Computational models for large-scale simulations of facilitated diffusion

The binding of site-specific transcription factors to their genomic target sites is a key step in gene regulation. While the genome is huge, transcription factors belong to the least abundant protein classes in the cell. It is therefore fascinating how short the time frame is that they require to home in on their target sites. The underlying search mechanism is called facilitated diffusion and assumes a combination of three-dimensional diffusion in the space around the DNA combined with one-dimensional random walk on it. In this review, we present the current understanding of the facilitated diffusion mechanism and identify questions that lack a clear or detailed answer. One way to investigate these questions is through stochastic simulation and, in this manuscript, we support the idea that such simulations are able to address them. Finally, we review which biological parameters need to be included in such computational models in order to obtain a detailed representation of the actual process. © 2012 The Royal Society of Chemistry

An integrated clinical program and crowdsourcing strategy for genomic sequencing and Mendelian disease gene discovery.

Despite major progress in defining the genetic basis of Mendelian disorders, the molecular etiology of many cases remains unknown. Patients with these undiagnosed disorders often have complex presentations and require treatment by multiple health care specialists. Here, we describe an integrated clinical diagnostic and research program using whole-exome and whole-genome sequencing (WES/WGS) for Mendelian disease gene discovery. This program employs specific case ascertainment parameters, a WES/WGS computational analysis pipeline that is optimized for Mendelian disease gene discovery with variant callers tuned to specific inheritance modes, an interdisciplinary crowdsourcing strategy for genomic sequence analysis, matchmaking for additional cases, and integration of the findings regarding gene causality with the clinical management plan. The interdisciplinary gene discovery team includes clinical, computational, and experimental biomedical specialists who interact to identify the genetic etiology of the disease, and when so warranted, to devise improved or novel treatments for affected patients. This program effectively integrates the clinical and research missions of an academic medical center and affords both diagnostic and therapeutic options for patients suffering from genetic disease. It may therefore be germane to other academic medical institutions engaged in implementing genomic medicine programs

Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

Inherited CHST11/MIR3922 deletion is associated with a novel recessive syndrome presenting with skeletal malformation and malignant lymphoproliferative disease

Glycosaminoglycans (GAGs) such as chondroitin are ubiquitous disaccharide carbohydrate chains that contribute to the formation and function of proteoglycans at the cell membrane and in the extracellular matrix. Although GAG-modifying enzymes are required for diverse cellular functions, the role of these proteins in human development and disease is less well understood. Here, we describe two sisters out of seven siblings affected by congenital limb malformation and malignant lymphoproliferative disease. Using Whole-Genome Sequencing (WGS), we identified in the proband deletion of a 55 kb region within chromosome 12q23 that encompasses part of CHST11 (encoding chondroitin-4-sulfotransferase 1) and an embedded microRNA (MIR3922). The deletion was homozygous in the proband but not in each of three unaffected siblings. Genotyping data from the 1000 Genomes Project suggest that deletions inclusive of both CHST11 and MIR3922 are rare events. Given that CHST11 deficiency causes severe chondrodysplasia in mice that is similar to human limb malformation, these results underscore the importance of chondroitin modification in normal skeletal development. Our findings also potentially reveal an unexpected role for CHST11 and/or MIR3922 as tumor suppressors whose disruption may contribute to malignant lymphoproliferative disease

Variants in STXBP3 are Associated with Very Early Onset Inflammatory Bowel Disease, Bilateral Sensorineural Hearing Loss and Immune Dysregulation

Background and aims: Very early onset inflammatory bowel disease [VEOIBD] is characterized by intestinal inflammation affecting infants and children less than 6 years of age. To date, over 60 monogenic aetiologies of VEOIBD have been identified, many characterized by highly penetrant recessive or dominant variants in underlying immune and/or epithelial pathways. We sought to identify the genetic cause of VEOIBD in a subset of patients with a unique clinical presentation. Methods: Whole exome sequencing was performed on five families with ten patients who presented with a similar constellation of symptoms including medically refractory infantile-onset IBD, bilateral sensorineural hearing loss and, in the majority, recurrent infections. Genetic aetiologies of VEOIBD were assessed and Sanger sequencing was performed to confirm novel genetic findings. Western analysis on peripheral blood mononuclear cells and functional studies with epithelial cell lines were employed. Results: In each of the ten patients, we identified damaging heterozygous or biallelic variants in the Syntaxin-Binding Protein 3 gene [STXBP3], a protein known to regulate intracellular vesicular trafficking in the syntaxin-binding protein family of molecules, but not associated to date with either VEOIBD or sensorineural hearing loss. These mutations interfere with either intron splicing or protein stability and lead to reduced STXBP3 protein expression. Knock-down of STXBP3 in CaCo2 cells resulted in defects in cell polarity. Conclusion: Overall, we describe a novel genetic syndrome and identify a critical role for STXBP3 in VEOIBD, sensorineural hearing loss and immune dysregulation.info:eu-repo/semantics/publishedVersio

