The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkie's association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie's transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie.

The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkie's association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie's transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie.

The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1_ encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.

EGFR and Hippo signaling pathways both control growth and, when dysregulated, contribute to tumorigenesis. We find that EGFR activates the Hippo pathway transcription factor Yorkie and demonstrate that Yorkie is required for the influence of EGFR on cell proliferation in Drosophila. EGFR regulates Yorkie through the influence of its Ras-MAPK branch on the Ajuba LIM protein Jub. Jub is epistatic to EGFR and Ras for Yorkie regulation, Jub is subject to MAPK-dependent phosphorylation, and EGFR-Ras-MAPK signaling enhances Jub binding to the Yorkie kinase Warts and the adaptor protein Salvador. An EGFR-Hippo pathway link is conserved in mammals, as activation of EGFR or RAS activates the Yorkie homolog YAP, and EGFR-RAS-MAPK signaling promotes phosphorylation of the Ajuba family protein WTIP and also enhances WTIP binding to the Warts and Salvador homologs LATS and WW45. Our observations implicate the Hippo pathway in EGFR-mediated tumorigenesis and identify a molecular link between these pathways.

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 base-pairs around the transcription start site, and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts, and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule Förster Resonance Energy Transfer. We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labelling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

The bacterial RNA polymerase holoenzyme consists of a catalytic core enzyme in complex with a sigma factor that is required for promoter-specific transcription initiation. Primary, or housekeeping, sigma factors are responsible for most of the gene expression that occurs during the exponential phase of growth. Primary sigma factors share four regions of conserved sequence, regions 1-4, which have been further subdivided. Many primary sigma factors also contain a nonconserved region (NCR) located between subregions 1.2 and 2.1, which can vary widely in length. Interactions between the NCR of the primary sigma factor of Escherichia coli, sigma(70), and the beta' subunit of the E. coli core enzyme have been shown to influence gene expression, suggesting that the NCR of primary sigma factors represents a potential target for transcription regulation. Here, we report the identification and characterization of a previously undocumented Chlamydia trachomatis transcription factor, designated GrgA (general regulator of genes A). We demonstrate in vitro that GrgA is a DNA-binding protein that can stimulate transcription from a range of sigma(66)-dependent promoters. We further show that GrgA activates transcription by contacting the NCR of the primary sigma factor of C. trachomatis, sigma(66). Our findings suggest GrgA serves as an important regulator of sigma(66)-dependent transcription in C. trachomatis. Furthermore, because GrgA is present only in chlamydiae, our findings highlight how nonconserved regions of the bacterial RNA polymerase can be targets of regulatory factors that are unique to particular organisms.

Plastids and mitochondria, the DNA-containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co-transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild-type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS-92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid-transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.

Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP beta subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP beta interactions demonstrates that the CarD/RNAP beta association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP beta interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP beta interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.

We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25-50 mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 x Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.

Metazoan cells are exposed to a multitude of signals, which they integrate to determine appropriate developmental or physiological responses. Although the Hippo pathway was only discovered recently, and our knowledge of Hippo signal transduction is far from complete, a wealth of interconnections amongst Hippo and other signaling pathways have already been identified. Hippo signaling is particularly important for growth control, and I describe how integration of Hippo and other pathways contributes to regulation of organ growth. Molecular links between Hippo signaling and other signal transduction pathways are summarized. Different types of mechanisms for signal integration are described, and examples of how the complex interconnections between pathways are used to guide developmental and physiological growth responses are discussed. Features of Hippo signaling appear to make it particularly well suited to signal integration, including its responsiveness to cell-cell contact and the mediation of its transcriptional output by transcriptional co-activator proteins that can interact with transcription factors of other pathways.

