Obstruction of BRAFV600E transcription by complementary PNA oligomers as a means to inhibit BRAF-mutant melanoma growth.

Peptide nucleic acid (PNA) oligomers are DNA mimics, which are capable of binding gene sequences 1000-fold more avidly than complementary native DNA by strand invasion and effectively obstruct transcription. Irreversibly obstructing the transcription or replication of a gene sequence, such as BRAFV600E, offers a potential route to specifically target the cancer cell itself. We have employed PNA oligomers to target BRAFV600E in a sequence-specific complementary manner. These PNAs have been modified by appending configurationally stabilizing cationic peptides in order to improve their cellular delivery and target avidity. Our results indicate that exposure of the melanoma cell lines to a modified PNA-peptide conjugate complementary to BRAFV600E mutation sequence results in a concentration-dependent and time-dependent inhibition of cell growth that is specific for the BRAFV600E-mutant melanoma cell lines with inhibition of mRNA and protein expression. Xenograft mouse trials show increased tumor growth delay and necrosis with the BRAFV600E-complementary PNA-peptide conjugates as compared with the saline and scrambled PNA sequence controls. Similarly, quantitative measurement shows a 2.5-fold decrease in Ki67 and a 3-fold increase in terminal deoxynucleotidyl transferase dUTP nick end labeling expression with this approach. PNA-delivery peptide conjugates represent a novel way to target BRAFV600E and represent a new approach in targeting selective oncogenes that induce tumor growth.

Regulation of developmental rate and germ cell proliferation in Caenorhabditis elegans by the p53 gene network.

Caenorhabditis elegans CEP-1 activates germline apoptosis in response to genotoxic stress, similar to its mammalian counterpart, tumor suppressor p53. In mammals, there are three p53 family members (p53, p63, and p73) that activate and repress many distinct and overlapping sets of genes, revealing a complex transcriptional regulatory network. Because CEP-1 is the sole p53 family member in C. elegans, analysis of this network is greatly simplified in this organism. We found that CEP-1 functions during normal development in the absence of stress to repress many (331) genes and activate only a few (28) genes. In response to genotoxic stress, 1394 genes are activated and 942 are repressed, many of which contain p53-binding sites. Comparison of the CEP-1 transcriptional network with transcriptional targets of the human p53 family reveals considerable overlap between CEP-1-regulated genes and homologues regulated by human p63 and p53, suggesting a composite p53/p63 action for CEP-1. We found that phg-1, the C. elegans Gas1 (growth arrest-specific 1) homologue, is activated by CEP-1 and is a negative regulator of cell proliferation in the germline in response to genotoxic stress. Further, we find that CEP-1 and PHG-1 mediate the decreased developmental rate and embryonic viability of mutations in the clk-2/TEL2 gene, which regulates lifespan and checkpoint responses.

ELT-5 and ELT-6 are required continuously to regulate epidermal seam cell differentiation and cell fusion in C. elegans.

The C. elegans epidermis is a simple epithelium comprised of three major cell types, the seam, syncytial and P cells. While specification of all major epidermal cells is known to require the ELT-1 GATA transcription factor, little is known about how the individual epidermal cell types are specified. We report that elt-5 and -6, adjacent genes encoding GATA factors, are essential for the development of the lateral epidermal cells, the seam cells. Inhibition of elt-5 and -6 function by RNA-mediated interference results in penetrant late embryonic and early larval lethality. Seam cells in affected animals do not differentiate properly: the alae, seam-specific cuticular structures, are generally absent and expression of several seam-specific markers is blocked. In addition, elt-3, which encodes another GATA factor normally expressed in non-seam epidermis, is often ectopically expressed in the seam cells of affected animals, demonstrating that ELT-5 and -6 repress elt-3 expression in wild-type seam cells. Seam cells in affected animals often undergo inappropriate fusion with the epidermal syncytia. Interference of elt-5 and -6 function during larval development can cause fusion of all seam cells with the surrounding syncytia and pronounced defects in molting. elt-5 and -6 are both expressed in seam cells and many other cells, and are apparently functionally interchangeable. Their expression is controlled by separable tissue-specific regulatory elements and the apportionment of monocistronic versus dicistronic transcription of both genes appears to be subject to cell-type-specific regulation. Collectively, these findings indicate that elt-5 and -6 function continuously throughout C. elegans development to regulate seam cell differentiation and cell fusion.

Distinct requirements for C.elegans TAF(II)s in early embryonic transcription.

