Hi-C/3C-seq Data Analysis for Prokaryotic Genomes with HiC-Pro.

Hi-C and 3C-seq are powerful tools to study the 3D genomes of bacteria and archaea, whose small cell sizes and growth conditions are often intractable to detailed microscopic analysis. However, the circularity of prokaryotic genomes requires a number of tricks for Hi-C/3C-seq data analysis. Here, I provide a practical guide to use the HiC-Pro pipeline for Hi-C/3C-seq data obtained from prokaryotes.

How Do Thermophiles Organize Their Genomes?

All cells must maintain the structural and functional integrity of the genome under a wide range of environments. High temperatures pose a formidable challenge to cells by denaturing the DNA double helix, causing chemical damage to DNA, and increasing the random thermal motion of chromosomes. Thermophiles, predominantly classified as bacteria or archaea, exhibit an exceptional capacity to mitigate these detrimental effects and prosper under extreme thermal conditions, with some species tolerating temperatures higher than 100°C. Their genomes are mainly characterized by the presence of reverse gyrase, a unique topoisomerase that introduces positive supercoils into DNA. This enzyme has been suggested to maintain the genome integrity of thermophiles by limiting DNA melting and mediating DNA repair. Previous studies provided significant insights into the mechanisms by which NAPs, histones, SMC superfamily proteins, and polyamines affect the 3D genomes of thermophiles across different scales. Here, I discuss current knowledge of the genome organization in thermophiles and pertinent research questions for future investigations.

Capturing chromosome conformation in Crenarchaea.

While there is a considerable body of knowledge regarding the molecular and structural biology and biochemistry of archaeal information processing machineries, far less is known about the nature of the substrate for these machineries-the archaeal nucleoid. In this article, we will describe recent advances in our understanding of the three-dimensional organization of the chromosomes of model organisms in the crenarchaeal phylum.

Facultative heterochromatin formation in rDNA is essential for cell survival during nutritional starvation.

During the cellular adaptation to nutrient starvation, cells temporarily decelerate translation processes including ribosomal biogenesis. However, the mechanisms repressing robust gene expression from the ribosomal gene cluster (rDNA) are unclear. Here, we demonstrate that fission yeast cells facing glucose starvation assemble facultative heterochromatin in rDNA leading to its transcriptional repression. Glucose starvation induces quick dissociation of the ATF/CREB-family protein Atf1 from rDNA, where in turn the histone chaperone FACT is recruited to promote H3K9 methylation and heterochromatinization. We also identify the histone acetyltransferase Gcn5 as a repressor of rDNA heterochromatinization in glucose-rich conditions, and this protein dissociates from rDNA upon glucose starvation. Facultative heterochromatin formation in rDNA requires histone deacetylases Clr3 and both the RNAi-dependent and -independent gene silencing pathways. This is essential in adaptation to starvation since mutants lacking heterochromatin formation in rDNA lead to untimely cell death during glucose starvation.

Chromosome conformation capture assay combined with biotin enrichment for hyperthermophilic archaea.

Chromosome organization in archaea has long been enigmatic due, in part, to the typically small cell size of archaea and the extremophilic nature of many of the model archaeal species studies, rendering live-cell imaging technically challenging. To circumvent these problems, we recently applied chromosome conformation capture combined with biotin enrichment and deep sequencing (Hi-C) to members of hyperthermophilic archaeal genus Sulfolobus. Our optimized Hi-C protocol described here permits delineation of how Sulfolobus species organize their chromosomes. For complete details on the use and execution of this protocol, please refer to Takemata et al. (2019).

High-resolution analysis of chromosome conformation in hyperthermophilic archaea.

Chromosome conformation capture (3C) techniques are emerging as promising approaches to study genome organization in Archaea, the least understood domain of life in terms of chromosome biology. Here, we describe a 3C technique combined with deep sequencing for the hyperthermophilic archaeal genus Sulfolobus. Instead of using restriction enzymes compatible with fill-in labeling, this protocol uses the 4-bp blunt cutter AluI to generate high-resolution (up to 2 kb) contact maps from Sulfolobus species. For complete details on the use and execution of this protocol, please refer to Takemata and Bell (2021).

Multi-scale architecture of archaeal chromosomes.

Chromosome conformation capture (3C) technologies have identified topologically associating domains (TADs) and larger A/B compartments as two salient structural features of eukaryotic chromosomes. These structures are sculpted by the combined actions of transcription and structural maintenance of chromosomes (SMC) superfamily proteins. Bacterial chromosomes fold into TAD-like chromosomal interaction domains (CIDs) but do not display A/B compartment-type organization. We reveal that chromosomes of Sulfolobus archaea are organized into CID-like topological domains in addition to previously described larger A/B compartment-type structures. We uncover local rules governing the identity of the topological domains and their boundaries. We also identify long-range loop structures and provide evidence of a hub-like structure that colocalizes genes involved in ribosome biogenesis. In addition to providing high-resolution descriptions of archaeal chromosome architectures, our data provide evidence of multiple modes of organization in prokaryotic chromosomes and yield insights into the evolution of eukaryotic chromosome conformation.

Emerging views of genome organization in Archaea.

Over the past decade, advances in methodologies for the determination of chromosome conformation have provided remarkable insight into the local and higher-order organization of bacterial and eukaryotic chromosomes. Locally folded domains are found in both bacterial and eukaryotic genomes, although they vary in size. Importantly, genomes of metazoans also possess higher-order organization into A- and B-type compartments, regions of transcriptionally active and inactive chromatin, respectively. Until recently, nothing was known about the organization of genomes of organisms in the third domain of life - the archaea. However, despite archaea possessing simple circular genomes that are morphologically reminiscent of those seen in many bacteria, a recent study of archaea of the genus Sulfolobus has revealed that it organizes its genome into large-scale domains. These domains further interact to form defined A- and B-type compartments. The interplay of transcription and localization of a novel structural maintenance of chromosomes (SMC) superfamily protein, termed coalescin, defines compartment identity. In this Review, we discuss the mechanistic and evolutionary implications of these findings.

