Germline DNA replication timing shapes mammalian genome composition.

Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.

Clonal allelic predetermination of immunoglobulin-κ rearrangement.

Although most genes are expressed biallelically, a number of key genomic sites--including immune and olfactory receptor regions--are controlled monoallelically in a stochastic manner, with some cells expressing the maternal allele and others the paternal allele in the target tissue. Very little is known about how this phenomenon is regulated and programmed during development. Here, using mouse immunoglobulin-κ (Igκ) as a model system, we demonstrate that although individual haematopoietic stem cells are characterized by allelic plasticity, early lymphoid lineage cells become committed to the choice of a single allele, and this decision is then stably maintained in a clonal manner that predetermines monoallelic rearrangement in B cells. This is accompanied at the molecular level by underlying allelic changes in asynchronous replication timing patterns at the κ locus. These experiments may serve to define a new concept of stem cell plasticity.

GTP exchange factor Vav regulates guided cell migration by coupling guidance receptor signalling to local Rac activation.

Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Here, using a multidisciplinary approach, we identify the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). We show that elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. Our results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements.

Oct-3/4 regulates stem cell identity and cell fate decisions by modulating Wnt/ß-catenin signalling.

Although the transcriptional regulatory events triggered by Oct-3/4 are well documented, understanding the proteomic networks that mediate the diverse functions of this POU domain homeobox protein remains a major challenge. Here, we present genetic and biochemical studies that suggest an unexpected novel strategy for Oct-3/4-dependent regulation of embryogenesis and cell lineage determination. Our data suggest that Oct-3/4 specifically interacts with nuclear ß-catenin and facilitates its proteasomal degradation, resulting in the maintenance of an undifferentiated, early embryonic phenotype both in Xenopus embryos and embryonic stem (ES) cells. Our data also show that Oct-3/4-mediated control of ß-catenin stability has an important function in regulating ES cell motility. Down-regulation of Oct-3/4 increases ß-catenin protein levels, enhancing Wnt signalling and initiating invasive cellular activity characteristic of epithelial-mesenchymal transition. Our data suggest a novel mode of regulation by which a delicate balance between ß-catenin, Tcf3 and Oct-3/4 regulates maintenance of stem cell identity. Altering the balance between these proteins can direct cell fate decisions and differentiation.

The type III secretion effector NleE inhibits NF-kappaB activation.

The complex host-pathogen interplay involves the recognition of the pathogen by the host's innate immune system and countermeasures taken by the pathogen. Detection of invading bacteria by the host leads to rapid activation of the transcription factor NF-kappaB, followed by inflammation and eradication of the intruders. In response, some pathogens, including enteropathogenic Escherichia coli (EPEC), acquired means of blocking NF-kappaB activation. We show that inhibition of NF-kappaB activation by EPEC involves the injection of NleE into the host cell. Importantly, we show that NleE inhibits NF-kappaB activation by preventing activation of IKKbeta and consequently the degradation of the NF-kappaB inhibitor, IkappaB. This NleE activity is enhanced by, but is not dependent on, a second injected effector, NleB. In conclusion, this study describes two effectors, NleB and NleE, with no similarity to other known proteins, used by pathogens to manipulate NF-kappaB signaling pathways.

Vav1 accelerates Ras-driven lung cancer and modulates its tumor microenvironment.

The potential impact of Vav1 on human cancer was only recently acknowledged, as it is detected as a mutant or an overexpressed gene in various cancers, including lung cancer. Vav1, which is normally and exclusively expressed in the hematopoietic system functions as a specific GDP/GTP nucleotide exchange factor (GEF), strictly regulated by tyrosine phosphorylation. To investigate whether Vav1 plays a causative or facilitating role in-vivo in lung cancer development and to examine whether it co-operates with other oncogenes, such as mutant K-Ras, we generated novel mouse strains that express: Vav1 or K-RasG12D in type II pneumocytes, as well as a transgenic mouse line that expresses both Vav1 and K-RasG12D in these cells. Coexpression of Vav1 and K-RasG12D in the lungs dramatically increased malignant lung cancer lesions, and did so significantly faster than K-RasG12D alone, strongly suggesting that these two oncogenes synergize to enhance lung tumor development. Vav1 expression alone had no apparent effects on lung tumorigenesis. The increase in lung cancer in K-RasG12D/Vav1 mice was accompanied by an increase in B-cell, T-cells, and monocyte infiltration in the tumor microenvironment. Concomitantly, ERK phosphorylation was highly elevated in the lungs of K-RasG12 D/Vav1 mice. Also, several cytokines such as IL-4 and IL-13 which play a significant role in the immune system, were elevated in lungs of Vav1 and K-RasG12 D/Vav1 mice. Our findings emphasize the contribution of Vav1 to lung tumor development through its signaling properties.

