Genome-wide analysis of alternative pre-mRNA splicing and RNA-binding specificities of the Drosophila hnRNP A/B family members.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been traditionally seen as proteins packaging RNA nonspecifically into ribonucleoprotein particles (RNPs), but evidence suggests specific cellular functions on discrete target pre-mRNAs. Here we report genome-wide analysis of alternative splicing patterns regulated by four Drosophila homologs of the mammalian hnRNP A/B family (hrp36, hrp38, hrp40, and hrp48). Analysis of the global RNA-binding distributions of each protein revealed both small and extensively bound regions on target transcripts. A significant subset of RNAs were bound and regulated by more than one hnRNP protein, revealing a combinatorial network of interactions. In vitro RNA-binding site selection experiments (SELEX) identified distinct binding motif specificities for each protein, which were overrepresented in their respective regulated and bound transcripts. These results indicate that individual heterogeneous ribonucleoproteins have specific affinities for overlapping, but distinct, populations of target pre-mRNAs controlling their patterns of RNA processing.

Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1.

Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER- AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER- AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis.

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

DNA regions bound at low occupancy by transcription factors do not drive patterned reporter gene expression in Drosophila.

In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound at high levels in Drosophila blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the cis-regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional cis-regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.

FGFR2 variants and breast cancer risk: fine-scale mapping using African American studies and analysis of chromatin conformation.

Genome-wide association studies have identified FGFR2 as a breast cancer (BC) susceptibility gene in populations of European and Asian descent, but a causative variant has not yet been conclusively identified. We hypothesized that the weaker linkage disequilibrium across this associated region in populations of African ancestry might help refine the set of candidate-causal single nucleotide polymorphisms (SNPs) previously identified by our group. Eight candidate-causal SNPs were evaluated in 1253 African American invasive BC cases and 1245 controls. A significant association with BC risk was found with SNP rs2981578 (unadjusted per-allele odds ratio = 1.20, 95% confidence interval 1.03-1.41, P(trend) = 0.02), with the odds ratio estimate similar to that reported in European and Asian subjects. To extend the fine-mapping, genotype data from the African American studies were analyzed jointly with data from European (n = 7196 cases, 7275 controls) and Asian (n = 3901 cases, 3205 controls) studies. In the combined analysis, SNP rs2981578 was the most strongly associated. Five other SNPs were too strongly correlated to be excluded at a likelihood ratio of < 1/100 relative to rs2981578. Analysis of DNase I hypersensitive sites indicated that only two of these map to highly accessible chromatin, one of which, SNP rs2981578, has previously been implicated in up-regulating FGFR2 expression. Our results demonstrate that the association of SNPs in FGFR2 with BC risk extends to women of African American ethnicity, and illustrate the utility of combining association analysis in datasets of diverse ethnic groups with functional experiments to identify disease susceptibility variants.

Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm.

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets.

The androgen receptor induces a distinct transcriptional program in castration-resistant prostate cancer in man.

The androgen receptor (AR) regulates prostate cell growth in man, and prostate cancer is the commonest cancer in men in the UK. We present a comprehensive analysis of AR binding sites in human prostate cancer tissues, including castrate-resistant prostate cancer (CRPC). We identified thousands of AR binding sites in CRPC tissue, most of which were not identified in PC cell lines. Many adjacent genes showed AR regulation in xenografts but not in cultured LNCaPs, demonstrating an in-vivo-restricted set of AR-regulated genes. Functional studies support a model of altered signaling in vivo that directs AR binding. We identified a 16 gene signature that outperformed a larger in-vitro-derived signature in clinical data sets, showing the importance of persistent AR signaling in CRPC.

The opposing transcriptional functions of Sin3a and c-Myc are required to maintain tissue homeostasis.

How the proto-oncogene c-Myc balances the processes of stem-cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc's transcriptional roles at the epidermal differentiation complex, a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovol-1) and displace (Cebpa, Mxi1 and Sin3a) specific sets of differentiation-specific transcriptional regulators to epidermal differentiation complex genes. We found that Sin3a causes deacetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3a, genomic recruitment of c-Myc to the epidermal differentiation complex is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3a reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis through a negative feedback loop.

Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions.

BACKGROUND: We previously established that six sequence-specific transcription factors that initiate anterior/posterior patterning in Drosophila bind to overlapping sets of thousands of genomic regions in blastoderm embryos. While regions bound at high levels include known and probable functional targets, more poorly bound regions are preferentially associated with housekeeping genes and/or genes not transcribed in the blastoderm, and are frequently found in protein coding sequences or in less conserved non-coding DNA, suggesting that many are likely non-functional. RESULTS: Here we show that an additional 15 transcription factors that regulate other aspects of embryo patterning show a similar quantitative continuum of function and binding to thousands of genomic regions in vivo. Collectively, the 21 regulators show a surprisingly high overlap in the regions they bind given that they belong to 11 DNA binding domain families, specify distinct developmental fates, and can act via different cis-regulatory modules. We demonstrate, however, that quantitative differences in relative levels of binding to shared targets correlate with the known biological and transcriptional regulatory specificities of these factors. CONCLUSIONS: It is likely that the overlap in binding of biochemically and functionally unrelated transcription factors arises from the high concentrations of these proteins in nuclei, which, coupled with their broad DNA binding specificities, directs them to regions of open chromatin. We suggest that most animal transcription factors will be found to show a similar broad overlapping pattern of binding in vivo, with specificity achieved by modulating the amount, rather than the identity, of bound factor.

