A transforming growth factor beta type I receptor that signals to activate gene expression.

Transforming growth factor beta (TGF-beta) is a multifunctional factor that regulates many aspects of cellular functions. TGF-beta signals through a heteromeric complex of the type I and type II TGF-beta receptors. However, the molecular mechanism of signal transduction by this receptor complex remains unresolved. The type II receptor belongs to a transmembrane receptor serine-threonine kinase family. A new member of this receptor family (R4) was identified and shown to be a functional TGF-beta type I receptor on the basis of its ability to restore a TGF-beta-induced gene response in mutant cell lines lacking endogenous type I receptor. Both ligand binding and signaling of the R4 protein were dependent on the presence of a functional type II receptor. The type I receptor has an intrinsic serine-threonine kinase activity, which was essential for signal transduction.

Cloning and functional characterization of the early-lymphocyte-specific Pb99 gene.

The Pb99 gene is specifically expressed in pre-B cells and thymocytes and not in mature B and T cells or nonlymphoid tissues, implying that it may function in early lymphoid development. We have previously described the cloning of an incomplete cDNA for Pb99. Here we report the isolation of full-length cDNAs and genomic clones for the murine Pb99 gene and the mapping of its location to mouse chromosome 8. Sequence analyses of different Pb99 cDNA clones suggest that there may be at least three forms of the Pb99 protein generated by differential processing of the Pb99 transcript. The cDNA with the longest open reading frame encodes a putative protein that has seven hydrophobic domains similar to those of seven membrane-spanning proteins, such as the classical G protein-coupled receptors. To directly address the role of the Pb99 protein in lymphoid development, Pb99-deficient mice were generated by gene targeting, and lymphocyte development in these mice was analyzed.

Overproduction, purification and characterization of M.HinfI methyltransferase and its deletion mutant.

We have used the polymerase chain reaction to alter transcriptional and translational signals surrounding the hinfIM gene [encoding M.HinfI methyltransferase (MTase)] so as to achieve overexpression in Escherichia coli. The PCR-generated hinfIM gene was subcloned in a high-expression vector under control of the hybrid trp-lac promoter. In addition, the positive retroregulator stem-loop sequence derived from the crystal protein-encoding gene of Bacillus thuringiensis was inserted downstream from hinfIM. Using a similar approach, we have also constructed overproducer clones of a deletion mutant of M.HinfI MTase that has 97 amino acids from the C terminus deleted. The plasmid from the mutant clones is fully protected from HinfI restriction endonuclease digestion. It appears that the functional properties (the recognition and catalytic functions) are encoded within this mutant gene. The overproducer clones yield the wild type (wt) and the mutant enzymes to about 10% of total cellular protein upon induction with 1 mM IPTG. The wt M.HinfI and the mutant MTase were purified to near electrophoretic homogeneity by phosphocellulose, DEAE and gel chromatography. Their monomer sizes by SDS/polyacrylamide-gel electrophoresis are 43 kDa and 31 kDa, respectively, in good agreement with that predicted from the nucleotide sequence. DNA methylation experiments with purified enzymes using single-strand and double-strand M13mp18 DNA substrates indicate that while wt enzyme methylates both forms of DNA substrates, the mutant enzyme appears to preferentially methylate ss DNA substrate.

ATM deficiency augments constitutively nuclear cyclin D1-driven genomic instability and lymphomagenesis.