Successful manipulation of the plastid genome (ptDNA) has been carried out so far only in tissue-culture cells, a limitation that prevents plastid transformation being applied in major agronomic crops. Our objective is to develop a tissue-culture independent protocol that enables manipulation of plastid genomes directly in plants to yield genetically stable seed progeny. We report that in planta excision of a plastid aurea bar gene (bar(au) ) is detectable in greenhouse-grown plants by restoration of the green pigmentation in tobacco leaves. The P1 phage Cre or PhiC31 phage Int site-specific recombinase was delivered on the Agrobacterium T-DNA injected at the axillary bud site, resulting in the excision of the target-site flanked marker gene. Differentiation of new apical meristems was forced by decapitating the plants above the injection site. The new shoot apex that differentiated at the injection site contained bar(au)-free plastids in 30-40% of the injected plants, of which 7% transmitted the bar(au)-free plastids to the seed progeny. The success of obtaining seed with bar(au)-free plastids depended on repeatedly forcing shoot development from axillary buds, a process that was guided by the size and position of green sectors in the leaves. The success of in planta plastid marker excision proved that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA encoding visual markers.

The Fat pathway controls both planar cell polarity (PCP) and organ growth [1, 2]. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP [1], whereas their slope can influence growth [3, 4]. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling [5-7]. Here we show that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, we show that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. We also identify a biochemical mechanism that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat.

Prokaryotic and eukaryotic RNA polymerases can use 2- to approximately 4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

A salient feature of genomes of higher organisms is the birth and death of gene copies. An example is the alpha prolamin genes, which encode seed storage proteins in grasses (Poaceae) and represent a medium-size gene family. To better understand the mechanism, extent, and pace of gene amplification, we compared prolamin gene copies in the genomes of two different tribes in the Panicoideae, the Paniceae and the Andropogoneae. We identified alpha prolamin (setarin) gene copies in the diploid foxtail millet (Paniceae) genome (490 Mb) and compared them with orthologous regions in diploid sorghum (730 Mb) and ancient allotetraploid maize (2,300 Mb) (Andropogoneae). Because sequenced genomes of other subfamilies of Poaceae like rice (389 Mb) (Ehrhartoideae) and Brachypodium (272 Mb) (Pooideae) do not have alpha prolamin genes, their collinear regions can serve as "empty" reference sites. A pattern emerged, where genes were copied and inserted into other chromosomal locations followed by additional tandem duplications (clusters). We observed both recent (species-specific) insertion events and older ones that are shared by these tribes. Many older copies were deleted by unequal crossing over of flanking sequences or damaged by truncations. However, some remain intact with active and inactive alleles. These results indicate that genomes reflect only a snapshot of the gene content of a species and are far less static than conventional genetics has suggested. Nucleotide substitution rates for active alpha prolamins genes were twice as high as for low copy number beta, gamma, and delta prolamin genes, suggesting that gene amplification accelerates the pace of divergence.

During meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly towards the poles is not known. To address these questions, we focused on the chromosomal passenger complex (CPC). We have found that the CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods which dramatically increase the effectiveness of RNAi in the germline, we show that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome bi-orientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data we propose that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the bi-orientation of homologous chromosomes.

During cell division, a bipolar array of microtubules forms the spindle through which the forces required for chromosome segregation are transmitted. Interestingly, the spindle as a whole is stable enough to support these forces even though it is composed of dynamic microtubules, which are constantly undergoing periods of growth and shrinkage. Indeed, the regulation of microtubule dynamics is essential to the integrity and function of the spindle. We show here that a member of an important class of microtubule-depolymerizing kinesins, KLP10A, is required for the proper organization of the acentrosomal meiotic spindle in Drosophila melanogaster oocytes. In the absence of KLP10A, microtubule length is not controlled, resulting in extraordinarily long and disorganized spindles. In addition, the interactions between chromosomes and spindle microtubules are disturbed and can result in the loss of contact. These results indicate that the regulation of microtubule dynamics through KLP10A plays a critical role in restricting the length and maintaining bipolarity of the acentrosomal meiotic spindle and in promoting the contacts that the chromosomes make with microtubules required for meiosis I segregation.

Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end-joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218 that is required for crossover formation and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C) that physically interact and promote homologous recombination. We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.