Association of cohesin and Nipped-B with transcriptionally active regions of the Drosophila melanogaster genome.

The cohesin complex is a chromosomal component required for sister chromatid cohesion that is conserved from yeast to man. The similarly conserved Nipped-B protein is needed for cohesin to bind to chromosomes. In higher organisms, Nipped-B and cohesin regulate gene expression and development by unknown mechanisms. Using chromatin immunoprecipitation, we find that Nipped-B and cohesin bind to the same sites throughout the entire non-repetitive Drosophila genome. They preferentially bind transcribed regions and overlap with RNA polymerase II. This contrasts sharply with yeast, where cohesin binds almost exclusively between genes. Differences in cohesin and Nipped-B binding between Drosophila cell lines often correlate with differences in gene expression. For example, cohesin and Nipped-B bind the Abd-B homeobox gene in cells in which it is transcribed, but not in cells in which it is silenced. They bind to the Abd-B transcription unit and downstream regulatory region and thus could regulate both transcriptional elongation and activation. We posit that transcription facilitates cohesin binding, perhaps by unfolding chromatin, and that Nipped-B then regulates gene expression by controlling cohesin dynamics. These mechanisms are likely involved in the etiology of Cornelia de Lange syndrome, in which mutation of one copy of the NIPBL gene encoding the human Nipped-B ortholog causes diverse structural and mental birth defects.

Expected rates and modes of evolution of enhancer sequences.

The evolution of new functions takes place partially through changes in the way transcription is controlled. Transcriptional control is brought about by the interactions of transcription factors with short target motifs in the DNAs of promoters and enhancers. One way in which changes in gene expression can evolve is through the acquisition of new transcription factor targets in enhancer sequences. Since such target sites are simple, they can be produced rapidly from random DNA by mutation and selection. Here we consider a population of organisms that finds itself in an ecological situation where bringing a particular target gene under the control of a particular transcription factor would be favored by natural selection. What will be the time required for such a process, as a function of the selection for the new target, the mutation rate, and the population size? The starting sequences considered are either real enhancers from the Drosophila melanogaster genome, or randomized versions of these. We find that the time required to find binding sites is strongly dependent on the existence in the starting sequence of sites that differ from binding sites by single substitutions (presites). The process of converting presites to binding sites is driven by natural selection, and thus the time required typically reduces with the strength of selection. However, if there is a strongly distorted G:C ratio in the starting sequence, presites will typically be absent, and the finding of binding sites will be preceded by a long time period of neutral evolution, however strong is the selection favoring sites. The positions of presites largely determine where binding sites will evolve. One result of this is that any incremental selective benefits that result from the relative positioning of sites have a surprisingly small impact on the final binding-site positions.

Evolution of developmental genes: molecular microevolution of enhancer sequences at the Ubx locus in Drosophila and its impact on developmental phenotypes.

Homeotic genes, which function to specify segment identity along the anterior-posterior axis of embryos, are controlled by extensive batteries of enhancer sequences. We have investigated patterns of interspecific and intraspecific molecular variation in three enhancers of the Ultrabithorax (Ubx) locus, which are bx-32.8, pbx32.7, and bxd4.1, from the Drosophila melanogaster species group. These enhancer sequences control Ubx expression by binding to multiple transcription factors encoded by gap, pair-rule, and dorsoventrally expressed genes. Sequence comparisons reveal purifying selection acting on all three enhancers, both in bases binding transcription factors and in bases whose functions are as yet unknown. Neutrality tests largely fail to reject a neutral evolution model. However, using a matrix similarity value to reflect the binding affinity of the protein-binding sites, interspecific and intraspecific variation that may have potential to affect the binding affinity of the sequences homologous to those binding transcription factors in D. melanogaster are discovered, suggesting evolutionary flexibility in the way in which these sequences function in the control of development. As a means of measuring the impact of intraspecific variation on observable phenotypes, we have induced Ubx mutant phenocopies with embryonic ether treatment, and find strong and highly significant variation between D. melanogaster strains in their phenocopy frequencies. This variation shows no significant correlation with the strengths of the mutant phenotypes when the strains are heterozygous with a Ubx null mutation. Estimated phylogenetic trees have been constructed for the three enhancer regions investigated. Neither of the two phenotypic traits investigated shows any significant associations with the phylogeny of any of the three enhancers.