Lance R. Thomas

Tumor-derived Mutations in the TRAIL Receptor DR5 Inhibit TRAIL Signaling through the DR4 Receptor by Competing for Ligand Binding

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine that preferentially induces apoptosis in tumor cells compared with normal cells through two receptors (DR4 and DR5). Somatic mutations in these receptors have been found in different kinds of cancer; however, it is poorly understood how the mutations affect signaling. We found that point mutations (L334F, E326K, E338K, and K386N) that were identified in human tumors result in the DR5 receptor losing its ability to form a functional death-inducing signaling complex and induce apoptosis. The mutant receptors also have a “dominant negative” effect whereby they inhibit the ability of TRAIL to induce apoptosis through functional DR4 receptors. This dominant negative mechanism is achieved through competition for TRAIL binding as shown by experiments where the ability of the mutant DR5 receptor to bind with the ligand was abolished, thus restoring TRAIL signaling through DR4. The inhibitory effect on signaling through the wild-type DR4 protein can be overcome if the inhibitory mechanism is bypassed by using a DR4-agonistic antibody that is not subject to this competition. This study provides a molecular basis for the use of specific therapeutic agonists of TRAIL receptors in people whose tumors harbor somatic DR5 mutations.

Proteolytic Control of the Oncoprotein Transcription Factor Myc

The c-Myc oncogene encodes a multifunctional transcription factor that directs the expression of genes required for cell growth and proliferation. Consistent with its potent growth-promoting properties, cells have evolved numerous mechanisms that limit the expression and activity of Myc. One of the most prominent of these mechanisms is proteolysis, which destroys Myc within minutes of its synthesis. The rapid and controlled destruction of Myc keeps its levels low and precisely tied to processes that regulate Myc production. In this review, we discuss how Myc protein stability is regulated and the influence of Myc proteolysis on its function. We describe what is known about how Myc is destroyed by ubiquitin (Ub)-mediated proteolysis, attempt to rationalize the role of different Ub-protein ligases and deubiquitylating enzymes (dUbs) in the regulation of Myc stability, and detail how these processes go awry in cancer. Finally, we discuss how our understanding of Myc regulation by the ubiquitin-proteasome system (UPS) can expose strategies for therapeutic intervention in human malignancies.

The adaptor protein TRADD activates distinct mechanisms of apoptosis from the nucleus and the cytoplasm

TNFR1 associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain along with nuclear import and export sequences that cause shuttling between the cytoplasm and nucleus. The death domain of TRADD contains the nuclear import sequence and expression of the core death domain (nuclear TRADD) results in exclusive nuclear localization and activation of a distinct apoptotic pathway. Cytoplasmic TRADD activates apoptosis through Fas-associated death domain protein (FADD) and caspase-8 activation that was blocked by caspase inhibitors or dominant-negative FADD. These inhibitors did not inhibit death induced by nuclear TRADD, which could only be inhibited by combining caspase inhibitors and a serine protease inhibitor. The pathway activated by nuclear TRADD requires caspase-9 catalytic activity. However, apoptosis activating factor deficiency confers only partial protection from death. This pathway represents an alternate means by which TRADD can regulate cell death independently of FADD and caspase-8 that occurs from the nucleus rather than the cytoplasm.