TAF(II)s are conserved components of the TFIID, TFTC and SAGA-related mRNA transcription complexes. In yeast (y), yTAF(II)17 is required broadly for transcription, but various other TAF(II)s appear to have more specialized functions. It is important to determine how TAF(II)s contribute to transcription in metazoans, which have larger and more diverse genomes. We have examined TAF(II) functions in early Caenorhabditis elegans embryos, which can survive without transcription for several cell generations. We show that taf-10 (yTAF(II)17) and taf-11 (yTAF(II)25) are required for a significant fraction of transcription, but apparently are not needed for expression of multiple developmental and other metazoan-specific genes. In contrast, taf-5 (yTAF(II)48; human TAF(II)130) seems to be required for essentially all early embryonic mRNA transcription. We conclude that TAF-10 and TAF-11 have modular functions in metazoans, and can be bypassed at many metazoan-specific genes. The broad involvement of TAF-5 in mRNA transcription in vivo suggests a requirement for either TFIID or a TFTC-like complex.

Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance.

We have identified a homolog of the mammalian p53 tumor suppressor protein in the nematode Caenorhabditis elegans that is expressed ubiquitously in embryos. The gene encoding this protein, cep-1, promotes DNA damage-induced apoptosis and is required for normal meiotic chromosome segregation in the germ line. Moreover, although somatic apoptosis is unaffected, cep-1 mutants show hypersensitivity to hypoxia-induced lethality and decreased longevity in response to starvation-induced stress. Overexpression of CEP-1 promotes widespread caspase-independent cell death, demonstrating the critical importance of regulating p53 function at appropriate levels. These findings show that C. elegans p53 mediates multiple stress responses in the soma, and mediates apoptosis and meiotic chromosome segregation in the germ line.

The zinc finger protein DIE-1 is required for late events during epithelial cell rearrangement in C. elegans.

The mechanism by which epithelial cells undergo directed rearrangement is central to morphogenesis, yet the regulation of these movements remains poorly understood. We have investigated epithelial cell rearrangement (intercalation) in the dorsal hypodermis, or embryonic epidermis, of the C. elegans embryo by analyzing the die-1(w34) mutant, which fails to undergo normal intercalation. Dorsal hypodermal cells of die-1(w34) homozygous embryos initiate but fail to complete the process of intercalation. Multiphoton microscopy reveals that intercalating cells extend monopolar, basolateral protrusions in their direction of migration; posterior dorsal hypodermal cells in die-1(w34) mutants appear to extend protrusions normally, but fail to translocate their cell bodies to complete rearrangement. Despite abnormal intercalation, the subsequent morphogenetic movements that enclose the embryo with epithelial cells and the process of dorsal cell fusion still occur. However, elongation of the embryo into a wormlike shape is disrupted in die-1(w34) embryos, suggesting that intercalation may be necessary for subsequent elongation of the embryo. Actin filaments are not properly organized within the dorsal hypodermis of die-1(w34) embryos, consistent with intercalation's being a necessary prerequisite for elongation. The die-1 gene encodes a C2H2 zinc finger protein containing four fingers, which likely acts as a transcriptional regulator. DIE-1 is present in the nuclei of hypodermal, muscle, gut, and pharyngeal cells; its distribution suggests that DIE-1 acts in each of these tissues to regulate morphogenetic movements. die-1(w34) mutants display morphogenetic defects in the pharynx, gut, and muscle quadrants, in addition to the defects in the dorsal hypodermis, consistent with the DIE-1 expression pattern. Mosaic analysis indicates that DIE-1 is autonomously required in the posterior dorsal hypodermis for intercalation. Our analysis documents for the first time the dynamics of protrusive activity during epithelial cell rearrangement. Moreover, our analysis of die-1 shows that the events of epithelial cell rearrangement are under transcriptional control, and that early and later phases of epithelial cell rearrangement are genetically distinguishable.

Restriction of mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta homolog is mediated by MED-1 and -2 in C. elegans.

The endoderm and much of the mesoderm arise from the EMS cell in the four-cell C. elegans embryo. We report that the MED-1 and -2 GATA factors specify the entire fate of EMS, which otherwise produces two C-like mesectodermal progenitors. The meds are direct targets of the maternal SKN-1 transcription factor; however, their forced expression can direct SKN-1-independent reprogramming of non-EMS cells into mesendodermal progenitors. We find that SGG-1/GSK-3beta kinase acts both as a Wnt-dependent activator of endoderm in EMS and an apparently Wnt-independent repressor of the meds in the C lineage, indicating a dual role for this kinase in mesendoderm development. Our results suggest that a broad tissue territory, mesendoderm, in vertebrates has been confined to a single cell in nematodes through a common gene regulatory network.