Cyclin D1 deregulation is implicated in the genesis of multiple human cancers. Importantly, nuclear cyclin D1 retention during S-phase promotes DNA re-replication and subsequent genomic instability, providing a direct correlation between aberrant cyclin D1/CDK4 activity, transcriptional regulation and double strand DNA break (DSB) induction. Together, these molecular events catalyze the genomic instability necessary for neoplastic transformation. Given that replication-associated DNA damage is central to cyclin D1-driven neoplasia, inactivation of critical checkpoint mediators should augment cyclin D1-dependent tumorigenesis in vivo. To interrogate potential synergy between constitutively nuclear cyclin D1 expression and impaired DSB-induced checkpoint integrity, Ataxia Telangiectasia Mutated (ATM)-deficient mice harboring the Eµ-D1T286A transgene were generated and evaluated for tumor onset. Eµ-D1T286A/ATM-/- mice exhibit dramatically accelerated incidence of both B- and T-cell lymphomas relative to Eµ-D1T286A or ATM-/- control cohorts. Lymphomas exhibit clonal chromosomal alterations distinct from ATM-/- mice, which typically acquire translocations involving the Tcrα/δ locus during V(D)J recombination, and instead harbor alterations at the c-Myc locus. Collectively, these findings reveal an intricate relationship wherein nuclear cyclin D1/CDK4 drives genomic instability in the absence of ATM function and clonal selection of cells harboring alterations within the murine c-Myc locus, ultimately facilitating transformation and tumor formation.

Tp53 deletion in B lineage cells predisposes mice to lymphomas with oncogenic translocations.

Inactivating Tp53 mutations are frequent genetic lesions in human tumors that harbor genomic instability, including B lineage lymphomas with IG translocations. Antigen receptor genes are assembled and modified in developing lymphocytes by RAG/AID-initiated genomic rearrangements that involve the induction of DNA double strand breaks (DSBs). Although TP53 inhibits the persistence of DSBs and induces apoptosis to protect cells from genomic instability and transformation, the development of spontaneous tumors harboring clonal translocations has not been reported in mice that only lack wild-type Tp53 protein or express Tp53 mutants. Tp53-deficient (Tp53(-/-)) mice succumb to T lineage lymphomas lacking clonal translocations but develop B lymphoid tumors containing immunoglobulin (Ig) translocations upon combined inactivation of DSB repair factors, RAG mutation or AID overexpression; mice expressing apoptosis-defective Tp53 mutants develop B cell lymphomas that have not been characterized for potential genomic instability. As somatic rather than germline inactivating mutations of TP53 are typically associated with human cancers and Tp53 deletion has cellular context dependent effects upon lymphocyte transformation, we generated mice with conditional Tp53 deletion in lineage-committed B lymphocytes to avoid complications associated with defective Tp53 responses during embryogenesis and/or in multi-lineage potential cells and, thereby, directly evaluate the potential physiological role of Tp53 in suppressing translocations in differentiated cells. These mb1-cre:Tp53(flox/flox) mice succumbed to lymphoid tumors containing Ig gene rearrangements and immunophenotypes characteristic of B cells from various developmental stages. Most mb1-cre:Tp53(flox/flox) tumors harbored clonal translocations, including Igh/c-myc or other oncogenic translocations generated by the aberrant repair of RAG/AID-generated DSBs. Our data indicate that Tp53 serves critical functions in B lineage lymphocytes to prevent transformation caused by translocations in cell populations experiencing physiological levels of RAG/AID-initiated DSB intermediates, and provide evidence that the somatic TP53 mutations found in diffuse large B-cell lymphoma and Burkitt's lymphoma may contribute to the development of these human malignancies.

Disruption of cyclin D1 nuclear export and proteolysis accelerates mammary carcinogenesis.

Cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and polyubiquitination by SCF(FBX4-alphaB crystallin). Inhibition of cyclin D1 proteolysis has been implicated as a causative factor leading to its overexpression in breast and esophageal carcinomas; however, the contribution of stable cyclin D1 to the genesis of such carcinomas has not been evaluated. We therefore generated transgenic mice wherein expression of either wild-type or a stable cyclin D1 allele (D1T286A) is regulated by MMTV-LTR. MMTV-D1T286A mice developed mammary adenocarcinomas at an increased rate relative to MMTV-D1 mice. Similar to human cancers that overexpress cyclin D1, D1T286A tumors were estrogen receptor-positive and exhibited estrogen-dependent growth. Collectively, these results suggest that temporal control of cyclin D1 subcellular localization and proteolysis is critical for maintenance of homeostasis within the mammary epithelium.

