Alterations of redox and iron metabolism accompany the development of HIV latency.

HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac-infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML NBs are hyper-SUMOylated and that PML protein is degraded via the ubiquitin-proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV-1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.

4q-D4Z4 chromatin architecture regulates the transcription of muscle atrophic genes in facioscapulohumeral muscular dystrophy.

Despite increasing insights in genome structure organization, the role of DNA repetitive elements, accounting for more than two thirds of the human genome, remains elusive. Facioscapulohumeral muscular dystrophy (FSHD) is associated with deletion of D4Z4 repeat array below 11 units at 4q35.2. It is known that the deletion alters chromatin structure in cis, leading to gene up-regulation. Here we show a genome-wide role of 4q-D4Z4 array in modulating gene expression via 3D nuclear contacts. We have developed an integrated strategy of 4q-D4Z4-specific 4C-seq and chromatin segmentation analyses, showing that 4q-D4Z4 3D interactome and chromatin states of interacting genes are impaired in FSHD1 condition; in particular, genes that have lost the 4q-D4Z4 interaction and with a more active chromatin state are enriched for muscle atrophy transcriptional signature. Expression level of these genes is restored by the interaction with an ectopic 4q-D4Z4 array, suggesting that the repeat directly modulates the transcription of contacted targets. Of note, the up-regulation of atrophic genes is a common feature of several FSHD1 and FSHD2 patients, indicating that we have identified a core set of deregulated genes involved in FSHD pathophysiology.

Mantra 2.0: an online collaborative resource for drug mode of action and repurposing by network analysis.

SUMMARY: Elucidation of molecular targets of a compound [mode of action (MoA)] and its off-targets is a crucial step in drug development. We developed an online collaborative resource (MANTRA 2.0) that supports this process by exploiting similarities between drug-induced transcriptional profiles. Drugs are organized in a network of nodes (drugs) and edges (similarities) highlighting 'communities' of drugs sharing a similar MoA. A user can upload gene expression profiles before and after drug treatment in one or multiple cell types. An automated processing pipeline transforms the gene expression profiles into a unique drug 'node' embedded in the drug-network. Visual inspection of the neighbouring drugs and communities helps in revealing its MoA and to suggest new applications of known drugs (drug repurposing). MANTRA 2.0 allows storing and sharing user-generated network nodes, thus making MANTRA 2.0 a collaborative ever-growing resource. AVAILABILITY AND IMPLEMENTATION: The web tool is freely available for academic use at http://mantra.tigem.it.

Transcriptional gene network inference from a massive dataset elucidates transcriptome organization and gene function.

We collected a massive and heterogeneous dataset of 20 255 gene expression profiles (GEPs) from a variety of human samples and experimental conditions, as well as 8895 GEPs from mouse samples. We developed a mutual information (MI) reverse-engineering approach to quantify the extent to which the mRNA levels of two genes are related to each other across the dataset. The resulting networks consist of 4 817 629 connections among 20 255 transcripts in human and 14 461 095 connections among 45 101 transcripts in mouse, with a inter-species conservation of 12%. The inferred connections were compared against known interactions to assess their biological significance. We experimentally validated a subset of not previously described protein-protein interactions. We discovered co-expressed modules within the networks, consisting of genes strongly connected to each other, which carry out specific biological functions, and tend to be in physical proximity at the chromatin level in the nucleus. We show that the network can be used to predict the biological function and subcellular localization of a protein, and to elucidate the function of a disease gene. We experimentally verified that granulin precursor (GRN) gene, whose mutations cause frontotemporal lobar degeneration, is involved in lysosome function. We have developed an online tool to explore the human and mouse gene networks.

Segmentation, 3D Reconstruction, and Analysis of PcG Proteins in Fluorescence Microscopy Images in Different Cell Culture Conditions.

Polycomb-group (PcG) of proteins are evolutionarily conserved transcription factors necessary for the regulation of gene expression during the development and the safeguard of cell identity in adulthood. In the nucleus, they form aggregates whose positioning and dimension are fundamental for their function. We present an algorithm, and its MATLAB implementation, based on mathematical methods to detect and analyze PcG proteins in fluorescence cell image z-stacks. Our algorithm provides a method to measure the number, the size, and the relative positioning of the PcG bodies in the nucleus for a better understanding of their spatial distribution, and thus of their role for a correct genome conformation and function.

An Algorithm for the Analysis of the 3D Spatial Organization of the Genome.

We present an algorithm, and its MATLAB implementation, based on mathematical methods to detect and localize 3D multicolor DNA FISH spots in fluorescence cell image z-stacks. This algorithm provides a method to measure the relative positioning of spots in the nucleus and inter-spot distances with the aim to enrich our understanding of the 3D spatial organization of the genome within the cell nucleus.

SAMMY-seq reveals early alteration of heterochromatin and deregulation of bivalent genes in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome is a genetic disease caused by an aberrant form of Lamin A resulting in chromatin structure disruption, in particular by interfering with lamina associated domains. Early molecular alterations involved in chromatin remodeling have not been identified thus far. Here, we present SAMMY-seq, a high-throughput sequencing-based method for genome-wide characterization of heterochromatin dynamics. Using SAMMY-seq, we detect early stage alterations of heterochromatin structure in progeria primary fibroblasts. These structural changes do not disrupt the distribution of H3K9me3 in early passage cells, thus suggesting that chromatin rearrangements precede H3K9me3 alterations described at later passages. On the other hand, we observe an interplay between changes in chromatin accessibility and Polycomb regulation, with site-specific H3K27me3 variations and transcriptional dysregulation of bivalent genes. We conclude that the correct assembly of lamina associated domains is functionally connected to the Polycomb repression and rapidly lost in early molecular events of progeria pathogenesis.

Dysfunctional polycomb transcriptional repression contributes to lamin A/C-dependent muscular dystrophy.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

MLL4-associated condensates counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki syndrome.

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.

An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes.

Beyond its role in providing structure to the nuclear envelope, lamin A/C is involved in transcriptional regulation. However, its cross talk with epigenetic factors--and how this cross talk influences physiological processes--is still unexplored. Key epigenetic regulators of development and differentiation are the Polycomb group (PcG) of proteins, organized in the nucleus as microscopically visible foci. Here, we show that lamin A/C is evolutionarily required for correct PcG protein nuclear compartmentalization. Confocal microscopy supported by new algorithms for image analysis reveals that lamin A/C knock-down leads to PcG protein foci disassembly and PcG protein dispersion. This causes detachment from chromatin and defects in PcG protein-mediated higher-order structures, thereby leading to impaired PcG protein repressive functions. Using myogenic differentiation as a model, we found that reduced levels of lamin A/C at the onset of differentiation led to an anticipation of the myogenic program because of an alteration of PcG protein-mediated transcriptional repression. Collectively, our results indicate that lamin A/C can modulate transcription through the regulation of PcG protein epigenetic factors.

Reverse engineering and analysis of genome-wide gene regulatory networks from gene expression profiles using high-performance computing.

Regulation of gene expression is a carefully regulated phenomenon in the cell. "Reverse-engineering" algorithms try to reconstruct the regulatory interactions among genes from genome-scale measurements of gene expression profiles (microarrays). Mammalian cells express tens of thousands of genes; hence, hundreds of gene expression profiles are necessary in order to have acceptable statistical evidence of interactions between genes. As the number of profiles to be analyzed increases, so do computational costs and memory requirements. In this work, we designed and developed a parallel computing algorithm to reverse-engineer genome-scale gene regulatory networks from thousands of gene expression profiles. The algorithm is based on computing pairwise Mutual Information between each gene-pair. We successfully tested it to reverse engineer the Mus Musculus (mouse) gene regulatory network in liver from gene expression profiles collected from a public repository. A parallel hierarchical clustering algorithm was implemented to discover "communities" within the gene network. Network communities are enriched for genes involved in the same biological functions. The inferred network was used to identify two mitochondrial proteins.

A parallel implementation of the network identification by multiple regression (NIR) algorithm to reverse-engineer regulatory gene networks.

The reverse engineering of gene regulatory networks using gene expression profile data has become crucial to gain novel biological knowledge. Large amounts of data that need to be analyzed are currently being produced due to advances in microarray technologies. Using current reverse engineering algorithms to analyze large data sets can be very computational-intensive. These emerging computational requirements can be met using parallel computing techniques. It has been shown that the Network Identification by multiple Regression (NIR) algorithm performs better than the other ready-to-use reverse engineering software. However it cannot be used with large networks with thousands of nodes--as is the case in biological networks--due to the high time and space complexity. In this work we overcome this limitation by designing and developing a parallel version of the NIR algorithm. The new implementation of the algorithm reaches a very good accuracy even for large gene networks, improving our understanding of the gene regulatory networks that is crucial for a wide range of biomedical applications.