karplus@cse.ucsc.edu; http://www.cse.ucsc.edu/karplus

No abstract

Bacterial genomes are organized in terms of structural and functional components. These components include promoters, transcription start and termination sites, open reading frames, regulatory non-coding regions, untranslated regions and transcription units, that together comprise the functional organization of a genome. Here, we use a systems approach that iteratively integrates multiple high-throughput measurements at a genome-scale to identify the organizational structure of the Escherichia coli K-12 MG1655 genome. Integration of the organizational components provides experimentally annotated transcription unit (TU) architecture, including alternative transcription start sites, promoter structures, boundaries and open reading frames. A total of 4,661 TUs were identified, representing an increase of > 530% over current knowledge. This comprehensive TU architecture allows for the elucidation of condition-specific uses of alternative sigma factors at the genome-scale. Furthermore, the TU architecture provides a foundation on which genome-scale transcriptional and translational regulatory networks are based.

Broad-acting transcription factors (TFs) in bacteria form regulons. Here, we present a 4-step method to fully reconstruct the leucineresponsive protein (Lrp) regulon in Escherichia coli K-12 MG 1655 that regulates nitrogen metabolism.Step 1 is composed of obtaining high-resolution ChIP-chip data for Lrp, the RNA polymerase and expression profiles under multiple environmental conditions. We identified 138 unique and reproducible Lrp-binding regions and classified their binding state under different conditions. In the second step, the analysis of these data revealed 6 distinct regulatory modes for individual ORFs. In the third step, we used the functional assignment of the regulated ORFs to reconstruct 4 types of regulatory network motifs around the metabolites that are affected by the corresponding gene products. In the fourth step, we determined how leucine, as a signaling molecule, shifts the regulatory motifs for particular metabolites. The physiological structure that emerges shows the regulatory motifs for different amino acid fall into the traditional classification of amino acid families, thus elucidating the structure and physiological functions of the Lrp-regulon. The same procedure can be applied to other broad-acting TFs, opening the way to full bottom-up reconstruction of the transcriptional regulatory network in bacterial cells. ChIP-chip ͉ transcription factorT ranscriptional regulatory systems often regulate the formation rates and the concentration of small molecules by 2 feedback loops that regulate the transporters and metabolic enzymes. In many cases, these 2 feedback loops are connected by a common transcription factor (TF) that senses the concentration of the small molecule (1). Little is known at present about the transition between the regulatory modes in the feedback loop motifs for global TFs in bacteria. One such transcription factor is the leucineresponsive protein (Lrp), which is a global transcription regulator widely distributed throughout the bacteria including Escherichia coli (2-4). The Lrp regulon includes genes involved in amino acid biosynthesis and degradation, small molecule transport, pili synthesis, and other cellular functions including 1-carbon metabolism (2, 4-6). The regulatory action of Lrp on target genes is often modulated by the binding of the small effector molecule leucine and in effect endows Lrp with the ability to affect transcriptional regulation in all possible ways. That is, upon addition of leucine to the environment, the activity of Lrp can be enhanced, reversed, or unaffected (2, 4, 7). Little is known about in vivo Lrp-binding events at the genome scale in the presence or absence of leucine and the extent to which the different modes of regulation are used for different metabolites. Such information is needed to reconstruct the Lrp regulon and the understanding of nitrogen metabolism.In this study, we applied a systems approach by integrating genome-scale data from chromatin immunoprecipitation followed by microarray hybridization (ChIP-chip) for Lrp and...

Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3’ splice sites (3’SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3’ SSs and propose that cryptic 3’SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3’SSs are present at low frequency (<10%) relative to nearby canonical 3’SSs, we identified ten genes that preferred out-of-frame cryptic 3’SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3’SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation.

Escherichia coli strains that have evolved in the laboratory in response to lactate minimal media show a wide range of different genetic adaptations.

We determined the genome-wide distribution of the nucleoid-associated protein Fis in Escherichia coli using chromatin immunoprecipitation coupled with high-resolution whole genome-tiling microarrays. We identified 894 Fis-associated regions across the E. coli genome. A significant number of these binding sites were found within open reading frames (33%) and between divergently transcribed transcripts (5%). Analysis indicates that A-tracts and AT-tracts are an important signal for preferred Fis-binding sites, and that A6-tracts in particular constitute a high-affinity signal that dictates Fis phasing in stretches of DNA containing multiple and variably spaced A-tracts and AT-tracts. Furthermore, we find evidence for an average of two Fis-binding regions per supercoiling domain in the chromosome of exponentially growing cells. Transcriptome analysis shows that ∼21% of genes are affected by the deletion of fis; however, the changes in magnitude are small. To address the differential Fis bindings under growth environment perturbation, ChIP-chip analysis was performed using cells grown under aerobic and anaerobic growth conditions. Interestingly, the Fis-binding regions are almost identical in aerobic and anaerobic growth conditions—indicating that the E. coli genome topology mediated by Fis is superficially identical in the two conditions. These novel results provide new insight into how Fis modulates DNA topology at a genome scale and thus advance our understanding of the architectural bases of the E. coli nucleoid.

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, wholetranscriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.