FKBP12 is not required for the modulation of transforming growth factor beta receptor I signaling activity in embryonic fibroblasts and thymocytes.

Transforming growth factor beta (TGF-beta) signals through a heteromeric complex of type I and type II transmembrane serine-threonine kinases. Recent evidence suggests that the immunophilin FKBP12 modulates the activity of the type I receptor, based on data that immunosuppressive drugs that disrupt FKBP12 binding to the type I receptor enhance TGF-beta signaling in mink lung epithelial cells, and overexpression of FKBP12 inhibits type I receptor phosphorylation by the type II receptor. To determine the physiological relevance of the FKBP12-TGF-beta receptor I interaction, we investigated whether disruption of this interaction affects TGF-beta-signaling in primary mouse embryo fibroblasts and thymocytes. We found that the addition of excess drugs had no effect on either TGF-beta-mediated transcriptional responses or growth inhibition. Dose-response curves for TGF-beta-mediated signaling in primary fibroblasts and thymocytes isolated from either wild-type or FKBP12-deficient mice were identical. Taken together, our results indicate that FKBP12 does not play a unique physiological role in TGF-beta signaling in primary fibroblasts and thymocytes.

A single heteromeric receptor complex is sufficient to mediate biological effects of transforming growth factor-beta ligands.

Transforming growth factor beta (TGF-beta), a multifunctional cytokine that regulates a variety of biological functions, signals through a heteromeric receptor complex of the type I and type II TGF-beta receptors. The type II receptor, a transmembrane serine-threonine kinase, was cloned based on its ability to directly bind TGF-beta. Recently, a number of candidate type I TGF-beta receptors have been isolated. Although only one of these transmembrane kinases (R4) has been shown to mediate TGF-beta-dependent gene activation, others bind TGF-beta when overexpressed in COS cells. Consequently, it has been postulated that the diversity of TGF-beta responses is generated through the association of distinct type I receptors with the type II TGF-beta receptor, thus creating receptor complexes of differential signaling capacities. In contrast to this model, we demonstrate that stable expression of only the R4 type I TGF-beta receptor in a mutant cell line lacking endogenous type I TGF-beta receptor was able to complex with the endogenous type II TGF-beta receptor and restore the effects of TGF-beta on inhibition of cell proliferation and activation of specific genes, regardless of which of the three mammalian isoforms of TGF-beta was used as the ligand. Therefore, R4 acts as a fully functional type I TGF-beta receptor, and the differential effects of TGF-beta are likely mediated by a single receptor complex consisting of R4 and the type II receptor.

Accessibility control of variable region gene assembly during T-cell development.

T-cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an alpha beta or gamma delta T-cell antigen receptor (TCR). The genes that encode the TCR beta and gamma chains lie in distinct loci, whereas the genes that encode the TCR alpha and delta chains lie in a single locus (TCR alpha/delta locus). Assembly of TCR variable region genes is mediated by a site-specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR beta, gamma and delta variable region genes are assembled prior to TCR alpha variable region genes. Furthermore, assembly of TCR beta variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement and expression of genes within the TCR alpha/delta locus presents a complicated problem. TCR alpha and delta variable region genes are assembled at different stages of T-cell development, and fully assembled TCR alpha and delta variable region genes must be expressed in distinct lineages of T cells, alpha beta and gamma delta, respectively. We have developed several experimental approaches to assess the role of cis-acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR beta and TCR alpha/delta loci during T-cell development.

Expression of the E2F1 transcription factor overcomes type beta transforming growth factor-mediated growth suppression.