Many genomic regions are required for normal embryonic programmed cell death in Caenorhabditis elegans.

To identify genes involved in programmed cell death (PCD) in Caenorhabditis elegans, we screened a comprehensive set of chromosomal deficiencies for alterations in the pattern of PCD throughout embryonic development. From a set of 58 deficiencies, which collectively remove approximately 74% of the genome, four distinct classes were identified. In class I (20 deficiencies), no significant deviation from wild type in the temporal pattern of cell corpses was observed, indicating that much of the genome does not contain zygotic genes that perform conspicuous roles in embryonic PCD. The class II deficiencies (16 deficiencies defining at least 11 distinct genomic regions) led to no or fewer-than-normal cell corpses. Some of these cause premature cell division arrest, probably explaining the diminution in cell corpse number; however, others have little effect on cell proliferation, indicating that the reduced cell corpse number is not a direct result of premature embryonic arrest. In class III (18 deficiencies defining at least 16 unique regions), an excess of cell corpses was observed. The developmental stage at which the extra corpses were observed varied among the class III deficiencies, suggesting the existence of genes that perform temporal-specific functions in PCD. The four deficiencies in class IV (defining at least three unique regions), showed unusually large corpses that were, in some cases, attributable to extremely premature arrest in cell division without a concomitant block in PCD. Deficiencies in this last class suggest that the cell death program does not require normal embryonic cell proliferation to be activated and suggest that while some genes required for cell division might also be required for cell death, others are not. Most of the regions identified by these deficiencies do not contain previously identified zygotic cell death genes. There are, therefore, a substantial number of as yet unidentified genes required for normal PCD in C. elegans.

The TBP-like factor CeTLF is required to activate RNA polymerase II transcription during C. elegans embryogenesis.

Metazoans possess two TATA-binding protein homologs, the general transcription factor TBP and a related factor called TLF. Four models have been proposed for the role of TLF in RNA polymerase II (Pol II) transcription: (1) TLF and TBP function redundantly, (2) TLF antagonizes TBP, (3) TLF is a tissue-specific TBP, or (4) TLF and TBP have distinct activities. Here we report that CeTLF is required to express a subset of Pol II genes and associates with at least one of these genes in vivo. CeTLF is also necessary to establish bulk transcription during early embryogenesis. Since CeTLF and CeTBP are expressed at comparable levels in the same cells, these findings suggest CeTLF performs a unique function in activating Pol II transcription distinct from that of CeTBP.

Action of the Caenorhabditis elegans GATA factor END-1 in Xenopus suggests that similar mechanisms initiate endoderm development in ecdysozoa and vertebrates.

In ecdysozoan protostomes, including arthropods and nematodes, transcription factors of the GATA family specify the endoderm: Drosophila dGATAb (ABF/Serpent) and Caenorhabditis elegans END-1 play important roles in generating this primary germ layer. end-1 is the earliest expressed endoderm-specific gene known in C. elegans and appears to initiate the program of gene expression required for endoderm differentiation, including a cascade of GATA factors required for development and maintenance of the intestine. Among vertebrate GATA proteins, the GATA-4/5/6 subfamily regulates aspects of late endoderm development, but a role for GATA factors in establishing the endoderm is unknown. We show here that END-1 binds to the canonical target DNA sequence WGATAR with specificity similar to that of vertebrate GATA-1 and GATA-4, and that it functions as a transcriptional activator. We exploited this activity of END-1 to demonstrate that establishment of the vertebrate endoderm, like that of invertebrate species, also appears to involve GATA transcriptional activity. Like the known vertebrate endoderm regulators Mixer and Sox17, END-1 is a potent activator of endoderm differentiation in isolated Xenopus ectoderm. Moreover, a dominant inhibitory GATA-binding fusion protein abrogates endoderm differentiation in intact embryos. By examining these effects in conjunction with those of Mixer- and Sox17beta-activating and dominant inhibitory constructs, we further establish the likely relationships between GATA activity and these regulators in early development of the vertebrate endoderm. These results suggest that GATA factors may function sequentially to regulate endoderm differentiation in both protostomes and deuterostomes.

A new generation of fluorescent chemosensors demonstrate improved analyte detection sensitivity and photobleaching resistance.