Vav1 Promotes B-Cell Lymphoma Development.

Vav1 is normally and exclusively expressed in the hematopoietic system where it functions as a specific GDP/GTP nucleotide exchange factor (GEF), firmly regulated by tyrosine phosphorylation. Mutations and overexpression of Vav1 in hematopoietic malignancies, and in human cancers of various histologic origins, are well documented. To reveal whether overexpression of Vav1 in different tissues suffices for promoting the development of malignant lesions, we expressed Vav1 in transgenic mice by using the ubiquitous ROSA26 promoter (Rosa Vav1). We detected Vav1 expression in epithelial tissues of various organs including pancreas, liver, and lung. While carcinomas did not develop in these organs, surprisingly, we noticed the development of B-cell lymphomas. Rac1-GTP levels did not change in tissues from Rosa Vav1 mice expressing the transgenic Vav1, while ERK phosphorylation increased in the lymphomas, suggesting that signaling pathways are evoked. One of the growth factors analyzed by us as a suspect candidate to mediate paracrine stimulation in the lymphocytes was CSF-1, which was highly expressed in the epithelial compartment of Rosa Vav1 mice. The expression of its specific receptor, CSF-1R, was found to be highly expressed in the B-cell lymphomas. Taken together, our results suggest a potential cross-talk between epithelial cells expressing Vav1, that secrete CSF-1, and the lymphocytes that express CSF-1R, thus leading to the generation of B-cell lymphomas. Our findings provide a novel mechanism by which Vav1 contributes to tumor propagation.

Mitotic H3K9ac is controlled by phase-specific activity of HDAC2, HDAC3, and SIRT1.

Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3, and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers, and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process-at prophase, HDAC2 modulates most transcription-associated H3K9ac-marked loci, and at metaphase, HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.

Tyrosine residues at the carboxyl terminus of Vav1 play an important role in regulation of its biological activity.

The guanine nucleotide exchange factor (GEF) Vav1 is an essential signal transducer protein in the hematopoietic system, where it is expressed physiologically. It is also involved in several human malignancies. Tyrosine phosphorylation at the Vav1 amino terminus plays a central role in regulating its activity; however, the role of carboxyl terminal tyrosine residues is unknown. We found that mutation of either Tyr-826 (Y826F) or Tyr-841 (Y841F) to phenylalanine led to loss of Vav1 GEF activity. When these Vav1 mutants were ectopically expressed in pancreatic cancer cells lacking Vav1, they failed to induce growth in agar, indicating loss of transforming potential. Furthermore, although Y841F had no effect on Vav1-stimulated nuclear factor of activated T cells (NFAT) activity, Y826F doubled NFAT activity when compared with Vav1, suggesting that Tyr-826 mediates an autoinhibitory effect on NFAT activity. SH2 profiling revealed that Shc, Csk, Abl, and Sap associate with Tyr-826, whereas SH2-B, Src, Brk, GTPase-activating protein, and phospholipase C-gamma associate with Tyr-841. Although the mutations in the Tyr-826 and Tyr-841 did not affect the binding of the carboxyl SH3 of Vav1 to other proteins, binding to several of the proteins identified by the SH2 profiling was lost. Of interest is Csk, which associates with wild-type Vav1 and Y841F, yet it fails to associate with Y826F, suggesting that loss of binding between Y826F and Csk might relieve an autoinhibitory effect, leading to increased NFAT. Our data indicate that GEF activity is critical for the function of Vav1 as a transforming protein but not for NFAT stimulation. The association of Vav1 with other proteins, detected by SH2 profiling, might affect other Vav1-dependent activities, such as NFAT stimulation.

Chromosomal coordination and differential structure of asynchronous replicating regions.

