No abstract

Many developmental control genes contain stalled RNA Polymerase II (Pol II) in the early Drosophila embryo, including four of the eight Hox genes. Here, we present evidence that the stalled Hox promoters possess an intrinsic insulator activity. The enhancer-blocking activities of these promoters are dependent on general transcription factors that inhibit Pol II elongation, including components of the DSIF and NELF complexes. The activities of conventional insulators are also impaired in embryos containing reduced levels of DSIF and NELF. Thus, promoter-proximal stalling factors might help promote insulator-promoter interactions. We propose that stalled promoters help organize gene complexes within chromosomal loop domains.Supplemental material is available at http://www.genesdev.org.

The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the trisnRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. Highresolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.

Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.

Epigenetic inheritance to maintain the expression state of the genome is essential during development. In Drosophila, the cis regulatory elements, called the Polycomb Response Elements (PREs) function to mark the epigenetic cellular memory of the corresponding genomic region with the help of PcG and trxG proteins. While the PcG genes code for the repressor proteins, the trxG genes encode activator proteins. The observations that some proteins may function both as PcG and trxG member and that both these group of proteins act upon common cis elements indicate at least a partial functional overlap among these proteins. Trl-GAGA was initially identified as a trxG member but later was shown to be essential for PcG function on several PREs. In order to understand how Trl-GAGA functions in PcG context, we have looked for the interactors of this protein. We identified lola like, aka batman, as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene. These observations suggest a possible mechanism of how Trl-GAGA plays a role in maintaining the repressed state of target genes involving lolal, which may function as a mediator to recruit PcG complexes.

Modifications of cis-regulatory DNAs, particularly enhancers, underlie changes in gene expression during animal evolution. Here, we present evidence for a distinct mechanism of regulatory evolution, whereby a novel pattern of gene expression arises from altered gene targeting of a conserved enhancer. The tinman gene complex (Tin-C) controls the patterning of dorsal mesodermal tissues, including the dorsal vessel or heart in Drosophila. Despite broad conservation of Tin-C gene expression patterns in the flour beetle (Tribolium castaneum), the honeybee (Apis mellifera) and the fruit fly (Drosophila melanogaster), the expression of a key pericardial determinant, ladybird, is absent from the dorsal mesoderm of Tribolium embryos. Evidence is presented that this loss in expression is replaced by expression of C15, the neighboring gene in the complex. This switch in expression from ladybird to C15 appears to arise from an inversion within the tinman complex, which redirects a conserved ladybird 3Ј enhancer to regulate C15. In Drosophila, this enhancer fails to activate C15 expression owing to the activity of an insulator at the intervening ladybird early promoter. By contrast, a chromosomal inversion allows the cardiac enhancer to bypass the ladybird insulator in Tribolium. Given the high frequency of genome rearrangements in insects, it is possible that such enhancer switching might be widely used in the diversification of the arthropods.

SUMMARY Hox genes control the anterior-posterior patterning of most metazoan embryos. Their sequential expression is initially established by the segmentation gene cascade in the early Drosophila embryo [1]. The maintenance of these patterns depends on the Polycomb group (PcG) and trithorax group (trxG) complexes during the remainder of the life cycle [2]. We provide both genetic and molecular evidence that the Hox genes are subject to an additional tier of regulation, i.e. at the level of transcription elongation. Both Ultrabithorax (Ubx) and Abdominal-B (Abd-B) genes contain stalled or paused RNA Polymerase II (Pol II) even when silent [3, 4]. The Pol II elongation factors Elongin-A and Cdk9 are essential for optimal Ubx and Abd-B expression. Mitotic recombination assays suggest that these elongation factors are also important for the regulation of Notch, EGF, and Dpp signaling genes. Stalled Pol II persists in tissues where Ubx and Abd-B are silenced by the PcG complex. We propose that stalling fosters both the rapid induction and precise silencing of Hox gene expression during development.

The GAGA factor (GAF), encoded by the Trithorax like gene (Trl) is a multifunctional protein involved in gene activation, Polycomb-dependent repression, chromatin remodeling and is a component of chromatin domain boundaries. Although first isolated as transcriptional activator of the Drosophila homeotic gene Ultrabithorax (Ubx), the molecular basis of this GAF activity is unknown. Here we show that dmTAF3 (also known as BIP2 and dTAF(II)155), a component of TFIID, interacts directly with GAF. We generated mutations in dmTAF3 and show that, in Trl mutant background, they affect transcription of Ubx leading to enhancement of Ubx phenotype. These results reveal that the gene activation pathway involving GAF is through its direct interaction with dmTAF3.