Larry D. Mesner

Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Bubble-chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription

We have used a novel bubble-trapping procedure to construct nearly pure and comprehensive human origin libraries from early S- and log-phase HeLa cells, and from log-phase GM06990, a karyotypically normal lymphoblastoid cell line. When hybridized to ENCODE tiling arrays, these libraries illuminated 15.3%, 16.4%, and 21.8% of the genome in the ENCODE regions, respectively. Approximately half of the origin fragments cluster into zones, and their signals are generally higher than those of isolated fragments. Interestingly, initiation events are distributed about equally between genic and intergenic template sequences. While only 13.2% and 14.0% of genes within the ENCODE regions are actually transcribed in HeLa and GM06990 cells, 54.5% and 25.6% of zonal origin fragments overlap transcribed genes, most with activating chromatin marks in their promoters. Our data suggest that cell synchronization activates a significant number of inchoate origins. In addition, HeLa and GM06990 cells activate remarkably different origin populations. Finally, there is only moderate concordance between the log-phase HeLa bubble map and published maps of small nascent strands for this cell line.

A winding road to origin discovery

Studies in our laboratory over the last three decades have shown that the Chinese hamster dihydrofolate reductase (DHFR) origin of replication corresponds to a broad zone of inefficient initiation sites distributed throughout the spacer between the convergently transcribed DHFR and 2BE2121 genes. It is clear from mutational analysis that none of these sites is genetically required for controlling origin activity. However, the integrity of the promoter of the DHFR gene is needed to activate the downstream origin, while the 3′ processing signals prevent invasion and inactivation of the downstream origin by transcription forks. Several other origins in metazoans have been shown to correspond to zones of inefficient sites, while a different subset appears to be similar to the fixed replicators that characterize origins in S. cerevisiae and lower organisms. These observations have led us to suggest a model in which the mammalian genome is dotted with a hierarchy of degenerate, redundant, and inefficient replicators at intervals of a kilobase or less, some of which may have evolved to be highly circumscribed and efficient. The activities of initiation sites are proposed to be largely regulated by local transcription and chromatin architecture. Recently, we and others have devised strategies for identifying active origins on a genome-wide scale in order to define their distributions between fixed and dispersive origin types and to detect relationships among origins, genes, and epigenetic markers. The global pictures emerging are suggestive but far from complete and appear to be plagued by some of the same uncertainties that have led to conflicting views of individual origins in the past (particularly DHFR). In this paper, we will trace the history of origin discovery in mammalian genomes, primarily using the well-studied DHFR origin as a model, because it has been analyzed by nearly every available origin mapping technique in several different laboratories, while many origins have been identified by only one. We will address the strengths and shortcomings of the various methods utilized to identify and characterize origins in complex genomes and will point out how we and others were sometimes led astray by false assumptions and biases, as well as insufficient information. The goal is to help guide future experiments that will provide a truly comprehensive and accurate portrait of origins and their regulation. After all, in the words of George Santayana, “Those who do not learn from history are doomed to repeat it.”

The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.

Bicc1 is a genetic determinant of osteoblastogenesis and bone mineral density.

Patient bone mineral density (BMD) predicts the likelihood of osteoporotic fracture. While substantial progress has been made toward elucidating the genetic determinants of BMD, our understanding of the factors involved remains incomplete. Here, using a systems genetics approach in the mouse, we predicted that bicaudal C homolog 1 (Bicc1), which encodes an RNA-binding protein, is responsible for a BMD quantitative trait locus (QTL) located on murine chromosome 10. Consistent with this prediction, mice heterozygous for a null allele of Bicc1 had low BMD. We used a coexpression network-based approach to determine how Bicc1 influences BMD. Based on this analysis, we inferred that Bicc1 was involved in osteoblast differentiation and that polycystic kidney disease 2 (Pkd2) was a downstream target of Bicc1. Knock down of Bicc1 and Pkd2 impaired osteoblastogenesis, and Bicc1 deficiency-dependent osteoblast defects were rescued by Pkd2 overexpression. Last, in 2 human BMD genome-wide association (GWAS) meta-analyses, we identified SNPs in BICC1 and PKD2 that were associated with BMD. These results, in both mice and humans, identify Bicc1 as a genetic determinant of osteoblastogenesis and BMD and suggest that it does so by regulating Pkd2 transcript levels.

Integrating GWAS and Co-expression Network Data Identifies Bone Mineral Density Genes SPTBN1 and MARK3 and an Osteoblast Functional Module

Bone mineral density (BMD) is a highly heritable predictor of osteoporotic fracture. Genome-wide association studies (GWAS) for BMD have identified dozens of associations; yet, the genes responsible for most associations remain elusive. Here, we used a bone co-expression network to predict causal genes at BMD GWAS loci based on the premise that genes underlying a disease are often functionally related and functionally related genes are often co-expressed. By mapping genes implicated by BMD GWAS onto a bone co-expression network, we predicted and inferred the function of causal genes for 30 of 64 GWAS loci. We experimentally confirmed that two of the genes predicted to be causal, SPTBN1 and MARK3, are potentially responsible for the effects of GWAS loci on chromosomes 2p16.2 and 14q32.32, respectively. This approach provides a roadmap for the dissection of additional BMD GWAS associations. Furthermore, it should be applicable to GWAS data for a wide range of diseases.

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

BackgroundStudies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei.FindingsHere we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates.ConclusionsWe have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.

Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis.

In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation - whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated napthoylated DEAE (BND)-cellulose in high salt conCentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA and are thus suitable for analysis on 2-D gels.

Network Analysis Implicates Alpha-Synuclein ( Snca ) in the Regulation of Ovariectomy-Induced Bone Loss

The postmenopausal period in women is associated with decreased circulating estrogen levels, which accelerate bone loss and increase the risk of fracture. Here, we gained novel insight into the molecular mechanisms mediating bone loss in ovariectomized (OVX) mice, a model of human menopause, using co-expression network analysis. Specifically, we generated a co-expression network consisting of 53 gene modules using expression profiles from intact and OVX mice from a panel of inbred strains. The expression of four modules was altered by OVX, including module 23 whose expression was decreased by OVX across all strains. Module 23 was enriched for genes involved in the response to oxidative stress, a process known to be involved in OVX-induced bone loss. Additionally, module 23 homologs were co-expressed in human bone marrow. Alpha synuclein (Snca) was one of the most highly connected “hub” genes in module 23. We characterized mice deficient in Snca and observed a 40% reduction in OVX-induced bone loss. Furthermore, protection was associated with the altered expression of specific network modules, including module 23. In summary, the results of this study suggest that Snca regulates bone network homeostasis and ovariectomy-induced bone loss.

A Systems-Level Understanding of Cardiovascular Disease through Networks

Tremendous insight into the molecular basis of cardiovascular disease (CVD) has been generated using traditional, reductionist lines of investigation. However, CVD is a multifactorial disorder influenced by the complex interplay of genetic and environmental factors. Network-based approaches are showing great promise in elucidating these interactions and how they influence cellular function and ultimately, CVD. In this chapter, we provide a general overview of network biology, highlighting the power of network theory to address questions regarding the etiology of CVD. We also discuss ways in which network and genetic analysis can be combined to understand how genetic information is integrated, coordinated, and transmitted through cellular networks to impact CVD.