Catechol 2,3-dioxygenase-assisted cleavage of aromatics by “anaerobic” termite gut spirochetes and genomic evidence of a complete meta-pathway

The termite hindgut microbial ecosystem functions like a miniature lignocellulose-metabolizing natural bioreactor, has significant implications to nutrient cycling in the terrestrial environment, and represents an array of microbial metabolic diversity. Deciphering the intricacies of this microbial community to obtain as complete a picture as possible of how it functions as a whole, requires a combination of various traditional and cutting-edge bioinformatic, molecular, physiological, and culturing approaches. Isolates from this ecosystem, including Treponema primitia str. ZAS-1 and ZAS-2 as well as T. azotonutricium str. ZAS-9, have been significant resources for better understanding the termite system. While not all functions predicted by the genomes of these three isolates are demonstrated in vitro, these isolates do have the capacity for several metabolisms unique to spirochetes and critical to the termite system’s reliance upon lignocellulose. In this thesis, work culturing, enriching for, and isolating diverse microorganisms from the termite hindgut is discussed. Additionally, strategies of members of the termite hindgut microbial community to defend against O₂-stress and to generate acetate, the “biofuel” of the termite system, are proposed. In particular, catechol 2,3-dioxygenase and other meta-cleavage catabolic pathway genes are described in the “anaerobic” termite hindgut spirochetes T. primitia str. ZAS-1 and ZAS-2, and the first evidence for aromatic ring cleavage in the phylum (division) Spirochetes is also presented. These results suggest that the potential for O₂-dependent, yet nonrespiratory, metabolisms of plant-derived aromatics should be re-evaluated in termite hindgut communities. Potential future work is also illustrated.

Functional genomic studies of the structure and regulation of eukaryotic transcriptomes

The main focus of this thesis is the use of high-throughput sequencing technologies in functional genomics (in particular in the form of ChIP-seq, chromatin immunoprecipitation coupled with sequencing, and RNA-seq) and the study of the structure and regulation of transcriptomes. Some parts of it are of a more methodological nature while others describe the application of these functional genomic tools to address various biological problems. A significant part of the research presented here was conducted as part of the ENCODE (ENCyclopedia Of DNA Elements) Project.

The first part of the thesis focuses on the structure and diversity of the human transcriptome. Chapter 1 contains an analysis of the diversity of the human polyadenylated transcriptome based on RNA-seq data generated for the ENCODE Project. Chapter 2 presents a simulation-based examination of the performance of some of the most popular computational tools used to assemble and quantify transcriptomes. Chapter 3 includes a study of variation in gene expression, alternative splicing and allelic expression bias on the single-cell level and on a genome-wide scale in human lymphoblastoid cells; it also brings forward a number of critical to the practice of single-cell RNA-seq measurements methodological considerations.

The second part presents several studies applying functional genomic tools to the study of the regulatory biology of organellar genomes, primarily in mammals but also in plants. Chapter 5 contains an analysis of the occupancy of the human mitochondrial genome by TFAM, an important structural and regulatory protein in mitochondria, using ChIP-seq. In Chapter 6, the mitochondrial DNA occupancy of the TFB2M transcriptional regulator, the MTERF termination factor, and the mitochondrial RNA and DNA polymerases is characterized. Chapter 7 consists of an investigation into the curious phenomenon of the physical association of nuclear transcription factors with mitochondrial DNA, based on the diverse collections of transcription factor ChIP-seq datasets generated by the ENCODE, mouseENCODE and modENCODE consortia. In Chapter 8 this line of research is further extended to existing publicly available ChIP-seq datasets in plants and their mitochondrial and plastid genomes.

The third part is dedicated to the analytical and experimental practice of ChIP-seq. As part of the ENCODE Project, a set of metrics for assessing the quality of ChIP-seq experiments was developed, and the results of this activity are presented in Chapter 9. These metrics were later used to carry out a global analysis of ChIP-seq quality in the published literature (Chapter 10). In Chapter 11, the development and initial application of an automated robotic ChIP-seq (in which these metrics also played a major role) is presented.

The fourth part presents the results of some additional projects the author has been involved in, including the study of the role of the Piwi protein in the transcriptional regulation of transposon expression in Drosophila (Chapter 12), and the use of single-cell RNA-seq to characterize the heterogeneity of gene expression during cellular reprogramming (Chapter 13).

The last part of the thesis provides a review of the results of the ENCODE Project and the interpretation of the complexity of the biochemical activity exhibited by mammalian genomes that they have revealed (Chapters 15 and 16), an overview of the expected in the near future technical developments and their impact on the field of functional genomics (Chapter 14), and a discussion of some so far insufficiently explored research areas, the future study of which will, in the opinion of the author, provide deep insights into many fundamental but not yet completely answered questions about the transcriptional biology of eukaryotes and its regulation.

The LIN-3 gene of the nematode C. elegans is the vulval-inducing signal

The development of the vulva of the nematode Caenorhabditis elegans is induced by a signal from the anchor cell of the somatic gonad. Activity of the gene lin-3 is required for the Vulval Precursor Cells (VPCs) to assume vulval fates. It is shown here that lin-3 encodes the vulval-inducing signal.

lin-3 was molecularly cloned by transposon-tagging and shown to encode a nematode member ofthe Epidermal Growth Factor (EGF) family. Genetic epistasis experiments indicate that lin-3 acts upstream of let-23, which encodes a homologue of the EGF-Receptor.

lin-3 transgenes that contain multiple copies of wild-type lin-3 genomic DNA clones confer a dominant multivulva phenotype in which up to all six of the VPCs assume vulval fates. The properties of these trans genes suggest that lin-3 can act in the anchor cell to induce vulval fates. Ablation of the gonadal precursors, which prevents the development of the AC, strongly reduces the ability of lin-3 transgenes to stimulate vulval development. A lin-3 recorder transgene that retains the ability to stimulate vulval development is expressed specifically in the anchor cell at the time of vulval induction.

Expression of an obligate secreted form of the EGF domain of Lin-S from a heterologous promoter is sufficient to induce vulval fates in the absence of the normal source of the inductive signal. This result suggests that Lin-S may act as a secreted factor, and that Lin-S may be the sole vulval-inducing signal made by the anchor cell.

lin-3 transgenes can cause adjacent VPCs to assume the 1° vulval fate and thus can override the action of the lateral signal mediated by lin-12 that normally prevents adjacent 1° fates. This indicates that the production of Lin-3 by the anchor cell must be limited to allow the VPCs to assume the proper pattern of fates of so 3° 3° 2° 1° 2° 3°.

Characterization and developmental regulation of a gene expressed specifically in the skeletogenic lineage of the sea urchin embryo

The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.

The important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.

Regulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.