Large-scale genetic perturbations reveal regulatory networks and an abundance of gene-specific repressors.

To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.

Functional overlap and regulatory links shape genetic interactions between signaling pathways.

To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control.

The specificity and topology of chromatin interaction pathways in yeast.

Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.

Donor KIR2DL1 Allelic Polymorphism Influences Posthematopoietic Progenitor Cell Transplantation Outcomes in the T Cell Depleted and Reduced Intensity Conditioning Setting.

The majority of established KIR clinical assessment algorithms used for donor selection for hematopoietic progenitor cell transplantation (HPCT) evaluate gene content (presence/absence) of the KIR gene complex. In comparison, relatively little is known about the impact of KIR allelic polymorphism. By analyzing donors of T cell depleted (TcD) reduced intensity conditioning (RIC) HPCT, this study investigated the influence on post-transplant outcome of 2 polymorphic residues of the inhibitory KIR2DL1. The aim of this study was to expand upon existing research into the influence of KIR2DL1 allelic polymorphism upon post-transplant outcome. The effects of allele groups upon transplant outcomes were investigated within a patient cohort using a defined treatment protocol of RIC with TcD. Using phylogenetic data, KIR2DL1 allelic polymorphism was categorized into groups on the basis of variation within codons 114 and 245 (positive or negative for the following groups: KIR2DL1*002/001g, KIR2DL1*003, KIR2DL1*004g) and the identification of null alleles. The influence of these KIR2DL1 allele groups in hematopoietic progenitor cell transplantation (HPCT) donors was assessed in the post-transplant data of 86 acute myelogenous leukemia patients receiving RIC TcD HPCT at a single center. KIR2DL1 allele groups in the donor significantly impacted upon 5-year post-transplant outcomes in RIC TcD HPCT. Donor KIR2DL1*003 presented the greatest influence upon post-transplant outcomes, with KIR2DL1*003 positive donors severely reducing 5-year post-transplant overall survival (OS) compared to those receiving a transplant from a KIR2DL1*003 negative donor (KIR2DL1*003 pos versus neg: 27.0% versus 60.0%, P = .008, pc = 0.024) and disease-free survival (DFS) (KIR2DL1*003 pos versus neg: 23.5% versus 60.0%, P = .004, pc = 0.012), and increasing 5-year relapse incidence (KIR2DL1*003 pos versus neg: 63.9% versus 27.2%, P = .009, pc = 0.027). KIR2DL1*003 homozygous and KIR2DL1*003 heterozygous grafts did not present significantly different post-transplant outcomes. Donors possessing the KIR2DL1*002/001 allele group were found to significantly improve post-transplant outcomes, with donors positive for the KIR2DL1*004 allele group presenting a trend towards improvement. KIR2DL1*002/001 allele group (KIR2DL1*002/001g) positive donors improved 5-year OS (KIR2DL1*002/001g pos versus neg: 56.4% versus 27.2%, P = .009, pc = 0.024) and DFS (KIR2DL1*002/001g pos versus neg: 53.8% versus 25.5%, P = .018, pc = 0.036). KIR2DL1*004 allele group (KIR2DL1*004g) positive donors trended towards improving 5-year OS (KIR2DL1*004g pos versus neg: 53.3% versus 35.5%, P = .097, pc = 0.097) and DFS (KIR2DL1*004g pos versus neg: 50.0% versus 33.9%, P = .121, pc = 0.121), and reducing relapse incidence (KIR2DL1*004g pos versus neg: 33.1% versus 54.0%, P = .079, pc = 0.152). The presented findings suggest donor selection algorithms for TcD RIC HPCT should consider avoiding KIR2DL1*003 positive donors, where possible, and contributes to the mounting evidence that KIR assessment in donor selection algorithms should reflect the conditioning regime protocol used.

Yeast glucose pathways converge on the transcriptional regulation of trehalose biosynthesis.

BACKGROUND: Cellular glucose availability is crucial for the functioning of most biological processes. Our understanding of the glucose regulatory system has been greatly advanced by studying the model organism Saccharomyces cerevisiae, but many aspects of this system remain elusive. To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of the different glucose signalling and metabolic pathways in Saccharomyces cerevisiae using DNA microarrays. RESULTS: In general, the mutations do not induce pathway-specific transcriptional responses. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. Detailed analysis uncovers established and new relationships within and between individual pathways and their members. In contrast to signalling components, metabolic components of the glucose regulatory system are transcriptionally more frequently affected. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system. CONCLUSIONS: The tight interconnection between the different pathways of the glucose regulatory system is reflected by the main transcriptional response observed. Tps2 and Tsl1, two enzymes involved in the biosynthesis of the storage carbohydrate trehalose, are predicted to be the most downstream transcriptional components. Epistasis analysis of tps2Δ double mutants supports this prediction. Although based on transcriptional changes only, these results suggest that all changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis.

Development of an immunogenicity score for HLA-DQ eplets: A conceptual study.

Eplets are defined as distinct amino acid configurations on the surface of HLA molecules. The aim of this study was to estimate the immunogenicity of HLA-DQ eplets in a cohort of 221 pregnancies with HLA-DQ mismatches. We defined the immunogenicity of an eplet by the frequency of antibody responses against it. Around 90% of all listed DQB1 or DQA1 eplets were at least five times mismatched and thus included for the calculation of their immunogenicity. The DQB1 eplets with the five highest immunogenicity scores were 55PP, 52PR, 52PQ, 85VG and 45EV; 25% of all DQB1 eplets were not reacting. The DQA1 eplets with the five highest immunogenicity scores were 25YS, 47QL, 55RR, 187T and 18S; 17% of all DQA1 eplets were not reacting. The immunogenicity score had a slightly higher area under the curve to predict development of child-specific antibodies than various molecular mismatch scores (eg, eplet mismatch load, amino acid mismatch load). Overlapping eplets were identified as a barrier to unambiguously assign the immunogenicity score based on HLA antibody reaction patterns. In this conceptual study, we explored the immunogenicity of HLA-DQ eplets and created a map of potentially immunogenic regions on HLA-DQ molecules, which requires validation in clinical transplant cohorts.