Stochastic asynchronous replication timing (AS-RT) is a phenomenon in which the time of replication of each allele is different, and the identity of the early allele varies between cells. By taking advantage of stable clonal pre-B cell populations derived from C57BL6/Castaneous mice, we have mapped the genome-wide AS-RT loci, independently of genetic differences. These regions are characterized by differential chromatin accessibility, mono-allelic expression and include new gene families involved in specifying cell identity. By combining population level mapping with single cell FISH, our data reveal the existence of a novel regulatory program that coordinates a fixed relationship between AS-RT regions on any given chromosome, with some loci set to replicate in a parallel and others set in the anti-parallel orientation. Our results show that AS-RT is a highly regulated epigenetic mark established during early embryogenesis that may be used for facilitating the programming of mono-allelic choice throughout development.

Vav1 mutations: What makes them oncogenic?

Vav1 is physiologically active as a GDP/GTP nucleotide exchange factor (GEF) in the hematopoietic system. Its wild-type form was recently implicated in mammalian malignancies of hematologic and non-hematologic tissue origins. Moreover, it was recently identified as a mutated gene in human cancers of various origins. In this review we focus on the functional activities of several of the Vav1 mutants analyzed for their tumorigenic properties. We also discuss the relationship of the tested biochemical properties of Vav1 mutants, E59K, D517E and L801P, to their computer-based predicted properties. These comparisons further enhance the need for integration of computation-based structural analyses with experimental data in order to fully appreciate the activity of mutant proteins. Our comprehensive evaluation supports the classification of Vav1 as a bona fide oncogene in human cancers.

Vav1 and mutant K-Ras synergize in the early development of pancreatic ductal adenocarcinoma in mice.

To explore the contribution of Vav1, a hematopoietic signal transducer, to pancreatic ductal adenocarcinoma (PDAC) development, we generated transgenic mouse lines expressing, Vav1, K-RasG12D, or both K-RasG12D and Vav1 in pancreatic acinar cells. Co-expression of Vav1 and K-RasG12D synergistically enhanced acinar-to-ductal metaplasia (ADM) formation, far exceeding the number of lesions developed in K-RasG12D mice. Mice expressing only Vav1 did not develop ADM. Moreover, the incidence of PDAC in K-RasG12D/Vav1 was significantly higher than in K-RasG12D mice. Discontinuing Vav1 expression in K-RasG12D/Vav1 mice elicited a marked regression of malignant lesions in the pancreas, demonstrating Vav1 is required for generation and maintenance of ADM. Rac1-GTP levels in the K-RasG12D/Vav1 mice pancreas clearly demonstrated an increase in Rac1 activity. Treatment of K-RasG12D and K-RasG12D/Vav1 mice with azathioprine, an immune-suppressor drug which inhibits Vav1's activity as a GDP/GTP exchange factor, dramatically reduced the number of malignant lesions. These results suggest that Vav1 plays a role in the development of PDAC when co-expressed with K-RasG12D via its activity as a GEF for Rac1GTPase.

Vav1 mutations identified in human cancers give rise to different oncogenic phenotypes.

Vav1 is physiologically active as a GDP/GTP nucleotide exchange factor (GEF) in the hematopoietic system. Overexpression of Vav1 in multiple tumor types is known to enhance oncogenicity, yet whether or not Vav1 is a bona fide oncogene is still a matter of debate. Although mutations in Vav1 were recently identified in human cancers of various origins, the functional activities of these mutants are not known. We tested the transforming potential of three mutations identified in human lung adenocarcinoma: E59K, D517E, and L801P. Results from several assays indicative of transforming activities such as rate of proliferation, growth in agar, and generation of tumors in NOD/SCID mice clearly indicated that E59K and D517E are highly transforming but L801P at the SH3 domain is not. The acquired oncogenic activity of these mutants can be attributed to their enhanced activity as GEFs for Rho/Rac GTPases. Deciphering of the mechanisms leading to overactivity of the tested mutants revealed that the E59K mutation facilitates cleavage of a truncated protein that is uncontrollably active as a GEF, while D517E generates a highly stable overexpressed protein that is also more active as a GEF than wild-type Vav1. These findings support the classification of Vav1 as a bona fide oncogene in human cancer.