Functional Analysis of Histone Methyltransferase G9a in B and T Lymphocytes

Lymphocyte development is controlled by dynamic repression and activation of gene expression. These developmental programs include the ordered, tissue-specific assembly of Ag receptor genes by V(D)J recombination. Changes in gene expression and the targeting of V(D)J recombination are largely controlled by patterns of epigenetic modifications imprinted on histones and DNA, which alter chromatin accessibility to nuclear factors. An important component of this epigenetic code is methylation of histone H3 at lysine 9 (H3K9me), which is catalyzed by histone methyltransferases and generally leads to gene repression. However, the function and genetic targets of H3K9 methyltransferases during lymphocyte development remain unknown. To elucidate the in vivo function of H3K9me, we generated mice lacking G9a, a major H3K9 histone methyltransferase, in lymphocytes. Surprisingly, lymphocyte development is unperturbed in G9a-deficient mice despite a significant loss of H3K9me2 in precursor B cells. G9a deficiency is manifest as modest defects in the proliferative capacity of mature B cells and their differentiation into plasma cells following stimulation with LPS and IL-4. Precursor lymphocytes from the mutant mice retain tissue- and stage-specific control over V(D)J recombination. However, G9a deficiency results in reduced usage of Igλ L chains and a corresponding inhibition of Igλ gene assembly in bone marrow precursors. These findings indicate that the H3K9me2 epigenetic mark affects a highly restricted set of processes during lymphocyte development and activation.

Mutations in the Pho2 (Bas2) Transcription Factor That Differentially Affect Activation with Its Partner Proteins Bas1, Pho4, and Swi5

The yeast PHO2 gene encodes a homeodomain protein that exemplifies combinatorial control in transcriptional activation. Pho2 alone binds DNA in vitrowith low affinity, but in vivo it activates transcription with at least three disparate DNA-binding proteins: the zinc finger protein Swi5, the helix-loop-helix factor Pho4, and Bas1, an myb-like activator. Pho2 + Swi5 activates HO, Pho2 + Pho4 activatesPHO5, and Pho2 + Bas1 activates genes in the purine and histidine biosynthesis pathways. We have conducted a genetic screen and identified 23 single amino acid substitutions in Pho2 that differentially affect its ability to activate its specific target genes. Analysis of the mutations suggests that the central portion of Pho2 serves as protein-protein interactive surface, with a requirement for distinct amino acids for each partner protein.

Dynamic regulation of antigen receptor gene assembly.

A hallmark feature of adaptive immunity is the production of lymphocytes bearing an enormous repertoire of receptors for foreign antigens. This repertoire is generated early in B and T-cell development by the process of V(D)J recombination, which randomly assembles functional immunoglobulin (Ig) and T-cell receptor (TCR) genes from large arrays of DNA segments. Precursor lymphocytes must target then retarget a single V(D)J recombinase enzyme to distinct regions within antigen receptor loci to guide lymphocyte development and to ensure that each mature B and T-cell expresses only a single antigen receptor specificity. Proper targeting of V(D)J recombinase is also essential to avoid chromosomal aberrations that result in lymphoid malignancies. Early studies suggested that changes in the specificity of V(D)J recombination are achieved by differentially opening or closing chromatin associated with Ig and TCR gene segments at the proper developmental time point. This accessibility model has been extended significantly in recent years and it has become clear that control mechanisms governing antigen receptor gene assembly are multifaceted and vary from locus to locus. In this chapter we review how genetic and epigenetic mechanisms as well as widespread changes in chromosomal conformation synergize to orchestrate the diversification of genes encoding B and T-cell antigen receptors.

The MYC–WDR5 Nexus and Cancer

The MYC oncogenes encode a family of transcription factors that feature prominently in cancer. MYC proteins are overexpressed or deregulated in a majority of malignancies and drive tumorigenesis by inducing widespread transcriptional reprogramming that promotes cell proliferation, metabolism, and genomic instability. The ability of MYC to regulate transcription depends on its dimerization with MAX, which creates a DNA-binding domain that recognizes specific sequences in the regulatory elements of MYC target genes. Recently, we discovered that recognition of target genes by MYC also depends on its interaction with WDR5, a WD40-repeat protein that exists as part of several chromatin-regulatory complexes. Here, we discuss how interaction of MYC with WDR5 could create an avidity-based chromatin recognition mechanism that allows MYC to select its target genes in response to both genetic and epigenetic determinants. We rationalize how the MYC-WDR5 interaction provides plasticity in target gene selection by MYC and speculate on the biochemical and genomic contexts in which this interaction occurs. Finally, we discuss how properties of the MYC-WDR5 interface make it an attractive point for discovery of small-molecule inhibitors of MYC function in cancer cells.

