3D chromatin connectivity underlies replication origin efficiency in mouse embryonic stem cells.

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.

3D-GNOME 2.0: a three-dimensional genome modeling engine for predicting structural variation-driven alterations of chromatin spatial structure in the human genome.

Structural variants (SVs) that alter DNA sequence emerge as a driving force involved in the reorganisation of DNA spatial folding, thus affecting gene transcription. In this work, we describe an improved version of our integrated web service for structural modeling of three-dimensional genome (3D-GNOME), which now incorporates all types of SVs to model changes to the reference 3D conformation of chromatin. In 3D-GNOME 2.0, the default reference 3D genome structure is generated using ChIA-PET data from the GM12878 cell line and SVs data are sourced from the population-scale catalogue of SVs identified by the 1000 Genomes Consortium. However, users may also submit their own structural data to set a customized reference genome structure, and/or a custom input list of SVs. 3D-GNOME 2.0 provides novel tools to inspect, visualize and compare 3D models for regions that differ in terms of their linear genomic sequence. Contact diagrams are displayed to compare the reference 3D structure with the one altered by SVs. In our opinion, 3D-GNOME 2.0 is a unique online tool for modeling and analyzing conformational changes to the human genome induced by SVs across populations. It can be freely accessed at https://3dgnome.cent.uw.edu.pl/.

A systematic analyses of different bioinformatics pipelines for genomic data and its impact on deep learning models for chromatin loop prediction.

Genomic data analysis has witnessed a surge in complexity and volume, primarily driven by the advent of high-throughput technologies. In particular, studying chromatin loops and structures has become pivotal in understanding gene regulation and genome organization. This systematic investigation explores the realm of specialized bioinformatics pipelines designed specifically for the analysis of chromatin loops and structures. Our investigation incorporates two protein (CTCF and Cohesin) factor-specific loop interaction datasets from six distinct pipelines, amassing a comprehensive collection of 36 diverse datasets. Through a meticulous review of existing literature, we offer a holistic perspective on the methodologies, tools and algorithms underpinning the analysis of this multifaceted genomic feature. We illuminate the vast array of approaches deployed, encompassing pivotal aspects such as data preparation pipeline, preprocessing, statistical features and modelling techniques. Beyond this, we rigorously assess the strengths and limitations inherent in these bioinformatics pipelines, shedding light on the interplay between data quality and the performance of deep learning models, ultimately advancing our comprehension of genomic intricacies.

Super-resolution visualization of chromatin loop folding in human lymphoblastoid cells using interferometric photoactivated localization microscopy.

The three-dimensional (3D) genome structure plays a fundamental role in gene regulation and cellular functions. Recent studies in 3D genomics inferred the very basic functional chromatin folding structures known as chromatin loops, the long-range chromatin interactions that are mediated by protein factors and dynamically extruded by cohesin. We combined the use of FISH staining of a very short (33 kb) chromatin fragment, interferometric photoactivated localization microscopy (iPALM), and traveling salesman problem-based heuristic loop reconstruction algorithm from an image of the one of the strongest CTCF-mediated chromatin loops in human lymphoblastoid cells. In total, we have generated thirteen good quality images of the target chromatin region with 2-22 nm oligo probe localization precision. We visualized the shape of the single chromatin loops with unprecedented genomic resolution which allowed us to study the structural heterogeneity of chromatin looping. We were able to compare the physical distance maps from all reconstructed image-driven computational models with contact frequencies observed by ChIA-PET and Hi-C genomic-driven methods to examine the concordance between single cell imaging and population based genomic data.

Multilayer OMIC Data in Medullary Thyroid Carcinoma Identifies the STAT3 Pathway as a Potential Therapeutic Target in RETM918T Tumors.

Purpose: Medullary thyroid carcinoma (MTC) is a rare disease with few genetic drivers, and the etiology specific to each known susceptibility mutation remains unknown. Exploiting multilayer genomic data, we focused our interest on the role of aberrant DNA methylation in MTC development.Experimental Design: We performed genome-wide DNA methylation profiling assessing more than 27,000 CpGs in the largest MTC series reported to date, comprising 48 molecularly characterized tumors. mRNA and miRNA expression data were available for 33 and 31 tumors, respectively. Two human MTC cell lines and 101 paraffin-embedded MTCs were used for validation.Results: The most distinctive methylome was observed for RETM918T-related tumors. Integration of methylation data with mRNA and miRNA expression data identified genes negatively regulated by promoter methylation. These in silico findings were confirmed in vitro for PLCB2, DKK4, MMP20, and miR-10a, -30a, and -200c. The mutation-specific aberrant methylation of PLCB2, DKK4, and MMP20 was validated in 25 independent MTCs by bisulfite pyrosequencing. The methylome and transcriptome data underscored JAK/Stat pathway involvement in RETM918T MTCs. Immunostaining [immunohistochemistry (IHC)] for the active form of signaling effector STAT3 was performed in a series of 101 MTCs. As expected, positive IHC was associated with RETM918T-bearing tumors (P < 0.02). Pharmacologic inhibition of STAT3 activity increased the sensitivity to vandetanib of the RETM918T-positive MTC cell line, MZ-CRC-1.Conclusions: Multilayer OMIC data analysis uncovered methylation hallmarks in genetically defined MTCs and revealed JAK/Stat signaling effector STAT3 as a potential therapeutic target for the treatment of RETM918T MTCs. Clin Cancer Res; 23(5); 1334-45. ©2016 AACR.

A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells.

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.