Rpb4p, a subunit of RNA polymerase II, mediates mRNA export during stress.

Changes in gene expression represent a major mechanism by which cells respond to stress. We and other investigators have previously shown that the yeast RNA polymerase II subunit Rpb4p is required for transcription under various stress conditions, but not under optimal growth conditions. Here we show that, in addition to its role in transcription, Rpb4p is also required for mRNA export, but only when cells are exposed to stress conditions. The roles of Rpb4p in transcription and in mRNA export can be uncoupled genetically by specific mutations in Rpb4p. Both functions of Rpb4p are required to maintain cell viability during stress. We propose that Rpb4p participates in the cellular responses to stress at the interface of the transcription and the export machineries.

Kinase-inactive glycogen synthase kinase 3beta promotes Wnt signaling and mammary tumorigenesis.

Recent studies have implicated ectopic activation of the Wnt pathway in many human cancers, including breast cancer. beta-catenin is a critical coactivator in this signaling pathway and is regulated in a complex fashion by phosphorylation, degradation, and nuclear translocation. Glycogen synthase kinase 3beta (GSK3beta) phosphorylation of the NH2-terminal domain of beta-catenin targets it for ubiquitination and proteosomal degradation. We hypothesized that expression of kinase-inactive GSK3beta (KI-GSK3beta) in mammary glands would function in a dominant-negative fashion by antagonizing the endogenous activity of GSK3beta and promoting breast cancer development. Consistent with this, we find that KI-GSK3beta stabilizes beta-catenin expression, catalyzes its localization to the nucleus, and up-regulates the downstream target gene, cyclin D1, in vitro. In vivo, transgenic mice overexpressing the KI-GSK3beta under the control of the mouse mammary tumor virus-long terminal repeat develop mammary tumors with overexpression of beta-catenin and cyclin D1. Thus, antagonism of GSK3beta activity is oncogenic in the mammary epithelium; mutation or pharmacologic down-regulation of GSK3beta could promote mammary tumors.

Programming asynchronous replication in stem cells.

Many regions of the genome replicate asynchronously and are expressed monoallelically. It is thought that asynchronous replication may be involved in choosing one allele over the other, but little is known about how these patterns are established during development. We show that, unlike somatic cells, which replicate in a clonal manner, embryonic and adult stem cells are programmed to undergo switching, such that daughter cells with an early-replicating paternal allele are derived from mother cells that have a late-replicating paternal allele. Furthermore, using ground-state embryonic stem (ES) cells, we demonstrate that in the initial transition to asynchronous replication, it is always the paternal allele that is chosen to replicate early, suggesting that primary allelic choice is directed by preset gametic DNA markers. Taken together, these studies help define a basic general strategy for establishing allelic discrimination and generating allelic diversity throughout the organism.

Direct detection and quantitation of a distinct T-cell epitope derived from tumor-specific epithelial cell-associated mucin using human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.

The recent characterization of MHC-displayed tumor-associated antigens that recognize effector cells of the immune system has created new perspectives for cancer therapy. Antibodies that recognize these tumor-associated MHC-peptide complexes with the same specificity as the T-cell antigen receptor will therefore be valuable tools for immunotherapy, as well as for studying antigen presentation in human cancers. Most tumor-associated antigens are expressed in only one or a few tumor types; however, specific T-cell epitopes derived from the Mucin-1 tumor-associated antigen (MUC1) that are widely expressed in many cancers were identified and shown to be recognized by CTLs. We selected a large nonimmune repertoire of phage Fab antibodies on recombinant human class I HLA-A2 complexes displaying an antigenic T-cell epitope derived from MUC1. High frequency of anti-MHC-peptide binders was observed (84%), and surprisingly, a high percentage (80%) of antibodies was fully specific for the MUC1 epitope. We isolated a surprisingly large panel of 16 different high-affinity human recombinant Fab antibodies that exhibited peptide-specific, MHC-restricted binding characteristics of T cells. The analyzed Fabs not only recognize the cognate MHC-peptide complex in a recombinant soluble form but also the native complex as displayed on the surface of antigen-presenting cells and breast tumor cells. Therefore, these findings demonstrate the ability to transform the unique fine specificity but low intrinsic affinity of T-cell receptors on T cells into high-affinity soluble antibody molecules endowed with a T-cell antigen receptor-like specificity. These molecules may prove to be very important and widely applicable for monitoring the expression of specific MHC-peptide complexes on the surface of tumor and immune cells for structure-function studies of T-cell receptor-peptide-MHC interactions, as well as for developing new targeting agents for immunotherapy.