Extensive regions of the FADD death domain are required for binding to the TRAIL receptor DR5

Extensive regions of the FADD death domain are required for binding to the TRAIL receptor DR5

pRS yeast vectors with a LYS2 marker

Vol. 36, No. 2 (2004) BioTechniques 213 We constructed two new general purpose cloning vectors for use in Saccharomyces cerevisiae. The new plasmids are pRS307, an integrating (YIp) vector with a LYS2 selectable marker, and pRS327, a multicopy (YEp) vector with a LYS2 marker and a 2-μm origin of replication. These plasmids, along with the previously described pRS317 plasmid, a single copy (YCp) vector with a LYS2 marker and a (centromeric) CEN element, provide a complete set of LYS2 plasmids with the Bluescript® polylinker that complement the widely used existing pRS vectors. Studies in yeast have been facilitated by sets of plasmid vectors with polylinkers that allow easy cloning. The YCplac/YIplac/YEplac set of vectors (1) contain the pUC polylinker (2) and a nutritional marker for selection in yeast. Thus, any fragment inserted into one of these vectors can be easily transferred to other versions, such as YCp, YIp, or YEp, or to a vector with a different selectable marker, either LEU2, TRP1, or URA3. The pRS set of plasmids (3,4) used the polylinker from the Bluescript plasmids (Stratagene, La Jolla, CA, USA) with YCp, YIp, and YEp versions constructed. Subsequently, pRS plasmids with the MET15 and ADE2 markers were constructed (5). Many commonly used yeast strains, including the BY strains (5) used to generate the complete collection of gene knockouts (6) have a lys2 mutation. Although the YCp plasmid with a LYS2 marker, pRS317, has been described (7), YIp and YEp versions have not been available. Here we describe the construction of YIp and YEp vectors with a LYS2 marker with the Bluescript polylinker. The integrating LYS2 plasmid pRS307 was constructed by ligation as follows. pRS317 was cleaved at the ApaLI site common to the pRS vectors, the ends were blunted with the Klenow fragment of DNA polymerase, the DNA was cleaved with EcoRI, and the 5.57-kb ApaLI (blunt)/EcoRI fragment was purified. pRS306 (4) was cleaved with NdeI, the ends were blunted with the Klenow fragment of DNA polymerase, cleaved with EcoRI, and the

A common functional consequence of tumor-derived mutations within c-MYC

The relevance of changes to the coding sequence of the c-MYC oncogene to malignancy is controversial. Overexpression of a pristine form of MYC is observed in many cancers and is sufficient to drive tumorigenesis in most contexts. Yet missense changes to MYC are found in ~50% of Burkitt’s lymphomas, aggregate within an amino-terminal degron important for proteasomal destruction of MYC, and where examined profoundly enhance the tumorigenic properties of MYC in vitro and in vivo. Much of the controversy surrounding these mutants stems from the limited number of mutations that have been evaluated and their clustering within a single region of the MYC protein; the highly-conserved Myc box I (MbI) element. Here, by analysis of extant genomic data sets, we identify a previously unrecognized hotspot for tumor-associated MYC mutations, located in a conserved central portion of the protein. We show that, despite their distal location in MYC, mutations in this region precisely phenocopy those in MbI in terms of stability, in vitro transformation, growth-promoting properties, in vivo tumorigenesis and ability to escape p53-dependent tumor surveillance mechanisms. The striking parallels between the behavior of tumor-derived mutations in disparate regions of the MYC protein reveals that a common molecular process is disrupted by these mutations, implying an active role for these mutations in tumorigenesis and suggesting that different therapeutic strategies may be needed for treatment of lymphomas expressing wild type versus mutant forms of MYC protein.