Paired-End Analysis of Transcription Start Sites in Arabidopsis Reveals Plant-Specific Promoter Signatures

Understanding plant gene promoter architecture has long been a challenge due to the lack of relevant large-scale data sets and analysis methods. Here we present a publicly available, large-scale transcription start site (TSS) dataset in plants using a high-resolution method for analysis of 5’ ends of mRNA transcripts. Our dataset is produced using the Paired-End Analysis of Transcription Start Sites (PEAT) protocol, providing millions of TSS locations from wild-type Col-0 Arabidopsis whole root samples. Using this dataset, we grouped TSS reads into “TSS tag clusters” and categorized clusters into three spatial initiation patterns: narrow peak, broad with peak, and weak peak. We then designed a machine learning model that predicts the presence of TSS tag clusters with outstanding sensitivity and specificity for all three initiation patterns. We used this model to analyze the transcription factor binding site content of promoters exhibiting these initiation patterns. In contrast to the canonical notions of TATA-containing and more broad “TATA-less” promoters, the model shows that, in plants, the vast majority of transcription start sites are TATA-free, and are defined by a large compendium of known DNA sequence binding elements. We present results on the usage of these elements, and provide our Plant PEAT Peaks (3PEAT) model that predicts the presence of TSSs directly from sequence.Keywords: Transcription factor, Start site, Arabidopsis, MicroRNA, Gene regulatio

VIS: the visible imager for Euclid

Euclid-VIS is the large format visible imager for the ESA Euclid space mission in their Cosmic Vision program, scheduled for launch in 2020. Together with the near infrared imaging within the NISP instrument, it forms the basis of the weak lensing measurements of Euclid. VIS will image in a single r+i+z band from 550-900 nm over a field of view of ~0.5 deg2. By combining 4 exposures with a total of 2260 sec, VIS will reach to V=24.5 (10σ) for sources with extent ~0.3 arcsec. The image sampling is 0.1 arcsec. VIS will provide deep imaging with a tightly controlled and stable point spread function (PSF) over a wide survey area of 15000 deg2 to measure the cosmic shear from nearly 1.5 billion galaxies to high levels of accuracy, from which the cosmological parameters will be measured. In addition, VIS will also provide a legacy dataset with an unprecedented combination of spatial resolution, depth and area covering most of the extra-Galactic sky. Here we will present the results of the study carried out by the Euclid Consortium during the period up to the Preliminary Design Review. © (2014) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Genome-wide identification of LEAFY target genes reveals novel insights into reproductive development in Arabidopsis thaliana

The transition from vegetative growth to flower formation is critical for the survival of flowering plants. The plant-specific transcription factor LEAFY (LFY) has central, evolutionarily conserved roles in this process, both in the formation of the first flower during the onset of reproduction and later in flower patterning. The genome-wide binding and expression studies presented here uncover largely distinct functions for direct LFY target genes at these two stages. LFY regulates a larger set of genes during flower development than previously anticipated, including upstream regulators of flowering time and genes in the auxin and gibberellin pathways that promote floral primordium outgrowth. We additionally show that LFY likely plays a role in the timing of floral homeotic gene expression through direct regulation of SEPALLATA3 . At the onset of reproduction, LFY directly represses the expression of genes regulating the plant\u27s response to external stimuli, which may facilitate resource allocation to flower development. Additionally, the computational pipeline for de novo cis motif analysis that we describe here uncovered stage-specific LFY consensus and cofactor motifs that likely contribute to differential LFY activity at both stages

LEAFY and Polar Auxin Transport Coordinately Regulate Arabidopsis Flower Development

The plant specific transcription factor LEAFY (LFY) plays a pivotal role in the developmental switch to floral meristem identity in Arabidopsis. Our recent study revealed that LFY additionally acts downstream of AUXIN RESPONSE FACTOR5/MONOPTEROS to promote flower primordium initiation. LFY also promotes initiation of the floral organ and floral organ identity. To further investigate the interplay between LFY and auxin during flower development, we examined the phenotypic consequence of disrupting polar auxin transport in lfy mutants by genetic means. Plants with compromised LFY activity exhibit increased sensitivity to disruption of polar auxin transport. Compromised polar auxin transport activity in the lfy mutant background resulted in formation of fewer floral organs, abnormal gynoecium development, and fused sepals. In agreement with these observations, expression of the auxin response reporter DR5rev::GFP as well as of the direct LFY target CUP-SHAPED COTYLEDON2 were altered in lfy mutant flowers. We also uncovered reduced expression of ETTIN, a regulator of gynoecium development and a direct LFY target. Our results suggest that LFY and polar auxin transport coordinately modulate flower development by regulating genes required for elaboration of the floral organs