Vav1 promotes lung cancer growth by instigating tumor-microenvironment cross-talk via growth factor secretion.

Vav1 is a signal transducer that functions as a scaffold protein and a regulator of cytoskeleton organization in the hematopoietic system, where it is exclusively expressed. Recently, Vav1 was shown to be involved in diverse human cancers, including lung cancer. We demonstrate that lung cancer cells that abnormally express Vav1 secrete growth factors in a Vav1-dependent manner. Transcriptome analysis demonstrated that Vav1 depletion results in a marked reduction in the expression of colony-stimulating-factor-1 (CSF1), a hematopoietic growth factor. The association between Vav1 expression and CSF1 was further supported by signal transduction experiments, supporting involvement of Vav1 in regulating lung cancer secretome. Blocking of ERK phosphorylation, led to a decrease in CSF1 transcription, thus suggesting a role for ERK, a downstream effector of Vav1, in CSF1 expression. CSF1-silenced cells exhibited reduced focus formation, proliferation abilities, and growth in NOD/SCID mice. CSF1-silenced H358 cells resulted in significantly smaller tumors, showing increased fibrosis and a decrease in tumor infiltrating macrophages. Finally, immunohistochemical analysis of primary human lung tumors revealed a positive correlation between Vav1 and CSF1 expression, which was associated with tumor grade. Additional results presented herein suggest a potential cross-talk between cancer cells and the microenvironment controlled by CSF1/Vav1 signaling pathways.

Vav1 fine tunes p53 control of apoptosis versus proliferation in breast cancer.

Vav1 functions as a signal transducer protein in the hematopoietic system, where it is exclusively expressed. Vav1 was recently implicated in several human cancers, including lung, pancreatic and neuroblasoma. In this study, we analyzed the expression and function of Vav1 in human breast tumors and breast cancer cell lines. Immunohistochemical analysis of primary human breast carcinomas indicated that Vav1 is expressed in 62% of 65 tumors tested and is correlated positively with estrogen receptor expression. Based on published gene profiling of 50 breast cancer cell lines, several Vav1-expressing cell lines were identified. RT-PCR confirmed Vav1 mRNA expression in several of these cell lines, yet no detectable levels of Vav1 protein were observed due to cbl-c proteasomal degradation. We used two of these lines, MCF-7 (Vav1 mRNA negative) and AU565 (Vav1 mRNA positive), to explore the effect of Vav1 expression on breast cell phenotype and function. Vav1 expression had opposite effects on function in these two lines: it reduced proliferation and enhanced cell death in MCF-7 cells but enhanced proliferation in AU565 cells. Consistent with these findings, transcriptome analysis revealed an increase in expression of proliferation-related genes in Vav1-expressing AU565 cells compared to controls, and an increase in apoptosis-related genes in Vav1-expressing MCF-7 cells compared with controls. TUNEL and γ-H2AX foci assays confirmed that expression of Vav1 increased apoptosis in MCF-7 cells but not AU565 cells and shRNA experiments revealed that p53 is required for this pro-apoptotic effect of Vav1 in these cells. These results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer and the p53 dependence of its pro-apoptotic effect in breast cells.

Direct proteasome binding and subsequent degradation of unspliced XBP-1 prevent its intracellular aggregation.

The non-canonical splicing of XBP-1 mRNA is a hallmark of the mammalian unfolded protein response (UPR). The proteasomal degradation of unspliced XBP-1 (XBP-1u) facilitates the termination of the UPR. Thus, understanding the mechanism of XBP-1u degradation may allow control over UPR duration and intensity. We show that XBP-1u interacts with purified 20S proteasomes through its unstructured C-terminus, which leads to its degradation in a manner that autonomously opens the proteasome gate. In living cells, the C-terminus of XBP-1u accumulates in aggresome structures in the presence of proteasome inhibitors. We propose that direct proteasomal degradation of XBP-1u prevents its intracellular aggregation.