Cross-regulation and interaction between eukaryotic gene regulatory processes

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references.Regulation of genes is fundamental to all living processes and can be exerted at many sequential steps. We studied several eukaryotic gene regulatory mechanisms with an emphasis on understanding the interplay between regulatory processes on a genome-wide scale. Gene splicing involves the joining of exonic RNA stretches from within a precursor messenger RNA (mRNA). Splicing typically occurs co-transcriptionally as the pre-mRNA is being produced from the DNA. We explored the relationship between the chromatin state of the gene-encoding DNA and the splicing machinery. We found a marked enrichment for nucleosomes at exonic DNA in human T cells, as compared to surrounding introns, an effect mostly explained by the biased nucleotide content of exons. The use of nucleosome positioning information improved splicing simulation models, suggesting nucleosome positioning may help determine cellular splicing patterns. Additionally, we found several histone marks enriched or depleted at exons compared to the background nucleosome levels, indicative of a histone code for splicing. These results connect the chromatin regulation and mRNA splicing processes in a genome-wide fashion. Another pre-mRNA processing step is cleavage and polyadenylation, which determines the 30 end of the mature mRNA. We found that 3P-Seq was able to quantify the levels of 30 end isoforms, in addition to the method's previous use for annotating mRNA 30 ends. Using 3P-Seq and a transcriptional shutoff experiment in mouse fibroblasts, we investigated the e?effect of nuclear alternative 30 end formation on mRNA stability, typically regulated in the cytoplasm. In genes with multiple, tandem 30 untranslated regions (30 UTRs) produced by alternative cleavage and polyadenylation, we found the shorter UTRs were significantly more stable in general than the longer isoforms. This di?difference was in part explained by the loss of cis-regulatory motifs, such as microRNA targets and PUF-binding sites, between the proximal and distal isoforms. Finally, we characterized the small interfering RNAs (siRNAs) produced from heterochromatic, silenced genomic regions in fission yeast. We observed a considerable bias for siRNAs with a 5' U, and used this bias to infer patterns of siRNA biogenesis. Furthermore, comparisons with between wild-type and the Cid14 non-canonical poly(A) polymerase mutant demonstrated that the exosome, the nuclear surveillance and processing complex, is required for RNA homeostasis. In the absence of a fully functional exosome complex, siRNAs are produced to normal exosome targets, including ribosomal and transfer RNAs, indicating these processes may compete for substrates and underscoring the interconnectedness of gene regulatory systems.by Noah Spies.Ph.D

Informatics for RNA sequencing: A web resource for analysis on the cloud

Massively parallel RNA sequencing (RNA-seq) has rapidly become the assay of choice for interrogating RNA transcript abundance and diversity. This article provides a detailed introduction to fundamental RNA-seq molecular biology and informatics concepts. We make available open-access RNA-seq tutorials that cover cloud computing, tool installation, relevant file formats, reference genomes, transcriptome annotations, quality-control strategies, expression, differential expression, and alternative splicing analysis methods. These tutorials and additional training resources are accompanied by complete analysis pipelines and test datasets made available without encumbrance at www.rnaseq.wiki

Die Ausgrabungen unter dem Ria-Felsdach, Salomonen 2014 – 2015

The Excavation at ‘Ria-rockshelter’, Solomon Islands 2014–2015. The dwelling site and burial place ‚Ria-rockshelter‘. The archaeological investigations at the ‘Ria-rockshelter’ within the research project “Settlement History of Melanesia – Prehistory of the Solomon Islands” are conducted in close cooperation with the National Museum Honiara and the Ministry of Culture and Tourism, Solomon Islands. The rock overhang ‘Ria’ is located in the province East Are Are in southern Malaita and was formed by an isolated natural limestone cliff and can serve as a shelter for one to two families. The archaeological potential of the shelter was suspected during a survey in the region in 2011 and finally confirmed through a first test sounding in 2013. The excavations continued in 2014 and 2015 and revealed new results. As primary raw material sources the limestone formations are bearing a quantity of embedded chert nodules waiting to be quarried out in suitable pieces for further working and the final tool production. As secondary raw material source the gravel bed of the small river by passing the ‘Ria-rockshelter’ contains pebbles and boulders made from chert. The raw material was brought as manuports to the site for further usage. The ‘Ria-rockshelter’ shows evidences of human presence in prehistoric times. Excavations under the rockshelter disclosed cultural deposits and features and a large collection of knapped stone tools, shells and faunal remains. In the upper layers besides of several fi re places a pavement made from accurately placed pebbles (hau poro) – all are affected of heat – was unearthed and indicating an earth oven (umu). The set of lithic implements consists of a great variety of flake adzes, serrated and denticulated pieces, unmodified flakes and cores. As ornaments diverse shell pectoral pendants were found. In the rear of the shelter two extended supine burials were discovered under a pebble pavement. During the excavation in 2015 remains of a third individual, an infant, came to light. The human remains can open the possibility for anthropological and genetic analysis. The latest radiocarbon dating from the 2014 and 2015 campaign showed the following results: 527 BP ± 40 (Labcode Erl-19176, carbon from quadrant Q8, layer 2). 601 ± 35 BP (Labcode Erl-20176; carbon from quadrant P8, layer 3). 140 ± 30 BP (Labcode Poz-80516; carbon from quadrant P8, layer 4b). 396 ± 35 BP (Labcode Erl-20178; carbon from quadrant P7, layer 5) and 464 ± 34 BP (Labcode Erl-20177; carbon from quadrant P7, layer 5). Individual I: 502 ± 37 BP (Labcode Erl-20179); Individual II: 460 ± 30 BP (Labcode Beta-433422); Individual III: 640 ± 30 BP (Labcode Beta-451930)