Physical and Functional Compartmentalization of Archaeal Chromosomes.

The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin."

Role of non-coding RNA transcription around gene regulatory elements in transcription factor recruitment.

Eukaryotic cells produce a variety of non-coding RNAs (ncRNAs), many of which have been shown to play pivotal roles in biological processes such as differentiation, maintenance of pluripotency of stem cells, and cellular response to various stresses. Genome-wide analyses have revealed that many ncRNAs are transcribed around regulatory DNA elements located proximal or distal to gene promoters, but their biological functions are largely unknown. Recently, it has been demonstrated in yeast and mouse that ncRNA transcription around gene promoters and enhancers facilitates DNA binding of transcription factors to their target sites. These results suggest universal roles of promoter/enhancer-associated ncRNAs in the recruitment of transcription factors to their binding sites.

Local potentiation of stress-responsive genes by upstream noncoding transcription.

It has been postulated that a myriad of long noncoding RNAs (lncRNAs) contribute to gene regulation. In fission yeast, glucose starvation triggers lncRNA transcription across promoter regions of stress-responsive genes including fbp1 (fructose-1,6-bisphosphatase1). At the fbp1 promoter, this transcription promotes chromatin remodeling and fbp1 mRNA expression. Here, we demonstrate that such upstream noncoding transcription facilitates promoter association of the stress-responsive transcriptional activator Atf1 at the sites of transcription, leading to activation of the downstream stress genes. Genome-wide analyses revealed that ∼50 Atf1-binding sites show marked decrease in Atf1 occupancy when cells are treated with a transcription inhibitor. Most of these transcription-enhanced Atf1-binding sites are associated with stress-dependent induction of the adjacent mRNAs or lncRNAs, as observed in fbp1 These Atf1-binding sites exhibit low Atf1 occupancy and high histone density in glucose-rich conditions, and undergo dramatic changes in chromatin status after glucose depletion: enhanced Atf1 binding, histone eviction, and histone H3 acetylation. We also found that upstream transcripts bind to the Groucho-Tup1 type transcriptional corepressors Tup11 and Tup12, and locally antagonize their repressive functions on Atf1 binding. These results reveal a new mechanism in which upstream noncoding transcription locally magnifies the specific activation of stress-inducible genes via counteraction of corepressors.

Dynamic transition of transcription and chromatin landscape during fission yeast adaptation to glucose starvation.

Shortage of glucose, the primary energy source for all organisms, is one of the most critical stresses influencing cell viability. Glucose starvation promptly induces changes in mRNA and noncoding RNA (ncRNA) transcription. We previously reported that glucose starvation induces long ncRNA (lncRNA) transcription in the 5' segment of a fission yeast gluconeogenesis gene (fbp1+), which leads to stepwise chromatin alteration around the fbp1+ promoter and to subsequent robust gene activation. Here, we analyzed genomewide transcription by strand-specific RNA sequencing, together with chromatin landscape by immunoprecipitation sequencing (ChIP-seq). Clustering analysis showed that distinct mRNAs and ncRNAs are induced at the early, middle and later stages of cellular response to glucose starvation. The starvation-induced transcription depends substantially on the stress-responsive transcription factor Atf1. Using a new computer program that examines dynamic changes in expression patterns, we identified ncRNAs with similar behavior to the fbp1+ lncRNA. We confirmed that there are continuous lncRNAs associated with local reduction of histone density. Overlapping with the regions for transcription of these lncRNAs, antisense RNAs are antagonistically transcribed under glucose-rich conditions. These results suggest that Atf1-dependent integrated networks of mRNA and lncRNA govern drastic changes in cell physiology in response to glucose starvation.

Antagonistic controls of chromatin and mRNA start site selection by Tup family corepressors and the CCAAT-binding factor.

The Tup family corepressors contribute to critical cellular responses, such as the stress response and differentiation, presumably by inducing repressive chromatin, though the precise repression mechanism remains to be elucidated. The Schizosaccharomyces pombe fission yeast Tup family corepressors Tup11 and Tup12 (Tup11/12), which are orthologs of Tup1 in Saccharomyces cerevisiae budding yeast and Groucho in Drosophila, negatively control chromatin and the transcriptional activity of some stress-responsive genes. Here, we demonstrate that Tup11/12 repress transcription of a gluconeogenesis gene, fbp1⁺, by three distinct mechanisms. First, Tup11/12 inhibit chromatin remodeling in the fbp1⁺ promoter region where the Atf1 and Rst2 transcriptional activators bind. Second, they repress the formation of an open chromatin configuration at the fbp1⁺ TATA box. Third, they repress mRNA transcription per se by regulating basic transcription factors. These inhibitory actions of Tup11/12 are antagonized by three different types of transcriptional activators: CREB/ATF-type Atf1, C2H2zinc finger-type Rst2, and CBF/NF-Y-type Php5 proteins. We also found that impaired chromatin remodeling and fbp1⁺ mRNA transcription in php5Δ strains are rescued by the double deletions of tup11⁺ and tup12⁺, although the distribution of the transcription start sites becomes broader than that in wild-type cells. These data reveal a new mechanism of precise determination of the mRNA start site by Tup family corepressors and CBF/NF-Y proteins.