The Effect of Macromolecular Crowding, Ionic Strength and Calcium Binding on Calmodulin Dynamics

The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells.Comment: Accepted to PLoS Comp Biol, 201

WGS-based telomere length analysis in Dutch family trios implicates stronger maternal inheritance and a role for RRM1 gene

Telomere length (TL) regulation is an important factor in ageing, reproduction and cancer development. Genetic, hereditary and environmental factors regulating TL are currently widely investigated, however, their relative contribution to TL variability is still understudied. We have used whole genome sequencing data of 250 family trios from the Genome of the Netherlands project to perform computational measurement of TL and a series of regression and genome-wide association analyses to reveal TL inheritance patterns and associated genetic factors. Our results confirm that TL is a largely heritable trait, primarily with mother’s, and, to a lesser extent, with father’s TL having the strongest influence on the offspring. In this cohort, mother’s, but not father’s age at conception was positively linked to offspring TL. Age-related TL attrition of 40 bp/year had relatively small influence on TL variability. Finally, we have identified TL-associated variations in ribonuclease reductase catalytic subunit M1 (RRM1 gene), which is known to regulate telomere maintenance in yeast. We also highlight the importance of multivariate approach and the limitations of existing tools for the analysis of TL as a polygenic heritable quantitative trait

Probing and quantifying DNA–protein interactions with asymmetrical flow field-flow fractionation

Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems

A framework for the detection of de novo mutations in family-based sequencing data

Germline mutation detection from human DNA sequence data is challenging due to the rarity of such events relative to the intrinsic error rates of sequencing technologies and the uneven coverage across the genome. We developed PhaseByTransmission (PBT) to identify de novo single nucleotide variants and short insertions and deletions (indels) from sequence data collected in parent-offspring trios. We compute the joint probability of the data given the genotype likelihoods in the individual family members, the known familial relationships and a prior probability for the mutation rate. Candidate de novo mutations (DNMs) are reported along with their posterior probability, providing a systematic way to prioritize them for validation. Our tool is integrated in the Genome Analysis Toolkit and can be used together with the ReadBackedPhasing module to infer the parental origin of DNMs based on phase-informative reads. Using simulated data, we show that PBT outperforms existing tools, especially in low coverage data and on the X chromosome. We further show that PBT displays high validation rates on empirical parent-offspring sequencing data for whole-exome data from 104 trios and X-chromosome data from 249 parent-offspring families. Finally, we demonstrate an association between father's age at conception and the number of DNMs in female offspring's X chromosome, consistent with previous literature reports

Skewed X-inactivation is common in the general female population

X-inactivation is a well-established dosage compensation mechanism ensuring that X-chromosomal genes are expressed at comparable levels in males and females. Skewed X-inactivation is often explained by negative selection of one of the alleles. We demonstrate that imbalanced expression of the paternal and maternal X-chromosomes is common in the general population and that the random nature of the X-inactivation mechanism can be sufficient to explain the imbalance. To this end, we analyzed blood-derived RNA and whole-genome sequencing data from 79 female children and their parents from the Genome of the Netherlands project. We calculated the median ratio of the paternal over total counts at all X-chromosomal heterozygous single-nucleotide variants with coverage ≥10. We identified two individuals where the same X-chromosome was inactivated in all cells. Imbalanced expression of the two X-chromosomes (ratios ≤0.35 or ≥0.65) was observed in nearly 50% of the population. The empirically observed skewing is explained by a theoretical model where X-inactivation takes place in an embryonic stage in which eight cells give rise to the hematopoietic compartment. Genes escaping X-inactivation are expressed from both alleles and therefore demonstrate less skewing than inactivated genes. Using this characteristic, we identified three novel escapee genes (SSR4, REPS2, and SEPT6), but did not find support for many previously reported escapee genes in blood. Our collective data suggest that skewed X-inactivation is common in the general population. This may contribute to manifestation of symptoms in carriers of recessive X-linked disorders. We recommend that X-inactivation results should not be used lightly in the interpretation of X-linked variants