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

<p>Abstract</p> <p>Background</p> <p>Studies 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.</p> <p>Findings</p> <p>Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, <it>Schizosaccharomyces pombe</it>. To preserve <it>in vivo </it>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.</p> <p>Conclusions</p> <p>We 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.</p

Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange

Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70–80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro

Mouse genome-wide association and systems genetics identifies Lhfp as a regulator of bone mass.

Bone mineral density (BMD) is a strong predictor of osteoporotic fracture. It is also one of the most heritable disease-associated quantitative traits. As a result, there has been considerable effort focused on dissecting its genetic basis. Here, we performed a genome-wide association study (GWAS) in a panel of inbred strains to identify associations influencing BMD. This analysis identified a significant (P = 3.1 x 10-12) BMD locus on Chromosome [email protected] Mbp that replicated in two separate inbred strain panels and overlapped a BMD quantitative trait locus (QTL) previously identified in a F2 intercross. The association mapped to a 300 Kbp region containing four genes; Gm2447, Gm20750, Cog6, and Lhfp. Further analysis found that Lipoma HMGIC Fusion Partner (Lhfp) was highly expressed in bone and osteoblasts. Furthermore, its expression was regulated by a local expression QTL (eQTL), which overlapped the BMD association. A co-expression network analysis revealed that Lhfp was strongly connected to genes involved in osteoblast differentiation. To directly evaluate its role in bone, Lhfp deficient mice (Lhfp-/-) were created using CRISPR/Cas9. Consistent with genetic and network predictions, bone marrow stromal cells (BMSCs) from Lhfp-/- mice displayed increased osteogenic differentiation. Lhfp-/- mice also had elevated BMD due to increased cortical bone mass. Lastly, we identified SNPs in human LHFP that were associated (P = 1.2 x 10-5) with heel BMD. In conclusion, we used GWAS and systems genetics to identify Lhfp as a regulator of osteoblast activity and bone mass

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

Specific signals at the 3′ end of the DHFR gene define one boundary of the downstream origin of replication

The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a 55-kb zone of potential initiation sites lying between the convergently transcribed DHFR and 2BE2121 genes. Two subregions within this zone (ori-β/ori-β′ and ori-γ) are preferred. In the DHFR-deficient variant, DR8, which has deleted a 14-kb sequence straddling the 3′ end of the DHFR gene, early-firing origin activity in the downstream ori-β/ori-β′ and ori-γ regions is completely suppressed. We show that the critical deleted sequences reside within a 168-bp segment encompassing the intron 5/exon 6 boundary, exon 6, 54 bp of the 3′ untranslated region (UTR), but not the three natural polyA sites. In wild-type cells, this sequence efficiently arrests transcription in a region a few kilobases downstream, which coincides with the 5′ boundary of the replication initiation zone. In DR8, DHFR-specific transcripts efficiently use an alternative sixth exon (6c) and polyA signals near the middle of the former intergenic region to process primary transcripts. However, transcription proceeds to a position almost 35 kb downstream from these signals, and replication initiation can only be detected beyond this point. When the wild-type 168-bp 3′ element is inserted into DR8 at the same position as alternative exon 6c, transcription is arrested efficiently and initiations occur almost immediately downstream. Thus, the normal 3′ end of the DHFR gene constitutes a boundary element not only for the gene but also for the local origin of replication

The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

The dihydrofolate reductase (DHFR) and 2BE2121 genes in the Chinese hamster are convergently transcribed in late G(1) and early S phase, and bracket an early-firing origin of replication that consists of a 55-kb zone of potential initiation sites. To test whether transcription through the DHFR gene is required to activate this origin in early S phase, we examined the two-dimension (2D) gel patterns of replication intermediates from several variants in which parts or all of the DHFR promoter had been deleted. In those variants in which transcription was undetectable, initiation in the intergenic spacer was markedly suppressed (but not eliminated) in early S phase. Furthermore, replication of the locus required virtually the entire S period, as opposed to the usual 3–4 h. However, restoration of transcription with either the wild-type Chinese hamster promoter or a Drosophila-based construct restored origin activity to the wild-type pattern. Surprisingly, 2D gel analysis of promoterless variants revealed that initiation occurs at a low level in early S phase not only in the intergenic region, but also in the body of the DHFR gene. The latter phenomenon has never been observed in the wild-type locus. These studies suggest that transcription through the gene normally increases the efficiency of origin firing in early S phase, but also suppresses initiation in the body of the gene, thus helping to define the boundaries of the downstream origin

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