Inhibition of cell growth by type beta transforming growth factor (TGF-beta) occurs in mid-G1 and is associated with decreased G1 cyclin-dependent kinase activity and maintenance of the retinoblastoma tumor suppressor protein Rb in an underphosphorylated, growth-suppressive state. A variety of recent experiments suggest that a functional target of Rb is the E2F transcription factor. In addition, the growth-suppressive effects of TGF-beta can be overcome by expression of viral oncogene products that dissociate E2F from Rb and Rb-related polypeptides. These results suggest the possibility that control of E2F may be a downstream event of TGF-beta action. Consistent with that possibility is the observation that E2F1 RNA levels are drastically reduced in TGF-beta-treated cells. We have also used a recombinant adenovirus containing the human E2F1 gene to overexpress the E2F1 product in mink lung epithelial cells that were growth arrested with TGF-beta. We find that overexpression of E2F1 can overcome the TGF-beta-mediated effect as measured by the activation of cellular DNA synthesis. These results suggest that a likely downstream target for the cyclin-dependent kinases, which are controlled by TGF-beta, is the activation of E2F.

Mechanisms that direct ordered assembly of T cell receptor beta locus V, D, and J gene segments.

T cell receptor (TCR) beta variable region genes are assembled in progenitor T cells from germ-line Vbeta, Dbeta, and Jbeta segments via an ordered two-step process in which Dbeta to Jbeta rearrangements occur on both alleles before appendage of a Vbeta to a preexisting DJbeta complex. Direct joining of Vbeta segments to nonrearranged Dbeta or Jbeta segments, while compatible with known restrictions on the V(D)J recombination mechanism, are infrequent within the endogenous TCRbeta locus. We have analyzed mechanisms that mediate ordered Vbeta, Dbeta, and Jbeta assembly via an approach in which TCRbeta minilocus recombination substrates were introduced into embryonic stem cells and then analyzed for rearrangement in normal thymocytes by recombinase-activating gene 2-deficient blastocyst complementation. These analyses demonstrated that Vbeta segments are preferentially targeted for rearrangement to Dbeta as opposed to Jbeta segments. In addition, we further demonstrated that Vbeta segments can be appended to nonrearranged endogenous Dbeta segments in which we have eliminated the ability of Dbeta segments to join to Jbeta segments. Our findings are discussed in the context of the mechanisms that regulate the ordered assembly and utilization of V, D, and J segments.

Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule.

The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments. V(D)J recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking recombination signal sequences (RSSs). Generally expressed DNA repair proteins then carry out the joining reaction. The conserved heptamer and nonamer sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting, specifies that V(D)J recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS. Vbeta segments are appended to DJbeta rearrangements, with little or no direct Vbeta to Jbeta joining, despite 12/23 compatibility of Vbeta 23-RSSs and Jbeta12-RSSs. Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)beta locus containing only one Dbeta (Dbeta1) gene segment and one Jbeta (Jbeta1) gene cluster to show that the 5' Dbeta1 12-RSS, but not the Jbeta1 12-RSSs, targets rearrangement of a diverse Vbeta repertoire. This targeting is precise and position-independent. This additional restriction on V(D)J recombination has important implications for the regulation of variable region gene assembly and repertoire development.

BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive human leukemias. Sequences within the first exon of BCR are required to activate the transforming potential of BCR-ABL. The SH2/SH3 domain-containing GRB-2 protein links tyrosine kinases to Ras signaling. We demonstrate that BCR-ABL exists in a complex with GRB-2 in vivo. Binding of GRB-2 to BCR-ABL is mediated by the direct interaction of the GRB-2 SH2 domain with a phosphorylated tyrosine, Y177, within the BCR first exon. The BCR-ABL-GRB-2 interaction is required for activation of the Ras signaling pathway. Mutation of Y177 to phenylalanine (Y177F) abolishes GRB-2 binding and abrogates BCR-ABL-induced Ras activation. The BCR-ABL (Y177F) mutant is unable to transform primary bone marrow cultures and is impaired in its ability to transform Rat1 fibroblasts. These findings implicate activation of Ras function as an important component in BCR-ABL-mediated transformation and demonstrate that GRB-2 not only functions in normal development and mitogenesis but also plays a role in oncogenesis.