Glucocorticoid receptor binds to genomic response elements and regulates gene transcription with cell- and gene-specificity. Within a response element, the precise sequence to which the receptor binds has been implicated in directing its structure and activity. We use NMR chemical shift difference mapping to show that non-specific interactions with particular base positions within the binding sequence, such as those of the “spacer”, affect the conformation of distinct regions of the rat glucocorticoid receptor DNA binding domain. These regions include the DNA-binding surface, the “lever arm” and the dimerization interface, suggesting an allosteric pathway that signals between the DNA binding sequence and the associated dimer partner. Disrupting this path by mutating the dimer interface alters sequence-specific conformations, DNA-binding kinetics and transcriptional activity. Our study demonstrates that glucocorticoid receptor dimer partners collaborate to read DNA shape and to direct sequence specific gene activity.

SUMMARY Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense.

BackgroundGlucocorticoid receptor (GR) is a hormone-activated, DNA-binding transcriptional regulatory factor that controls inflammation, metabolism, stress responses, and other physiological processes. In vitro, GR binds as an inverted dimer to a motif consisting of two imperfectly palindromic 6 bp half sites separated by 3 bp spacers. In vivo, GR employs different patterns of functional surfaces of GR to regulate different target genes. The relationships between GR genomic binding and functional surface utilization have not been defined.ResultsWe find that A477T, a GR mutant that disrupts the dimerization interface, differs from wild-type GRα in binding and regulation of target genes. Genomic regions strongly occupied by A477T are enriched for a novel half site motif. In vitro, GRα binds half sites as a monomer. Through the overlap between GRα- and A477T-bound regions, we identify GRα-bound regions containing only half sites. We further identify GR target genes linked with half sites and not with the full motif.ConclusionsGenomic regions bound by GR differ in underlying DNA sequence motifs and in the GR functional surfaces employed for regulation. Identification of GR binding regions that selectively utilize particular GR surfaces may discriminate sub-motifs, including the half site motif, that favor those surfaces. This approach may contribute to predictive models for GR activity and therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0418-y) contains supplementary material, which is available to authorized users.

By analysing mRNAs with the polymerase chain reaction (PCR) and by studying in vitro generated mutants we have identified an intron in the genome of wheat dwarf virus (WDV), a geminivirus of cereals. Polypeptides whose expression is essential for the replication of the viral DNA have been defined. They are encoded by two distinct overlapping open reading frames (ORFs). The joining of these two ORFs by deletion of the intron as well as the introduction of a frameshift mutation within the intron do not prevent replication of the viral genome in suspension culture cells. In contrast to WDV, the geminiviruses of dicotyledonous plants possess a single continuous ORF, highly homologous to the two individual ones of WDV. We propose that mRNA splicing is a common feature of all geminiviruses of the Gramineae and might contribute to their host class specificity. The existence of a functional intron is a novel finding for the plant viruses.

It is a long-held belief in evolutionary biology that the rate of molecular evolution for a given DNA sequence is inversely related to the level of functional constraint. This belief holds true for the protein-coding homeotic (Hox) genes originally discovered in Drosophila melanogaster. Expression of the Hox genes in Drosophila embryos is essential for body patterning and is controlled by an extensive array of cis-regulatory modules (CRMs). How the regulatory modules functionally evolve in different species is not clear. A comparison of the CRMs for the Abdominal-B gene from different Drosophila species reveals relatively low levels of overall sequence conservation. However, embryonic enhancer CRMs from other Drosophila species direct transgenic reporter gene expression in the same spatial and temporal patterns during development as their D. melanogaster orthologs. Bioinformatic analysis reveals the presence of short conserved sequences within defined CRMs, representing gap and pair-rule transcription factor binding sites. One predicted binding site for the gap transcription factor KRUPPEL in the IAB5 CRM was found to be altered in Superabdominal (Sab) mutations. In Sab mutant flies, the third abdominal segment is transformed into a copy of the fifth abdominal segment. A model for KRUPPEL-mediated repression at this binding site is presented. These findings challenge our current understanding of the relationship between sequence evolution at the molecular level and functional activity of a CRM. While the overall sequence conservation at Drosophila CRMs is not distinctive from neighboring genomic regions, functionally critical transcription factor binding sites within embryonic enhancer CRMs are highly conserved. These results have implications for understanding mechanisms of gene expression during embryonic development, enhancer function, and the molecular evolution of eukaryotic regulatory modules.