, many immunologically relevant cells are naturally non-adherent and have small cytoplasm-to-nuclear area ratios. Using the ImageStream imaging flow cytometer, we have developed a novel method that measures nuclear translocation in large populations using cross-correlation analysis of nuclear and NF-kappaB images from each cell. This approach accurately measures NF-kappaB translocation in cells with small cytoplasmic areas in dose- and time-dependent manners. Further, NF-kappaB translocation was accurately measured in a subset of cells contained in a mixed population and the technique was successfully employed to measure IRF-7 translocation in plasmacytoid dendritic cells (PDC) obtained from human peripheral blood. The techniques described here provide an objective and statistically robust method for measuring cytoplasmic to nuclear molecular translocation events in a variety of immunologically relevant cell types with characteristically low cytoplasm-to-nuclear area ratios.

The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation.

BACKGROUND: The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. RESULTS: By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. CONCLUSIONS: These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

Protein kinase D1 and the beta 1 integrin cytoplasmic domain control beta 1 integrin function via regulation of Rap1 activation.

The functional activity of integrins is dynamically regulated by T cell receptor stimulation and by protein kinase C (PKC). We report a novel function for the PKC effector protein kinase D1 (PKD1) in integrin activation. Constitutively active and kinase-inactive PKD1 mutants lacking the PKD1 pleckstrin homology (PH) domain block phorbol ester- and TCR-mediated activation and clustering of beta1 integrins. The PH domain of PKD1 mediates the association of PKD1 with the GTPase Rap1 and is central to Rap1 activation and membrane translocation in T cells. Furthermore, PKD1 and Rap1 associate with beta1 integrins in a manner that is dependent on the carboxy-terminal end of the beta1 integrin subunit cytoplasmic domain. beta1 integrin expression is required for Rap1 activation and membrane localization of the PKD1-Rap1 complex. Therefore, PKD1 promotes integrin activation in T cells by regulating Rap1 activation and membrane translocation via interactions with the beta1 integrin subunit cytoplasmic domain.

Expression of a viral polymerase-bound host factor turns human cell lines permissive to a plant- and insect-infecting virus.

Tospoviruses are the only plant-infecting members of the Bunyaviridae family of ambisense ssRNA viruses. Tomato spotted wilt tospovirus (TSWV), the type-member, also causes mild infection on its main insect vector, Frankliniella occidentalis. Herein, we identified an F. occidentalis putative transcription factor (FoTF) that binds to the TSWV RNA-dependent RNA polymerase and to viral RNA. Using in vitro RNA synthesis assays, we show that addition of purified FoTF improves viral replication, but not transcription. Expression of FoTF deletion mutants, unable to bind the RNA-dependent RNA polymerase or viral RNA, blocks TSWV replication in F. occidentalis cells. Finally, expression of FoTF wild-type turns human cell lines permissive to TSWV replication. These data indicate that FoTF is a host factor required for TSWV replication in vitro and in vivo, provide an experimental system that could be used to compare molecular defense mechanisms in plant, insect, and human cells against the same pathogen (TSWV), and could lead to a better understanding of evolutionary processes of ambisense RNA viruses.

The plant virus Tomato Spotted Wilt Tospovirus activates the immune system of its main insect vector, Frankliniella occidentalis.

Tospoviruses have the ability to infect plants and their insect vectors. Tomato spotted wilt virus (TSWV), the type species in the Tospovirus genus, infects its most important insect vector, Frankliniella occidentalis, the western flower thrips (WFT). However, no detrimental effects on the life cycle or cytopathological changes have been reported in the WFT after TSWV infection, and relatively few viral particles can be observed even several days after infection. We hypothesized that TSWV infection triggers an immune response in the WFT. Using subtractive cDNA libraries to probe WFT DNA macroarrays, we found that the WFT's immune system is activated by TSWV infection. The activated genes included (i) those encoding antimicrobial peptides, such as defensin and cecropin; (ii) genes involved in pathogen recognition, such as those encoding lectins; (iii) those encoding receptors that activate the innate immune response, such as Toll-3; and (iv) those encoding members of signal transduction pathways activated by Toll-like receptors, such as JNK kinase. Transcriptional upregulation of these genes after TSWV infection was confirmed by Northern analysis, and the kinetics of the immune response was measured over time. Several of the detected genes were activated at the same time that viral replication was first detected by reverse transcription-PCR. To our knowledge, this is the first report of the activation of an insect vector immune response by a plant virus. The results may lead to a better understanding of insects' immune responses against viruses and may help in the future development of novel control strategies against plant viruses, as well as human and animal viruses transmitted by insect vectors.