Molecular chemosensors have found increased utility in the development of precise and sensitive detection devices. However, chemosensors that report binding via fluorescence through UV excitation are susceptible to destruction via photodegradation of the fluorophore. In the following report, the dansyl fluorophore in a previously reported chemosensor for peptides is replaced with an acridone derivative that is highly resistant to photobleaching. Its spectral properties are closely matched to those of the original dansyl fluorophore, and although quite structurally dissimilar, the new more photostable acridone chemosensor analogue exhibits only minor differences in binding/detection characteristics.

Alterations in the conserved SL1 trans-spliced leader of Caenorhabditis elegans demonstrate flexibility in length and sequence requirements in vivo.

Approximately 70% of mRNAs in Caenorhabditis elegans are trans spliced to conserved 21- to 23-nucleotide leader RNAs. While the function of SL1, the major C. elegans trans-spliced leader, is unknown, SL1 RNA, which contains this leader, is essential for embryogenesis. Efforts to characterize in vivo requirements of the SL1 leader sequence have been severely constrained by the essential role of the corresponding DNA sequences in SL1 RNA transcription. We devised a heterologous expression system that circumvents this problem, making it possible to probe the length and sequence requirements of the SL1 leader without interfering with its transcription. We report that expression of SL1 from a U2 snRNA promoter rescues mutants lacking the SL1-encoding genes and that the essential embryonic function of SL1 is retained when approximately one-third of the leader sequence and/or the length of the leader is significantly altered. In contrast, although all mutant SL1 RNAs were well expressed, more severe alterations eliminate this essential embryonic function. The one non-rescuing mutant leader tested was never detected on messages, demonstrating that part of the leader sequence is essential for trans splicing in vivo. Thus, in spite of the high degree of SL1 sequence conservation, its length, primary sequence, and composition are not critical parameters of its essential embryonic function. However, particular nucleotides in the leader are essential for the in vivo function of the SL1 RNA, perhaps for its assembly into a functional snRNP or for the trans-splicing reaction.

Reprogramming of early embryonic blastomeres into endodermal progenitors by a Caenorhabditis elegans GATA factor.

The END-1 GATA factor has been implicated in specifying endoderm in Caenorhabditis elegans and is the earliest known zygotic protein expressed in the lineage of E, the clonal endoderm progenitor. We report that ubiquitous end-1 expression during a critical period in embryogenesis causes all non-endodermal lineages to produce endoderm instead of ectoderm and/or mesoderm. END-1 expression bypasses the requirement for maternal SKN-1 and the maternal Wnt signaling pathway in endoderm formation. This suggests that a primary function of these maternal factors is to regulate zygotic end-1 expression, which is then sufficient to initiate the entire program for endoderm development.

The maternal-to-zygotic transition in embryonic patterning of Caenorhabditis elegans.

Maternal factors laid down in the oocyte regulate blastomere identities in the early Caenorhabditis elegans embryo by activating zygotic patterning genes and restricting their expression to the appropriate lineages. A number of early-acting zygotic genes that specify various cell fates have been identified recently and their temporal and spatial regulation by maternal factors has begun to be elucidated.

Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4.

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele of zen-4, an MKLP1 homologue in the nematode Caenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.

Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans.

The tumor suppressor gene FHIT encompasses the common human chromosomal fragile site at 3p14.2 and numerous cancer cell biallelic deletions. To study Fhit function we cloned and characterized FHIT genes from Drosophila melanogaster and Caenorhabditis elegans. Both genes code for fusion proteins in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases; the D. melanogaster fusion protein exhibited diadenosine triphosphate (ApppA) hydrolase activity expected of an authentic Fhit homolog. In human and mouse, the nitrilase homologs and Fhit are encoded by two different genes: FHIT and NIT1, localized on chromosomes 3 and 1 in human, and 14 and 1 in mouse, respectively. We cloned and characterized human and murine NIT1 genes and determined their exon-intron structure, patterns of expression, and alternative processing of their mRNAs. The tissue specificity of expression of murine Fhit and Nit1 genes was nearly identical. Because fusion proteins with dual or triple enzymatic activities have been found to carry out specific steps in a given biochemical or biosynthetic pathway, we postulate that Fhit and Nit1 likewise collaborate in a biochemical or cellular pathway in mammalian cells.

end-1 encodes an apparent GATA factor that specifies the endoderm precursor in Caenorhabditis elegans embryos.

