Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin

Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization

The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1

Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo

The pipeline project:Pre-publication independent replications of a single laboratory's research pipeline

This crowdsourced project introduces a collaborative approach to improving the reproducibility of scientific research, in which findings are replicated in qualified independent laboratories before (rather than after) they are published. Our goal is to establish a non-adversarial replication process with highly informative final results. To illustrate the Pre-Publication Independent Replication (PPIR) approach, 25 research groups conducted replications of all ten moral judgment effects which the last author and his collaborators had "in the pipeline" as of August 2014. Six findings replicated according to all replication criteria, one finding replicated but with a significantly smaller effect size than the original, one finding replicated consistently in the original culture but not outside of it, and two findings failed to find support. In total, 40% of the original findings failed at least one major replication criterion. Potential ways to implement and incentivize pre-publication independent replication on a large scale are discussed. (C) 2015 The Authors. Published by Elsevier Inc.</p

Data from a pre-publication independent replication initiative examining ten moral judgement effects

We present the data from a crowdsourced project seeking to replicate findings in independent laboratories before (rather than after) they are published. In this Pre-Publication Independent Replication (PPIR) initiative, 25 research groups attempted to replicate 10 moral judgment effects from a single laboratory's research pipeline of unpublished findings. The 10 effects were investigated using online/lab surveys containing psychological manipulations (vignettes) followed by questionnaires. Results revealed a mix of reliable, unreliable, and culturally moderated findings. Unlike any previous replication project, this dataset includes the data from not only the replications but also from the original studies, creating a unique corpus that researchers can use to better understand reproducibility and irreproducibility in science

The pipeline project: Pre-publication independent replications of a single laboratory's research pipeline

This crowdsourced project introduces a collaborative approach to improving the reproducibility of scientific research, in which findings are replicated in qualified independent laboratories before (rather than after) they are published. Our goal is to establish a non-adversarial replication process with highly informative final results. To illustrate the Pre-Publication Independent Replication (PPIR) approach, 25 research groups conducted replications of all ten moral judgment effects which the last author and his collaborators had “in the pipeline” as of August 2014. Six findings replicated according to all replication criteria, one finding replicated but with a significantly smaller effect size than the original, one finding replicated consistently in the original culture but not outside of it, and two findings failed to find support. In total, 40% of the original findings failed at least one major replication criterion. Potential ways to implement and incentivize pre-publication independent replication on a large scale are discussed

Data from a pre-publication independent replication initiative examining ten moral judgement effects

We present the data from a crowdsourced project seeking to replicate findings in independent laboratories before (rather than after) they are published. In this Pre-Publication Independent Replication (PPIR) initiative, 25 research groups attempted to replicate 10 moral judgment effects from a single laboratory's research pipeline of unpublished findings. The 10 effects were investigated using online/lab surveys containing psychological manipulations (vignettes) followed by questionnaires. Results revealed a mix of reliable, unreliable, and culturally moderated findings. Unlike any previous replication project, this dataset includes the data from not only the replications but also from the original studies, creating a unique corpus that researchers can use to better understand reproducibility and irreproducibility in science.Link_to_subscribed_fulltex

The pipeline project: Pre-publication independent replications of a single laboratory's research pipeline

© 2015 The Authors This crowdsourced project introduces a collaborative approach to improving the reproducibility of scientific research, in which findings are replicated in qualified independent laboratories before (rather than after) they are published. Our goal is to establish a non-adversarial replication process with highly informative final results. To illustrate the Pre-Publication Independent Replication (PPIR) approach, 25 research groups conducted replications of all ten moral judgment effects which the last author and his collaborators had âin the pipelineâ as of August 2014. Six findings replicated according to all replication criteria, one finding replicated but with a significantly smaller effect size than the original, one finding replicated consistently in the original culture but not outside of it, and two findings failed to find support. In total, 40% of the original findings failed at least one major replication criterion. Potential ways to implement and incentivize pre-publication independent replication on a large scale are discussed.Link_to_subscribed_fulltex

