Sebastiaan van Heesch

Distribution and functional impact of DNA copy number variation in the rat

The abundance and dynamics of copy number variants (CNVs) in mammalian genomes poses new challenges in the identification of their impact on natural and disease phenotypes. We used computational and experimental methods to catalog CNVs in rat and found that they share important functional characteristics with those in human. In addition, 113 one-to-one orthologous genes overlap CNVs in both human and rat, 80 of which are implicated in human disease. CNVs are nonrandomly distributed throughout the genome. Chromosome 18 is a cold spot for CNVs as well as evolutionary rearrangements and segmental duplications, suggesting stringent selective mechanisms underlying CNV genesis or maintenance. By exploiting gene expression data available for rat recombinant inbred lines, we established the functional relationship of CNVs underlying 22 expression quantitative trait loci. These characteristics make the rat an excellent model for studying phenotypic effects of structural variation in relation to human complex traits and disease.

Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes.

BackgroundLong noncoding RNAs (lncRNAs) form an abundant class of transcripts, but the function of the majority of them remains elusive. While it has been shown that some lncRNAs are bound by ribosomes, it has also been convincingly demonstrated that these transcripts do not code for proteins. To obtain a comprehensive understanding of the extent to which lncRNAs bind ribosomes, we performed systematic RNA sequencing on ribosome-associated RNA pools obtained through ribosomal fractionation and compared the RNA content with nuclear and (non-ribosome bound) cytosolic RNA pools.ResultsThe RNA composition of the subcellular fractions differs significantly from each other, but lncRNAs are found in all locations. A subset of specific lncRNAs is enriched in the nucleus but surprisingly the majority is enriched in the cytosol and in ribosomal fractions. The ribosomal enriched lncRNAs include H19 and TUG1.ConclusionsMost studies on lncRNAs have focused on the regulatory function of these transcripts in the nucleus. We demonstrate that only a minority of all lncRNAs are nuclear enriched. Our findings suggest that many lncRNAs may have a function in cytoplasmic processes, and in particular in ribosome complexes.

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.

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.

Quantitative and Qualitative Proteome Characteristics Extracted from In-Depth Integrated Genomics and Proteomics Analysis

Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtained peptide evidence for 26,463 rat liver proteins. We validated 1,195 gene predictions, 83 splice events, 126 proteins with nonsynonymous variants, and 20 isoforms with nonsynonymous RNA editing. Quantitative RNA sequencing and proteomics data correlate highly between strains but poorly among each other, indicating extensive nongenetic regulation. Our multilevel analysis identified a genomic variant in the promoter of the most differentially expressed gene Cyp17a1, a previously reported top hit in genome-wide association studies for human hypertension, as a potential contributor to the hypertension phenotype in SHR rats. These results demonstrate the power of and need for integrative analysis for understanding genetic control of molecular dynamics and phenotypic diversity in a system-wide manner.

Nucleosomal DNA binding drives the recognition of H3K36-methylated nucleosomes by the PSIP1-PWWP domain

BackgroundRecognition of histone modifications by specialized protein domains is a key step in the regulation of DNA-mediated processes like gene transcription. The structural basis of these interactions is usually studied using histone peptide models, neglecting the nucleosomal context. Here, we provide the structural and thermodynamic basis for the recognition of H3K36-methylated (H3K36me) nucleosomes by the PSIP1-PWWP domain, based on extensive mutational analysis, advanced nuclear magnetic resonance (NMR), and computational approaches.ResultsThe PSIP1-PWWP domain binds H3K36me3 peptide and DNA with low affinity, through distinct, adjacent binding surfaces. PWWP binding to H3K36me nucleosomes is enhanced approximately 10,000-fold compared to a methylated peptide. Based on mutational analyses and NMR data, we derive a structure of the complex showing that the PWWP domain is bound to H3K36me nucleosomes through simultaneous interactions with both methylated histone tail and nucleosomal DNA.ConclusionConcerted binding to the methylated histone tail and nucleosomal DNA underlies the high- affinity, specific recognition of H3K36me nucleosomes by the PSIP1-PWWP domain. We propose that this bipartite binding mechanism is a distinctive and general property in the recognition of histone modifications close to the nucleosome core.

Titin-truncating variants affect heart function in disease cohorts and the general population

Titin-truncating variants (TTNtv) commonly cause dilated cardiomyopathy (DCM). TTNtv are also encountered in ∼1% of the general population, where they may be silent, perhaps reflecting allelic factors. To better understand TTNtv, we integrated TTN allelic series, cardiac imaging and genomic data in humans and studied rat models with disparate TTNtv. In patients with DCM, TTNtv throughout titin were significantly associated with DCM. Ribosomal profiling in rat showed the translational footprint of premature stop codons in Ttn, TTNtv-position-independent nonsense-mediated degradation of the mutant allele and a signature of perturbed cardiac metabolism. Heart physiology in rats with TTNtv was unremarkable at baseline but became impaired during cardiac stress. In healthy humans, machine-learning-based analysis of high-resolution cardiac imaging showed TTNtv to be associated with eccentric cardiac remodeling. These data show that TTNtv have molecular and physiological effects on the heart across species, with a continuum of expressivity in health and disease.

Systematic biases in DNA copy number originate from isolation procedures

BackgroundThe ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal.ResultsWhile GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing.ConclusionThese results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses.

Homozygous and Heterozygous p53 Knockout Rats Develop Metastasizing Sarcomas with High Frequency

The TP53 tumor suppressor gene is mutated in the majority of human cancers. Inactivation of p53 in a variety of animal models results in early-onset tumorigenesis, reflecting the importance of p53 as a gatekeeper tumor suppressor. We generated a mutant Tp53 allele in the rat using a target-selected mutagenesis approach. Here, we report that homozygosity for this allele results in complete loss of p53 function. Homozygous mutant rats predominantly develop sarcomas with an onset of 4 months of age with a high occurrence of pulmonary metastases. Heterozygous rats develop sarcomas starting at 8 months of age. Molecular analysis revealed that these tumors exhibit a loss-of-heterozygosity of the wild-type Tp53 allele. These unique features make this rat highly complementary to other rodent p53 knockout models and a versatile tool for investigating tumorigenesis processes as well as genotoxic studies.

The PLETHORA gene regulatory network guides growth and cell differentiation in Arabidopsis roots

PLETHORA transcription factor gradients regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation genes. Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis thaliana root, which contrasts with well-described transcription factor gradients in animals specifying distinct cell fates within an essentially static context. To understand the output of the PLT gradient, we studied the gene set transcriptionally controlled by PLTs. Our work reveals how the PLT gradient can regulate cell state by region-specific induction of cell proliferation genes and repression of differentiation. Moreover, PLT targets include major patterning genes and autoregulatory feedback components, enforcing their role as master regulators of organ development.