The endoderm in the nematode Caenorhabditis elegans is clonally derived from the E founder cell. We identified a single genomic region (the endoderm-determining region, or EDR) that is required for the production of the entire C. elegans endoderm. In embryos lacking the EDR, the E cell gives rise to ectoderm and mesoderm instead of endoderm and appears to adopt the fate of its cousin, the C founder cell. end-1, a gene from the EDR, restores endoderm production in EDR deficiency homozygotes. end-1 transcripts are first detectable specifically in the E cell, consistent with a direct role for end-1 in endoderm development. The END-1 protein is an apparent zinc finger-containing GATA transcription factor. As GATA factors have been implicated in endoderm development in other animals, our findings suggest that endoderm may be specified by molecularly conserved mechanisms in triploblastic animals. We propose that end-1, the first zygotic gene known to be involved in the specification of germ layer and founder cell identity in C. elegans, may link maternal genes that regulate the establishment of the endoderm to downstream genes responsible for endoderm differentiation.

A deficiency screen for zygotic loci required for establishment and patterning of the epidermis in Caenorhabditis elegans.

To identify genomic regions required for establishment and patterning of the epidermis, we screened 58 deficiencies that collectively delete at least approximately 67% of the Caenorhabditis elegans genome. The epidermal pattern of deficiency homozygous embryos was analyzed by examining expression of a marker specific for one of the three major epidermal cell types, the seam cells. The organization of the epidermis and internal organs was also analyzed using a monoclonal antibody specific for epithelial adherens junctions. While seven deficiencies had no apparent effect on seam cell production, 21 were found to result in subnormal, and five in excess numbers of these cells. An additional 23 deficiencies blocked expression of the seam cell marker, in some cases without preventing cell proliferation. Two deficiencies result in multinucleate seam cells. Deficiencies were also identified that result in subnormal numbers of epidermal cells, hyperfusion of epidermal cells into a large syncytium, or aberrant epidermal differentiation. Finally, analysis of internal epithelia revealed deficiencies that cause defects in formation of internal organs, including circularization of the intestine and bifurcation of the pharynx lumen. This study reveals that many regions of the C. elegans genome are required zygotically for patterning of the epidermis and other epithelia.

lin-12 and glp-1 are required zygotically for early embryonic cellular interactions and are regulated by maternal GLP-1 signaling in Caenorhabditis elegans.

Cell-cell interactions mediated by LIN-12 and GLP-1, members of the LNG (LIN-12, Notch, GLP-1) family of receptors, are required to specify numerous cell fates during development of the nematode Caenorhabditis elegans. Maternally expressed GLP-1 participates in two of at least four sequential inductive interactions that specify the fates of early embryonic descendants of the AB founder cell. We report that GLP-1 and LIN-12, and apparently their ligand, LAG-2, as well as a downstream component, LAG-1, are required in the latter two inductions. We find that LAG-2 is expressed in the signaling cells and LIN-12 is expressed in cells receiving the inductions, consistent with their proposed roles as ligand and receptor, respectively. Furthermore, we report that maternal GLP-1 activity is required (1) to repress early zygotic lag-2 expression and (2) to activate zygotic lin-12 expression in the early embryo. The patterning of both receptor and ligand expression by maternal GLP-1 signaling establishes competence for the zygotic LNG-mediated cellular interactions and localizes these interactions to the appropriate cells. We propose that activation of maternal GLP-1 regulates zygotic lin-12 and lag-2 expression by a regulatory mechanism analogous to that described for the post-embryonic gonad.

Novel Hoogsteen-like bases for configurational recognition of the T-A base pair by DNA triplex formation.

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides binding duplex A-T or G-C base-pair DNA sequences specifically at homopurine sites in the major groove as T-A-T and C+.G-C triplets. Here we report the successful modeling of novel unnatural nucleosides that recognize the T-A DNA base pair by Hoogsteen interaction. Since the DNA triplex can be considered to assume an A-type or B-type conformation, these novel Hoogsteen nucleotides are tested within model A-type and B-type conformation triplex structures. A triplet consisting of the T-A base pair and one of the novel Hoogsteen nucleotides replaces the central T.A-T triplet in the triplex using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration. The entire triplex is energy minimized and the presence of any structural or energetic perturbations due to the central triplet is assessed with respect to the unmodified energy-minimized (T.A-T)11 proposed starting structures. Incorporation of these novel triplets into both A-type and B-type natural tiplex structures provokes minimal change in the configuration of the central and adjacent triplets. The plan is to produce a series of Hoogsteen-like bases that preferentially bind the T-A major groove in either an A-type or B-type conformation. Selective recognition of the T-A major groove with respect to the G-C major groove, which presents similar keto and amine placement, is also assessed with configurational preference. Evaluation of the triplex solution structure by using these unnatural bases as binding conformational probes is a prerequisite to the further design of triplet forming bases.