Obtention of viable cell suspensions from breast cancer tumor biopsies for 3D chromatin conformation and single-cell transcriptome analysis.

Molecular and cellular characterization of tumors is essential due to the complex and heterogeneous nature of cancer. In recent decades, many bioinformatic tools and experimental techniques have been developed to achieve personalized characterization of tumors. However, sample handling continues to be a major challenge as limitations such as prior treatments before sample acquisition, the amount of tissue obtained, transportation, or the inability to process fresh samples pose a hurdle for experimental strategies that require viable cell suspensions.

Gene expression dynamics during temperature-dependent sex determination in a sea turtle.

Fifty years ago, researchers discovered a link between ambient temperature and the sex of turtle embryos. More recently, significant progress has been made in understanding the influence of temperature on freshwater turtles. However, our understanding of the key genetic factors in other turtle groups, such as sea turtles, remains limited.

A novel -regulatory element regulates αD and αA-globin gene expression in chicken erythroid cells.

Background: -regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis.

Methods: Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs.

RNA polymerase II pausing regulates chromatin organization in erythrocytes.

Chicken erythrocytes are nucleated cells often considered to be transcriptionally inactive, although the epigenetic changes and chromatin remodeling that would mediate transcriptional repression and the extent of gene silencing during avian terminal erythroid differentiation are not fully understood. Here, we characterize the changes in gene expression, chromatin accessibility, genome organization and chromatin nuclear disposition during the terminal stages of erythropoiesis in chicken and uncover complex chromatin reorganization at different genomic scales. We observe a robust decrease in transcription in erythrocytes, but a set of genes maintains their expression, including genes involved in RNA polymerase II (Pol II) promoter-proximal pausing.

Chromosome conformation capture technologies as tools to detect structural variations and their repercussion in chromatin 3D configuration.

3D genome organization regulates gene expression in different physiological and pathological contexts. Characterization of chromatin structure at different scales has provided information about how the genome organizes in the nuclear space, from chromosome territories, compartments of euchromatin and heterochromatin, topologically associated domains to punctual chromatin loops between genomic regulatory elements and gene promoters. In recent years, chromosome conformation capture technologies have also been used to characterize structural variations (SVs) in pathological conditions.

Chromatin Structure from Development to Ageing.

Nuclear structure influences genome architecture, which contributes to determine patterns of gene expression. Global changes in chromatin dynamics are essential during development and differentiation, and are one of the hallmarks of ageing. This chapter describes the molecular dynamics of chromatin structure that occur during development and ageing.

Down-Regulation-Resistant STAT4 Risk Haplotype Contributes to Lupus Nephritis Through CD4+ T Cell Interferon-γ Production.

Objective: Variants in STAT4 are associated with systemic lupus erythematosus (SLE) and other autoimmune diseases. We undertook this study to investigate how disease-associated variants affect STAT4 expression, in particular in CD4+ T cells where STAT4 plays an essential role.

Methods: We compared Th1 differentiation between naive CD4+ T cells from healthy donors homozygous for the risk (R/R) or nonrisk (NR/NR) alleles.

In-Nucleus Hi-C in Drosophila Cells.

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene.

Heterochromatin as an Important Driver of Genome Organization.

Heterochromatin is a constituent of eukaryotic genomes with functions spanning from gene expression silencing to constraining DNA replication and repair. Inside the nucleus, heterochromatin segregates spatially from euchromatin and is localized preferentially toward the nuclear periphery and surrounding the nucleolus. Despite being an abundant nuclear compartment, little is known about how heterochromatin regulates and participates in the mechanisms driving genome organization.

In situ dissection of domain boundaries affect genome topology and gene transcription in Drosophila.

Chromosomes are organized into high-frequency chromatin interaction domains called topologically associating domains (TADs), which are separated from each other by domain boundaries. The molecular mechanisms responsible for TAD formation are not yet fully understood. In Drosophila, it has been proposed that transcription is fundamental for TAD organization while the participation of genetic sequences bound by architectural proteins (APs) remains controversial.

RNA Interactions Are Essential for CTCF-Mediated Genome Organization.

The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms by which CTCF dynamically contributes to genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10).

Shaping Up the Embryo: The Role of Genome 3D Organization.

The spatial organization of the chromatinized genome inside the cell nucleus impacts genomic function. In transcription, the hierarchical genome structure creates spatial regulatory landscapes, in which modulating elements like enhancers can contact their target genes and activate their expression, as a result of restricting their exploration to a specific topological neighbourhood. Here we describe exciting recent findings obtained through "C" technologies in pluripotent cells and early embryogenesis and emphasize some of the key unanswered questions arising from them.

Long-Range Enhancer Interactions Are Prevalent in Mouse Embryonic Stem Cells and Are Reorganized upon Pluripotent State Transition.

Transcriptional enhancers, including super-enhancers (SEs), form physical interactions with promoters to regulate cell-type-specific gene expression. SEs are characterized by high transcription factor occupancy and large domains of active chromatin, and they are commonly assigned to target promoters using computational predictions. How promoter-SE interactions change upon cell state transitions, and whether transcription factors maintain SE interactions, have not been reported.

HiCUP: pipeline for mapping and processing Hi-C data.

HiCUP is a pipeline for processing sequence data generated by Hi-C and Capture Hi-C (CHi-C) experiments, which are techniques used to investigate three-dimensional genomic organisation. The pipeline maps data to a specified reference genome and removes artefacts that would otherwise hinder subsequent analysis. HiCUP also produces an easy-to-interpret yet detailed quality control (QC) report that assists in refining experimental protocols for future studies.

Individual and Sequential Chromatin Immunoprecipitation Protocols.

DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene expression making it relevant to determine the profiles of cohabitation of several proteins on the chromatin fiber. Chromatin immunoprecipitation (ChIP) has been broadly used to determine the profile of several histone posttranslational modifications as well as transcription factor occupancy in vivo. However, individual ChIP does not resolve whether the epitope under study is present at the same time on a given genomic location.

Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.

The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks.

3D genome architecture from populations to single cells.

Dominated by microscopy for decades the nuclear genome organization field has recently undergone a dramatic transition fuelled by new next generation sequencing technologies that are beginning to bridge the gap between microscopic observations and molecular scale studies. It is no longer in doubt that the nucleus is spatially compartmentalized and that the genome organization with respect to these compartments is cell type specific. However, it is still unclear if and how this organization contributes to genome function, or whether it is simply a consequence of it.

The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays.