TY - JOUR T1 - NanoCAGE-XL and CapFilter: an approach to genome wide identification of high confidence transcription start sites. JF - BMC Genomics Y1 - 2015 A1 - Cumbie, Jason S A1 - Ivanchenko, Maria G A1 - Megraw, Molly KW - Arabidopsis KW - Genes, Plant KW - Genome, Plant KW - Nanotechnology KW - Plant Roots KW - Promoter Regions, Genetic KW - Sequence Analysis, DNA KW - Software KW - Transcription Initiation Site AB -

BACKGROUND: Identifying the transcription start sites (TSS) of genes is essential for characterizing promoter regions. Several protocols have been developed to capture the 5' end of transcripts via Cap Analysis of Gene Expression (CAGE) or linker-ligation strategies such as Paired-End Analysis of Transcription Start Sites (PEAT), but often require large amounts of tissue. More recently, nanoCAGE was developed for sequencing on the Illumina GAIIx to overcome these difficulties.

RESULTS: Here we present the first publicly available adaptation of nanoCAGE for sequencing on recent ultra-high throughput platforms such as Illumina HiSeq-2000, and CapFilter, a computational pipeline that greatly increases confidence in TSS identification. We report excellent gene coverage, reproducibility, and precision in transcription start site discovery for samples from Arabidopsis thaliana roots.

CONCLUSION: nanoCAGE-XL together with CapFilter allows for genome wide identification of high confidence transcription start sites in large eukaryotic genomes.

[Link to Protocol, Additional Data, and Supplementary Materials]

[Link to CapFilter Software]

VL - 16 ER - TY - JOUR T1 - TIPR: transcription initiation pattern recognition on a genome scale. JF - Bioinformatics Y1 - 2015 A1 - Morton, Taj A1 - Wong, Weng-Keen A1 - Megraw, Molly KW - Algorithms KW - Genomics KW - Machine Learning KW - Molecular Sequence Annotation KW - Sequence Analysis, DNA KW - Software KW - Transcription Initiation Site KW - Transcription Initiation, Genetic AB -

MOTIVATION: The computational identification of gene transcription start sites (TSSs) can provide insights into the regulation and function of genes without performing expensive experiments, particularly in organisms with incomplete annotations. High-resolution general-purpose TSS prediction remains a challenging problem, with little recent progress on the identification and differentiation of TSSs which are arranged in different spatial patterns along the chromosome.

RESULTS: In this work, we present the Transcription Initiation Pattern Recognizer (TIPR), a sequence-based machine learning model that identifies TSSs with high accuracy and resolution for multiple spatial distribution patterns along the genome, including broadly distributed TSS patterns that have previously been difficult to characterize. TIPR predicts not only the locations of TSSs but also the expected spatial initiation pattern each TSS will form along the chromosome-a novel capability for TSS prediction algorithms. As spatial initiation patterns are associated with spatiotemporal expression patterns and gene function, this capability has the potential to improve gene annotations and our understanding of the regulation of transcription initiation. The high nucleotide resolution of this model locates TSSs within 10 nucleotides or less on average.

CONTACT: megrawm@science.oregonstate.edu.

[Software and Supplementary Materials Link]

VL - 31 IS - 23 ER - TY - JOUR T1 - Paired-end analysis of transcription start sites in Arabidopsis reveals plant-specific promoter signatures. JF - Plant Cell Y1 - 2014 A1 - Morton, Taj A1 - Petricka, Jalean A1 - Corcoran, David L A1 - Li, Song A1 - Winter, Cara M A1 - Carda, Alexa A1 - Benfey, Philip N A1 - Ohler, Uwe A1 - Megraw, Molly KW - Arabidopsis KW - Arabidopsis Proteins KW - Binding Sites KW - Cluster Analysis KW - DNA, Plant KW - Gene Expression Regulation, Plant KW - Genome, Plant KW - Models, Genetic KW - Nucleotide Motifs KW - Plant Roots KW - Promoter Regions, Genetic KW - RNA, Messenger KW - RNA, Plant KW - Sequence Analysis, DNA KW - Species Specificity KW - TATA Box KW - Transcription Factors KW - Transcription Initiation Site AB -

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.

[Link to Additional Data and Supplementary Materials]

VL - 26 IS - 7 ER - TY - JOUR T1 - A transcription factor affinity-based code for mammalian transcription initiation. JF - Genome Res Y1 - 2009 A1 - Megraw, Molly A1 - Pereira, Fernando A1 - Jensen, Shane T A1 - Ohler, Uwe A1 - Hatzigeorgiou, Artemis G KW - Base Composition KW - Databases, Genetic KW - DNA KW - Gene Expression Regulation KW - Genome, Human KW - Humans KW - Promoter Regions, Genetic KW - RNA Polymerase II KW - TATA Box KW - Transcription Factors KW - Transcription Initiation Site KW - Transcription, Genetic AB -

The recent arrival of large-scale cap analysis of gene expression (CAGE) data sets in mammals provides a wealth of quantitative information on coding and noncoding RNA polymerase II transcription start sites (TSS). Genome-wide CAGE studies reveal that a large fraction of TSS exhibit peaks where the vast majority of associated tags map to a particular location ( approximately 45%), whereas other active regions contain a broader distribution of initiation events. The presence of a strong single peak suggests that transcription at these locations may be mediated by position-specific sequence features. We therefore propose a new model for single-peaked TSS based solely on known transcription factors (TFs) and their respective regions of positional enrichment. This probabilistic model leads to near-perfect classification results in cross-validation (auROC = 0.98), and performance in genomic scans demonstrates that TSS prediction with both high accuracy and spatial resolution is achievable for a specific but large subgroup of mammalian promoters. The interpretable model structure suggests a DNA code in which canonical sequence features such as TATA-box, Initiator, and GC content do play a significant role, but many additional TFs show distinct spatial biases with respect to TSS location and are important contributors to the accurate prediction of single-peak transcription initiation sites. The model structure also reveals that CAGE tag clusters distal from annotated gene starts have distinct characteristics compared to those close to gene 5'-ends. Using this high-resolution single-peak model, we predict TSS for approximately 70% of mammalian microRNAs based on currently available data.

[Links to Tools and Supplementary Materials]

VL - 19 IS - 4 ER - TY - JOUR T1 - MicroRNA promoter element discovery in Arabidopsis. JF - RNA Y1 - 2006 A1 - Megraw, Molly A1 - Baev, Vesselin A1 - Rusinov, Ventsislav A1 - Jensen, Shane T A1 - Kalantidis, Kriton A1 - Hatzigeorgiou, Artemis G KW - Arabidopsis KW - Base Sequence KW - Binding Sites KW - Databases, Genetic KW - Feedback, Physiological KW - Genes, Plant KW - MicroRNAs KW - Promoter Regions, Genetic KW - TATA Box KW - Transcription Factors KW - Transcription Initiation Site AB -

In this study we present a method of identifying Arabidopsis miRNA promoter elements using known transcription factor binding motifs. We provide a comparative analysis of the representation of these elements in miRNA promoters, protein-coding gene promoters, and random genomic sequences. We report five transcription factor (TF) binding motifs that show evidence of overrepresentation in miRNA promoter regions relative to the promoter regions of protein-coding genes. This investigation is based on the analysis of 800-nucleotide regions upstream of 63 experimentally verified Transcription Start Sites (TSS) for miRNA primary transcripts in Arabidopsis. While the TATA-box binding motif was also previously reported by Xie and colleagues, the transcription factors AtMYC2, ARF, SORLREP3, and LFY are identified for the first time as overrepresented binding motifs in miRNA promoters.

VL - 12 IS - 9 ER -