Image files used for RICS and N&B analysis of 35S:GFP and UBQ10:mCherry

Images used for RICS and N&B analysis of 35S:GFP and UBQ10:mCherry lines. Images were acquired using a Zeiss 710 or Zeiss 780 confocal microscope. Images can be viewed using the Zen imaging software. Images were analyzed using the SimFCS software

Data from: Paired-end analysis of transcription start sites in Arabidopsis reveals plant-specific promoter signatures

Understanding plant gene promoter architecture has long been a challenge due to the lack of relevant large-scale data sets and analysis methods. Here, we present a publicly available, large-scale transcription start site (TSS) data set in plants using a high-resolution method for analysis of 5′ ends of mRNA transcripts. Our data set is produced using the paired-end analysis of transcription start sites (PEAT) protocol, providing millions of TSS locations from wild-type Columbia-0 Arabidopsis thaliana whole root samples. Using this data set, we grouped TSS reads into “TSS tag clusters” and categorized clusters into three spatial initiation patterns: narrow peak, broad with peak, and weak peak. We then designed a machine learning model that predicts the presence of TSS tag clusters with outstanding sensitivity and specificity for all three initiation patterns. We used this model to analyze the transcription factor binding site content of promoters exhibiting these initiation patterns. In contrast to the canonical notions of TATA-containing and more broad “TATA-less” promoters, the model shows that, in plants, the vast majority of transcription start sites are TATA free and are defined by a large compendium of known DNA sequence binding elements. We present results on the usage of these elements and provide our Plant PEAT Peaks (3PEAT) model that predicts the presence of TSSs directly from sequence

A Molecular Framework for Auxin-Mediated Initiation of Flower Primordia

SummaryA classical role of the hormone auxin is in the formation of flowers at the periphery of the reproductive shoot apex. Mutants in regulators of polar auxin transport or in the auxin-responsive transcription factor MONOPTEROS (MP) form naked inflorescence “pins” lacking flowers. How auxin maxima and MP direct initiation of flower primordia is poorly understood. Here, we identify three genes whose expression is directly induced by auxin-activated MP that furthermore jointly regulate flower primordium initiation. These three genes encode known regulators of flower development: LEAFY (LFY), which specifies floral fate, and two AINTEGUMENTA-LIKE/PLETHORA transcription factors, key regulators of floral growth. Our study thus reveals a mechanistic link between flower primordium initiation and subsequent steps in flower morphogenesis. Finally, we uncover direct positive feedback from LFY to the auxin pathway. The auxin LFY module we describe may have been recruited during evolution to pattern other plant organ systems

MegrawMollyBotanyPlantPathologyPairedEndAnalysis(Figures1-6).pdf

Understanding plant gene promoter architecture has long been a challenge due to the lack of relevant large-scale data sets and analysis methods. Here we present a publicly available, large-scale transcription start site (TSS) dataset in plants using a high-resolution method for analysis of 5’ ends of mRNA transcripts. Our dataset is produced using the Paired-End Analysis of Transcription Start Sites (PEAT) protocol, providing millions of TSS locations from wild-type Col-0 Arabidopsis whole root samples. Using this dataset, we grouped TSS reads into “TSS tag clusters” and categorized clusters into three spatial initiation patterns: narrow peak, broad with peak, and weak peak. We then designed a machine learning model that predicts the presence of TSS tag clusters with outstanding sensitivity and specificity for all three initiation patterns. We used this model to analyze the transcription factor binding site content of promoters exhibiting these initiation patterns. In contrast to the canonical notions of TATA-containing and more broad “TATA-less” promoters, the model shows that, in plants, the vast majority of transcription start sites are TATA-free, and are defined by a large compendium of known DNA sequence binding elements. We present results on the usage of these elements, and provide our Plant PEAT Peaks (3PEAT) model that predicts the presence of TSSs directly from sequence.Keywords: Start site, Transcription factor, Arabidopsis, MicroRNA, Gene regulationKeywords: Start site, Transcription factor, Arabidopsis, MicroRNA, Gene regulatio

Image files used for RICS and N&B analysis of 35S:GFP and UBQ10:mCherry

Images used for RICS and N&B analysis of 35S:GFP and UBQ10:mCherry lines. Images were acquired using a Zeiss 710 or Zeiss 780 confocal microscope. Images can be viewed using the Zen imaging software. Images were analyzed using the SimFCS software