Automated ROI-Based Labeling for Multi-Voxel Magnetic Resonance Spectroscopy Data Using FreeSurfer

Purpose: Advanced analysis methods for multi-voxel magnetic resonance spectroscopy (MRS) are crucial for neurotransmitter quantification, especially for neurotransmitters showing different distributions across tissue types. So far, only a handful of studies have used region of interest (ROI)-based labeling approaches for multi-voxel MRS data. Hence, this study aims to provide an automated ROI-based labeling tool for 3D-multi-voxel MRS data.Methods: MRS data, for automated ROI-based labeling, was acquired in two different spatial resolutions using a spiral-encoded, LASER-localized 3D-MRS imaging sequence with and without MEGA-editing. To calculate the mean metabolite distribution within selected ROIs, masks of individual brain regions were extracted from structural T1-weighted images using FreeSurfer. For reliability testing of automated labeling a comparison to manual labeling and single voxel selection approaches was performed for six different subcortical regions.Results: Automated ROI-based labeling showed high consistency [intra-class correlation coefficient (ICC) > 0.8] for all regions compared to manual labeling. Higher variation was shown when selected voxels, chosen from a multi-voxel grid, uncorrected for voxel composition, were compared to labeling methods using spatial averaging based on anatomical features within gray matter (GM) volumes.Conclusion: We provide an automated ROI-based analysis approach for various types of 3D-multi-voxel MRS data, which dramatically reduces hands-on time compared to manual labeling without any possible inter-rater bias

DGIdb 2.0: Mining clinically relevant drug-gene interactions

The Drug–Gene Interaction Database (DGIdb, www. dgidb.org) is a web resource that consolidates dis-parate data sources describing drug–gene interac-tions and gene druggability. It provides an intuitive graphical user interface and a documented applica-tion programming interface (API) for querying these data. DGIdb was assembled through an extensive manual curation effort, reflecting the combined in-formation of twenty-seven sources. For DGIdb 2.0, substantial updates have been made to increase content and improve its usefulness as a resource for mining clinically actionable drug targets. Specif-ically, nine new sources of drug–gene interactions have been added, including seven resources specifi-cally focused on interactions linked to clinical trials. These additions have more than doubled the over-all count of drug–gene interactions. The total num-ber of druggable gene claims has also increased by 30%. Importantly, a majority of the unrestricted, publicly-accessible sources used in DGIdb are now automatically updated on a weekly basis, providing the most current information for these sources. Fi-nally, a new web view and API have been developed to allow searching for interactions by drug identifiers to complement existing gene-based search function-ality. With these updates, DGIdb represents a com-prehensive and user friendly tool for mining the druggable genome for precision medicine hypothe-sis generation

Impacts of the Northwest Forest Plan on forest composition and bird populations

The Northwest Forest Plan (NWFP) initiated one of the most sweeping changes to forest management in the world, affecting 10 million hectares of federal land. The NWFP is a science-based plan incorporating monitoring and adaptive management and provides a unique opportunity to evaluate the influence of policy. We used >25 years of region-wide bird surveys, forest data, and landownership maps to test this policy's effect on biodiversity. Clearcutting decreased rapidly, and we expected populations of older-forest-associated birds to stabilize on federal land, but to continue declining on private industrial lands where clearcutting continued. In contrast, we expected declines in early-seral-associated species on federal land because of reduced anthropogenic disturbance since the NWFP. Bayesian hierarchical models revealed that bird species' population trends tracked changes in forest composition. However, against our expectations, declines of birds associated with older forests accelerated. These declines are partly explained by losses of older forests due to fire on federal land and continued clearcutting elsewhere. Indeed, the NWFP anticipated that reversing declines of older forests would take time. Overall, the early-seral ecosystem area was stable, but declined in two ecoregions-the Coast Range and Cascades-along with early-seral bird populations. Although the NWFP halted clearcutting on federal land, this has so far been insufficient to reverse declines in older-forest-associated bird populations. These findings underscore the importance of continuing to prioritize older forests under the NWFP and ensuring that the recently proposed creation of early-seral ecosystems does not impede the conservation and development of older-forest structure

CIViCdb 2022: Evolution of an open-access cancer variant interpretation knowledgebase

CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC\u27s functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing \u3e3200 variants in \u3e470 genes from \u3e3100 publications