Toward defining the immunogenicity of HLA epitopes: Impact of HLA class I eplets on antibody formation during pregnancy.

Eplets are functional units of structural epitopes on donor HLA, potentially recognized by complementarity-determining regions of the paratope of the recipients' B-cell receptors or antibodies (Ab). Their individual immunogenicity is poorly described, yet this feature would be of clinical importance for pretransplant risk assessment. The aim of this study was to determine the relative immunogenicity of HLA class I eplets in the pregnancy setting, where mismatched eplets are present on paternal HLA antigens of the unborn child. One hundred fifty-nine predominantly Caucasian mothers giving birth at the University Hospital Basel and their first newborns were HLA-typed at high-resolution by next-generation sequencing (NGS) (NGSgo Workflow and NGSengine from GenDx; sequencing with a Miseq from Illumina) and eplets were determined using HLAMatchmaker. HLA class I specific IgG Ab was assessed in maternal sera drawn immediately after full-term delivery, by OneLambda LABScreen single antigen ibeads. The Ab profile was subsequently evaluated for eplet-associated patterns. All 72 currently Ab-verified HLA class I eplets were examined for their immunogenicity according to the frequency of child-specific HLA Ab (CSA) directed against their structures. Four hundred twelve of 477 (86.4%) paternal HLA-A, -B or -C alleles were mismatched. CSA were present in 46 mothers (28.9%), directed against 80 (19.4%) of these mismatches. The 10 most immunogenic eplets were 62GK, 145KHA, 144TKH, 62GE, 107W, 80I, 82LR, 41T, 127K, 45KE with immunogenicity rates between 45.8% and 27.3%. This pregnancy study also identified five non-reactive eplets: 62RR, 76ESN, 80TLR, 156DA, 163RW. Based on our results, immunogenic hot and cold spots on the surface of HLA class I molecules were localized and visualized on 3D models. This study strengthens the presumption that different eplets represent different immunogenic potentials. Validation of these results in the clinical transplant setting is an essential next step in identifying those eplets representing a particularly high-risk potential.

Centromere binding and a conserved role in chromosome stability for SUMO-dependent ubiquitin ligases.

The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs). Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5Δ and slx8Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis.

Modulation of cell motility by spatial repositioning of enzymatic ATP/ADP exchange capacity.

ATP is the "principal energy currency" in metabolism and the most versatile small molecular regulator of cellular activities. Although already much is known about the role of ATP in fundamental processes of living systems, data about its compartmentalization are rather scarce, and we still have only very limited understanding of whether patterns in the distribution of intracellular ATP concentration ("ATP inhomogeneity") do exist and have a regulatory role. Here we report on the analysis of coupling of local ATP supply to regulation of actomyosin behavior, a widespread and dynamic process with conspicuous high ATP dependence, which is central to cell shape changes and cell motility. As an experimental model, we use embryonic fibroblasts from knock-out mice without major ATP-ADP exchange enzymes, in which we (re)introduce the ATP/ADP exchange enzyme adenylate kinase-1 (AK1) and deliberately manipulate its spatial positioning by coupling to different artificial location tags. By transfection-complementation of AK1 variants and comparison with yellow fluorescent protein controls, we found that motility and spreading were enhanced in cells with AK1 with a focal contact guidance tag. Intermediary enhancement was observed in cells with membrane-targeted or cytosolic AK1. Use of a heterodimer-inducing approach for transient translocation of AK1 to focal contacts under conditions of constant global AK1 activity in the cell corroborated these results. Based on our findings with these model systems, we propose that local ATP supply in the cell periphery and "on site" fuelling of the actomyosin machinery, when maintained via enzymes involved in phosphoryl transfer, are codetermining factors in the control of cell motility.

Structure-system correlation identifies a gene regulatory Mediator submodule.

A combination of crystallography, biochemistry, and gene expression analysis identifies the coactivator subcomplex Med8C/18/20 as a functionally distinct submodule of the Mediator head module. Med8C forms a conserved alpha-helix that tethers Med18/20 to the Mediator. Deletion of Med8C in vivo results in dissociation of Med18/20 from Mediator and in loss of transcription activity of extracts. Deletion of med8C, med18, or med20 causes similar changes in the yeast transcriptome, establishing Med8C/18/20 as a predominantly positive, gene-specific submodule required for low transcription levels of nonactivated genes, including conjugation genes. The presented structure-based system perturbation is superior to gene deletion analysis of gene regulation.

Genome-wide location of the coactivator mediator: Binding without activation and transient Cdk8 interaction on DNA.

Mediator is a general coactivator of RNA polymerase II (Pol II) transcription. Genomic location analyses of different Mediator subunits indicate a uniformly composed core complex upstream of active genes but unexpectedly also upstream of inactive genes and on the coding regions of some highly active genes. The repressive Cdk8 submodule is associated with core Mediator at all sites but with a lower degree of occupancy, indicating transient interaction, regardless of promoter activity. This suggests gene-specific regulation of Cdk8 activity, rather than regulated Cdk8 recruitment. Mediator presence is not necessarily linked to transcription. This goes beyond Cdk8-repressed genes, indicating that Mediator can mark some regulatory regions ahead of additional signals. Overlap with intergenic Pol II location in stationary phase points to a role as a binding platform for inactive Pol II during quiescence. These results shed light on Cdk8 repression, suggest additional roles for Mediator, and query models of recruitment-coupled regulation.