Fragilities Caused by Dosage Imbalance in Regulation of the Budding Yeast Cell Cycle

Cells can maintain their functions despite fluctuations in intracellular parameters, such as protein activities and gene expression levels. This commonly observed biological property of cells is called robustness. On the other hand, these parameters have different limitations, each reflecting the property of the subsystem containing the parameter. The budding yeast cell cycle is quite fragile upon overexpression of CDC14, but is robust upon overexpression of ESP1. The gene products of both CDC14 and ESP1 are regulated by 1∶1 binding with their inhibitors (Net1 and Pds1), and a mathematical model predicts the extreme fragility of the cell cycle upon overexpression of CDC14 and ESP1 caused by dosage imbalance between these genes. However, it has not been experimentally shown that dosage imbalance causes fragility of the cell cycle. In this study, we measured the quantitative genetic interactions of these genes by performing combinatorial “genetic tug-of-war” experiments. We first showed experimental evidence that dosage imbalance between CDC14 and NET1 causes fragility. We also showed that fragility arising from dosage imbalance between ESP1 and PDS1 is masked by CDH1 and CLB2. The masking function of CLB2 was stabilization of Pds1 by its phosphorylation. We finally modified Chen's model according to our findings. We thus propose that dosage imbalance causes fragility in biological systems

Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria harbor two [NiFe]-type hydrogenases consisting of a large and a small subunit, the Hup- and Hox-hydrogenase, respectively. Insertion of ligands and correct folding of nickel-iron hydrogenases require assistance of accessory maturation proteins (encoded by the <it>hyp</it>-genes). The intergenic region between the structural genes encoding the uptake hydrogenase (<it>hupSL</it>) and the accessory maturation proteins (<it>hyp </it>genes) in the cyanobacteria <it>Nostoc </it>PCC 7120 and <it>N. punctiforme </it>were analysed using molecular methods.</p> <p>Findings</p> <p>The five ORFs, located in between the uptake hydrogenase structural genes and the <it>hyp</it>-genes, can form a transcript with the <it>hyp</it>-genes. An identical genomic localization of these ORFs are found in other filamentous, N₂-fixing cyanobacterial strains. In <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120 the ORFs upstream of the <it>hyp</it>-genes showed similar transcript level profiles as <it>hupS </it>(hydrogenase structural gene), <it>nifD </it>(nitrogenase structural gene), <it>hypC </it>and <it>hypF </it>(accessory hydrogenase maturation genes) after nitrogen depletion. <it>In silico </it>analyzes showed that these ORFs in <it>N. punctiform</it>e harbor the same conserved regions as their homologues in <it>Nostoc </it>PCC 7120 and that they, like their homologues in <it>Nostoc </it>PCC 7120, can be transcribed together with the <it>hyp</it>-genes forming a larger extended <it>hyp-</it>operon. DNA binding studies showed interactions of the transcriptional regulators CalA and CalB to the promoter regions of the extended <it>hyp</it>-operon in <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120.</p> <p>Conclusions</p> <p>The five ORFs upstream of the <it>hyp</it>-genes in several filamentous N₂-fixing cyanobacteria have an identical genomic localization, in between the genes encoding the uptake hydrogenase and the maturation protein genes. In <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120 they are transcribed as one operon and may form transcripts together with the <it>hyp</it>-genes. The expression pattern of the five ORFs within the extended <it>hyp</it>-operon in both <it>Nostoc punctiforme </it>and <it>Nostoc </it>PCC 7120 is similar to the expression patterns of <it>hupS</it>, <it>nifD</it>, <it>hypF </it>and <it>hypC</it>. CalA, a known transcription factor, interacts with the promoter region between <it>hupSL </it>and the five ORFs in the extended <it>hyp</it>-operon in both <it>Nostoc </it>strains.</p

FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication

Differences in Cell Division Rates Drive the Evolution of Terminal Differentiation in Microbes

Multicellular differentiated organisms are composed of cells that begin by developing from a single pluripotent germ cell. In many organisms, a proportion of cells differentiate into specialized somatic cells. Whether these cells lose their pluripotency or are able to reverse their differentiated state has important consequences. Reversibly differentiated cells can potentially regenerate parts of an organism and allow reproduction through fragmentation. In many organisms, however, somatic differentiation is terminal, thereby restricting the developmental paths to reproduction. The reason why terminal differentiation is a common developmental strategy remains unexplored. To understand the conditions that affect the evolution of terminal versus reversible differentiation, we developed a computational model inspired by differentiating cyanobacteria. We simulated the evolution of a population of two cell types –nitrogen fixing or photosynthetic– that exchange resources. The traits that control differentiation rates between cell types are allowed to evolve in the model. Although the topology of cell interactions and differentiation costs play a role in the evolution of terminal and reversible differentiation, the most important factor is the difference in division rates between cell types. Faster dividing cells always evolve to become the germ line. Our results explain why most multicellular differentiated cyanobacteria have terminally differentiated cells, while some have reversibly differentiated cells. We further observed that symbioses involving two cooperating lineages can evolve under conditions where aggregate size, connectivity, and differentiation costs are high. This may explain why plants engage in symbiotic interactions with diazotrophic bacteria

Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1)

Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1) for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles

Copy Number Variation in Patients with Disorders of Sex Development Due to 46,XY Gonadal Dysgenesis

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases

Effects of episodic future thinking and self-projection on children’s prospective memory performance

The present study is the first to investigate the benefits of episodic future thinking (EFT) at encoding on prospective memory (PM) in preschool (age: M = 66.34 months, SD = 3.28)and primary school children (age: M = 88.36 months, SD = 3.12). A second aim was to examine if self-projection influences the possible effects of EFT instructions. PM was assessed using a standard PM paradigm in children with a picture-naming task as the ongoing activity in which the PM task was embedded. Further, two first- and two second-order ToM tasks were administered as indicator of children’s self-projection abilities. Forty-one preschoolers and 39 school-aged children were recruited. Half of the participants in each age group were instructed to use EFT as a strategy to encode the PM task, while the others received standard PM instructions. Results revealed a significant age effect, with school-aged children significantly outperforming preschoolers and a significant effect of encoding condition with overall better performance when receiving EFT instructions compared to the standard encoding condition. Even though the interaction between age group and encoding condition was not significant, planned comparisons revealed first evidence that compared to the younger age group, older children’s PM benefited more from EFT instructions during intention encoding. Moreover, results showed that although self-projection had a significant impact on PM performance, it did not influence the effects of EFT instructions. Overall, results indicate that children can use EFT encoding strategies to improve their PM performance once EFT abilities are sufficiently developed. Further, they provide first evidence that in addition to executive functions, which have already been shown to influence the development of PM across childhood, self-projection seems to be another key mechanism underlying this development

Low Dosage of Histone H4 Leads to Growth Defects and Morphological Changes in Candida albicans

Chromatin function depends on adequate histone stoichiometry. Alterations in histone dosage affect transcription and chromosome segregation, leading to growth defects and aneuploidies. In the fungal pathogen Candida albicans, aneuploidy formation is associated with antifungal resistance and pathogenesis. Histone modifying enzymes and chromatin remodeling proteins are also required for pathogenesis. However, little is known about the mechanisms that generate aneuploidies or about the epigenetic mechanisms that shape the response of C. albicans to the host environment. Here, we determined the impact of histone H4 deficit in the growth and colony morphology of C. albicans. We found that C. albicans requires at least two of the four alleles that code for histone H4 (HHF1 and HHF22) to grow normally. Strains with only one histone H4 allele show a severe growth defect and unstable colony morphology, and produce faster-growing, morphologically stable suppressors. Segmental or whole chromosomal trisomies that increased wild-type histone H4 copy number were the preferred mechanism of suppression. This is the first study of a core nucleosomal histone in C. albicans, and constitutes the prelude to future, more detailed research on the function of histone H4 in this important fungal pathogen

Defining the genotypic and phenotypic spectrum of X-linked MSL3-related disorder

PURPOSE: We sought to delineate the genotypic and phenotypic spectrum of female and male individuals with X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). METHODS: Twenty-five individuals (15 males, 10 females) with causative variants in MSL3 were ascertained through exome or genome sequencing at ten different sequencing centers. RESULTS: We identified multiple variant types in MSL3 (ten nonsense, six frameshift, four splice site, three missense, one in-frame-deletion, one multi-exon deletion), most proven to be de novo, and clustering in the terminal eight exons suggesting that truncating variants in the first five exons might be compensated by an alternative MSL3 transcript. Three-dimensional modeling of missense and splice variants indicated that these have a deleterious effect. The main clinical findings comprised developmental delay and intellectual disability ranging from mild to severe. Autism spectrum disorder, muscle tone abnormalities, and macrocephaly were common as well as hearing impairment and gastrointestinal problems. Hypoplasia of the cerebellar vermis emerged as a consistent magnetic resonance image (MRI) finding. Females and males were equally affected. Using facial analysis technology, a recognizable facial gestalt was determined. CONCLUSION: Our aggregated data illustrate the genotypic and phenotypic spectrum of X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). Our cohort improves the understanding of disease related morbidity and allows us to propose detailed surveillance guidelines for affected individuals