A Shift in Paradigms: Spatial Genomics Approaches to Reveal Single-Cell Principles of Genome Organization.

The genome tridimensional (3D) organization and its role towards the regulation of key cell processes such as transcription is currently a main question in biology. Interphase chromosomes are spatially segregated into "territories," epigenetically-defined large domains of chromatin that interact to form "compartments" with common transcriptional status, and insulator-flanked domains called "topologically associating domains" (TADs). Moreover, chromatin organizes around nuclear structures such as lamina, speckles, or the nucleolus to acquire a higher-order genome organization. Due to recent technological advances, the different hierarchies are being solved. Particularly, advances in microscopy technologies are shedding light on the genome structure at multiple levels. Intriguingly, more and more reports point to high variability and stochasticity at the single-cell level. However, the functional consequences of such variability in genome conformation are still unsolved. Here, I will discuss the implication of the cell-to-cell heterogeneity at the different scales in the context of newly developed imaging approaches, particularly multiplexed Fluorescence in situ hybridization methods that enabled "chromatin tracing." Extensions of these methods are now combining spatial information of dozens to thousands of genomic loci with the localization of nuclear features such as the nucleolus, nuclear speckles, or even histone modifications, creating the fast-moving field of "spatial genomics." As our view of genome organization shifts the focus from ensemble to single-cell, new insights to fundamental questions begin to emerge.

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development.

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.

Impairing activation of phospholipid synthesis by c-Fos interferes with glioblastoma cell proliferation.

Glioblastoma multiforme is the most aggressive type of tumor of the CNS with an overall survival rate of approximately one year. Since this rate has not changed significantly over the last 20 years, the development of new therapeutic strategies for the treatment of these tumors is peremptory. The over-expression of the proto-oncogene c-Fos has been observed in several CNS tumors including glioblastoma multiforme and is usually associated with a poor prognosis. Besides its genomic activity as an AP-1 transcription factor, this protein can also activate phospholipid synthesis by a direct interaction with key enzymes of their metabolic pathways. Given that the amino-terminal portion of c-Fos (c-Fos-NA: amino acids 1-138) associates to but does not activate phospholipid synthesizing enzymes, we evaluated if c-Fos-NA or some shorter derivatives are capable of acting as dominant-negative peptides of the activating capacity of c-Fos. The over-expression or the exogenous administration of c-Fos-NA to cultured T98G cells hampers the interaction between c-Fos and PI4K2A, an enzyme activated by c-Fos. Moreover, it was observed a decrease in tumor cell proliferation rates in vitro and a reduction in tumor growth in vivo when a U87-MG-generated xenograft on nude mice is intratumorally treated with recombinant c-Fos-NA. Importantly, a smaller peptide of 92 amino acids derived from c-Fos-NA retains the capacity to interfere with tumor proliferation in vitro and in vivo. Taken together, these results support the use of the N-terminal portion of c-Fos, or shorter derivatives as a novel therapeutic strategy for the treatment of glioblastoma multiforme.

Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M.

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.

TADs or no TADS: Lessons From Single-cell Imaging of Chromosome Architecture.

Eukaryotic genomes are folded in a hierarchical organization that reflects and possibly regulates their function. Genomewide studies revealed a new level of organization at the kilobase-to-megabase scale termed "topological associating domains" (TADs). TADs are characterized as stable units of chromosome organization that restrict the action of regulatory sequences within one "functional unit." Consequently, TADs are expected to appear as physical entities in most cells. Very recent single-cell studies have shown a notable variability in genome architecture at this scale, raising concerns about this model. Furthermore, the direct and simultaneous observation of genome architecture and transcriptional output showed the lack of stable interactions between regulatory sequences in transcribing cells. These findings are consistent with a large body of evidence suggesting that genome organization is highly heterogeneous at different scales. In this review, we discuss the main strategies employed to image chromatin organization, present the latest state-of-the-art developments, and propose an interpretation reconciling population-based findings with direct single-cell chromatin organization observations. All in all, we propose that TADs are made of multiple, low-frequency, low-affinity interactions that increase the probability, but are not deterministic, of regulatory interactions.

Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms.

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.

Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions.

At the kilo- to megabase pair scales, eukaryotic genomes are partitioned into self-interacting modules or topologically associated domains (TADs) that associate to form nuclear compartments. Here, we combine high-content super-resolution microscopies with state-of-the-art DNA-labeling methods to reveal the variability in the multiscale organization of the Drosophila genome. We find that association frequencies within TADs and between TAD borders are below ~10%, independently of TAD size, epigenetic state, or cell type. Critically, despite this large heterogeneity, we are able to visualize nanometer-sized epigenetic domains at the single-cell level. In addition, absolute contact frequencies within and between TADs are to a large extent defined by genomic distance, higher-order chromosome architecture, and epigenetic identity. We propose that TADs and compartments are organized by multiple, small-frequency, yet specific interactions that are regulated by epigenetics and transcriptional state.

The Catalytic Efficiency of Lipin 1ß Increases by Physically Interacting with the Proto-oncoprotein c-Fos.

Phosphatidic acid (PA) is a central precursor for membrane phospholipid biosynthesis. The lipin family is a magnesium-dependent type I PA phosphatase involved in de novo synthesis of neutral lipids and phospholipids. The regulation of lipin activity may govern the pathways by which these lipids are synthesized and control the cellular levels of important signaling lipids. Moreover, the proto-oncoprotein c-Fos has an emerging role in glycerolipid synthesis regulation; by interacting with key synthesizing enzymes it is able to increase overall phospho- and glycolipid synthesis. We studied the lipin 1ß enzyme activity in a cell-free system using PA/Triton X-100 mixed micelles as substrate, analyzing it in the presence/absence of c-Fos. We found that lipin 1ß kcat value increases around 40% in the presence of c-Fos, with no change in the lipin 1ß affinity for the PA/Triton X-100 mixed micelles. We also probed a physical interaction between both proteins. Although the c-Fos domain involved in lipin activation is its basic domain, the interaction domain is mapped to the N-terminal c-Fos. In conclusion, we provide evidence for a novel positive regulator of lipin 1ß PA phosphatase activity that is not achieved via altering its subcellular localization or affinity for membranes but rather through directly increasing its catalytic efficiency.

c-Fos: an AP-1 transcription factor with an additional cytoplasmic, non-genomic lipid synthesis activation capacity.

The mechanisms that co-ordinately activate lipid synthesis when high rates of membrane biogenesis are needed to support cell growth are largely unknown. c-Fos, a well known AP-1 transcription factor, has emerged as a unique protein with the capacity to associate to specific enzymes of the pathway of synthesis of phospholipids at the endoplasmic reticulum and activate their synthesis to accompany genomic decisions of growth. Herein, we discuss this cytoplasmic, non-genomic effect of c-Fos in the context of other mechanisms that have been proposed to regulate lipid synthesis.

Mechanistic insights into the nongenomic regulation of phospholipid synthesizing enzymes.

Lipid synthesis is a complex process regulated at multiple levels. Here, we will discuss nongenomic regulatory mechanisms, particularly the activation and/or recruitment of key enzymes to membranes. The phospholipid synthesis enzymes Lipin and CTP:phosphocholine cytidylyltransferase are taken as examples of these mechanisms that are mediated by posttranslational modifications or by an intrinsic property of the enzyme that senses lipid composition. In addition, special emphasis will be put on another relevant non genomic lipid synthesis regulation mechanism that is dependent on c-Fos, a protein that has deserved less attention so far. This latter regulatory mechanism is emerging as an important determinant for processes that require high rates of lipid synthesis such as those of growth and proliferation.

c-Fos activates and physically interacts with specific enzymes of the pathway of synthesis of polyphosphoinositides.

The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II ß was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.