Local and global analysis of the structure-function relation in chromatin

Every cell in the human body contains the identical hereditary information, encoded in the DNA. The sequence of the human genome was deciphered one decade ago as part of the Human Genome Project (International Human Genome Sequencing Consortium, 2001, 2004; Venter et al., 2001). Remarkably, knowledge of the sequence could not explain the spatial and temporal differences in gene expression, which result in the various tissues composing a human body. It becomes more and more evident that several epigenetic pathways are required to regulate transcription in a time- and context-dependent manner. This thesis investigates one particular mechanism in detail, the functional effect of chromatin architecture on gene regulation. In eukaryotes, DNA is compacted into chromatin, with nucleosomes being the basic packaging unit. In general, chromatin can be classified into two major states, the transcriptionally active euchromatin and the repressed heterochromatin (Woodcock and Ghosh, 2010). Gene expression is closely linked to the structural and spatial arrangement of the chromatin domains in the nucleus, with silent genomic loci tethered to the nuclear periphery and active regions located in the center (Takizawa et al., 2008). To allow access of DNA-binding proteins involved in transcription, replication or repair, the nucleoprotein complex is capable of dynamically changing its accessibility upon physiological cues. Chromatin dynamics are modulated by a number of different factors, e.g. ATP-dependent chromatin remodeling machines, histone chaperones and histone-modifying enzymes (Margueron and Reinberg, 2010). This study aimed to elucidate the structure-function relation of chromatin in mammalian cells on both a global and a local scale. First, chromatin dynamics were analyzed on a genome-wide level. Several high-throughput assays were developed, including formalde-hyde-assisted isolation of regulatory elements (FAIRE)-Seq to monitor the tissue-specific usage of regulatory elements in human blood cells. In addition, micrococcal nuclease (MNase)-Seq was applied to study global re-positioning of nucleosomes and to identify functionally different chromatin domains according to their accessibility. High-throughput sequencing of mono- and dinucleosomal DNA from HeLa cells reveals a condensed chromatin structure at promoters of repressed genes and a defined, open structure around the transcription start sites (TSSs) of highly expressed genes. Further characterization of the determined chromatin domains uncovers correlation of very accessible, open structures with increased occupancy of RNA polymerase (RNAP) II, CTCF and activating histone marks. In contrast, less accessible chromatin is associated with low gene density and expression. Remarkably, the two states differ in nucleosome spacing, directly reflecting accessibility and the respective higher-order organization. Furthermore, MNase-Seq of primary human cells treated with the proinflammatory cytokine TNFα reveals genome-wide re-positioning of nucleosomes. Strikingly, chromatin structures around responsive TSSs but also throughout the gene body open up gradually, as monitored at 10 and 30 min after stimulation. In addition, defined nucleosome positioning and a distinct histone PTM signature are observed at exon/intron junctions, in particular around novel recursive splicing sites. The global opening precedes transcription and is most pronounced at ‘hotspots’ carrying binding sites for NF-κB and associated proteins. Exposure of the binding sites is probably achieved by TNFα-specific chromatin remodeling and occurs predominantly at promoter regions, exons and Alu repeats. Finally, this thesis links altered chromatin structures in Down syndrome patients to upregulated expression of histone clusters and chromatin-associated proteins. In a second chapter, this thesis focuses on the local chromatin architecture of ribosomal RNA (rRNA) genes in mouse. Previous studies showed that actively transcribed or poised rRNA genes form promoter-terminator chromatin loops (Németh et al., 2008). Here, a hamster cell line harboring one repeat of mouse rDNA is introduced as a new model system to elucidate functional dynamics in chromatin looping on a single gene scale. Both promoter and terminator comprise binding sites for the Transcription Termination Factor for RNA polymerase I (TTF-I), therefore, the impact of this protein on chromatin looping is investigated in detail. Notably, TTF-I binds cooperatively to the target site cluster at the rRNA gene terminator in a chromatin-dependent manner. Furthermore, the cluster enhances gene expression directly by increasing the loading of RNAPI and transcription factors to the gene. In addition, the results indicate that TTF-I binding is indispensible for establishing the chromatin loop by mediating promoter-terminator interactions. According to the ribomotor model, the loop structure promotes efficient recycling of polymerases and associated proteins, explaining the observed effects in factor occupancy and gene expression. In summary, the chromatinized TTF-I binding site cluster at the rRNA gene terminator acts as an enhancer element by physically interacting with the promoter, up-regulating transcription levels by transcription factor recruitment and exhibiting a defined chromatin signature linked to active regulatory elements

Circular RNAs: Potential Applications as Therapeutic Targets and Biomarkers in Breast Cancer

Circular RNAs (circRNAs) are a class of non-coding RNAs that form a covalently closed loop. A number of functions and mechanisms of action for circRNAs have been reported, including as miRNA sponge, exerting transcriptional and translational regulation, interacting with proteins, and coding for peptides. CircRNA dysregulation has also been implicated in many cancers, such as breast cancer. Their relatively high stability and presence in bodily fluids makes cancer-associated circRNAs promising candidates as a new biomarker. In this review, we summarize the research undertaken on circRNAs associated with breast cancer, discuss circRNAs as biomarkers, and present circRNA-based therapeutic approaches