Combined Study of Gene Expression and Chromosome Three-Dimensional Structure in Escherichia coli During Growth Process.

The chromosome structure of different bacteria has its unique organization pattern, which plays an important role in maintaining the spatial location relationship between genes and regulating gene expression. Conversely, transcription also plays a global role in regulating the three-dimensional structure of bacterial chromosomes. Therefore, we combine RNA-Seq and Hi-C technology to explore the relationship between chromosome structure changes and transcriptional regulation in E. coli at different growth stages. Transcriptome analysis indicates that E. coli synthesizes many ribosomes and peptidoglycan in the exponential phase. In contrast, E. coli undergoes more transcriptional regulation and catabolism during the stationary phase, reflecting its adaptability to changes in environmental conditions during growth. Analyzing the Hi-C data shows that E. coli has a higher frequency of global chromosomal interaction in the exponential phase and more defined chromosomal interaction domains (CIDs). Still, the long-distance interactions at the replication termination region are lower than in the stationary phase. Combining transcriptome and Hi-C data analysis, we conclude that highly expressed genes are more likely to be distributed in CID boundary regions during the exponential phase. At the same time, most high-expression genes distributed in the CID boundary regions are ribosomal gene clusters, forming clearer CID boundaries during the exponential phase. The three-dimensional structure of chromosome and expression pattern is altered during the growth of E. coli from the exponential phase to the stationary phase, clarifying the synergy between the two regulatory aspects.

The roles of nucleoid-associated proteins and topoisomerases in chromosome structure, strand segregation, and the generation of phenotypic heterogeneity in bacteria.

How to adapt to a changing environment is a fundamental, recurrent problem confronting cells. One solution is for cells to organize their constituents into a limited number of spatially extended, functionally relevant, macromolecular assemblies or hyperstructures, and then to segregate these hyperstructures asymmetrically into daughter cells. This asymmetric segregation becomes a particularly powerful way of generating a coherent phenotypic diversity when the segregation of certain hyperstructures is with only one of the parental DNA strands and when this pattern of segregation continues over successive generations. Candidate hyperstructures for such asymmetric segregation in prokaryotes include those containing the nucleoid-associated proteins (NAPs) and the topoisomerases. Another solution to the problem of creating a coherent phenotypic diversity is by creating a growth-environment-dependent gradient of supercoiling generated along the replication origin-to-terminus axis of the bacterial chromosome. This gradient is modulated by transcription, NAPs, and topoisomerases. Here, we focus primarily on two topoisomerases, TopoIV and DNA gyrase in Escherichia coli, on three of its NAPs (H-NS, HU, and IHF), and on the single-stranded binding protein, SSB. We propose that the combination of supercoiling-gradient-dependent and strand-segregation-dependent topoisomerase activities result in significant differences in the supercoiling of daughter chromosomes, and hence in the phenotypes of daughter cells.

SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks.

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

Genome duplication in Leishmania major relies on persistent subtelomeric DNA replication.

DNA replication is needed to duplicate a cell's genome in S phase and segregate it during cell division. Previous work in Leishmania detected DNA replication initiation at just a single region in each chromosome, an organisation predicted to be insufficient for complete genome duplication within S phase. Here, we show that acetylated histone H3 (AcH3), base J and a kinetochore factor co-localise in each chromosome at only a single locus, which corresponds with previously mapped DNA replication initiation regions and is demarcated by localised G/T skew and G4 patterns. In addition, we describe previously undetected subtelomeric DNA replication in G2/M and G1-phase-enriched cells. Finally, we show that subtelomeric DNA replication, unlike chromosome-internal DNA replication, is sensitive to hydroxyurea and dependent on 9-1-1 activity. These findings indicate that Leishmania's genome duplication programme employs subtelomeric DNA replication initiation, possibly extending beyond S phase, to support predominantly chromosome-internal DNA replication initiation within S phase.

The RECQL helicase prevents replication fork collapse during replication stress.

Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy.

Mammalian histones facilitate antimicrobial synergy by disrupting the bacterial proton gradient and chromosome organization.

First proposed as antimicrobial agents, histones were later recognized for their role in condensing chromosomes. Histone antimicrobial activity has been reported in innate immune responses. However, how histones kill bacteria has remained elusive. The co-localization of histones with antimicrobial peptides (AMPs) in immune cells suggests that histones may be part of a larger antimicrobial mechanism in vivo. Here we report that histone H2A enters E. coli and S. aureus through membrane pores formed by the AMPs LL-37 and magainin-2. H2A enhances AMP-induced pores, depolarizes the bacterial membrane potential, and impairs membrane recovery. Inside the cytoplasm, H2A reorganizes bacterial chromosomal DNA and inhibits global transcription. Whereas bacteria recover from the pore-forming effects of LL-37, the concomitant effects of H2A and LL-37 are irrecoverable. Their combination constitutes a positive feedback loop that exponentially amplifies their antimicrobial activities, causing antimicrobial synergy. More generally, treatment with H2A and the pore-forming antibiotic polymyxin B completely eradicates bacterial growth.

Surveillance of cohesin-supported chromosome structure controls meiotic progression.

Chromosome movements and programmed DNA double-strand breaks (DSBs) promote homologue pairing and initiate recombination at meiosis onset. Meiotic progression involves checkpoint-controlled termination of these events when all homologue pairs achieve synapsis and form crossover precursors. Exploiting the temporo-spatial organisation of the C. elegans germline and time-resolved methods of protein removal, we show that surveillance of the synaptonemal complex (SC) controls meiotic progression. In nuclei with fully synapsed homologues and crossover precursors, removing different meiosis-specific cohesin complexes, which are individually required for SC stability, or a SC central region component causes functional redeployment of the chromosome movement and DSB machinery, triggering whole-nucleus reorganisation. This apparent reversal of the meiotic programme requires CHK-2 kinase reactivation via signalling from chromosome axes containing HORMA proteins, but occurs in the absence of transcriptional changes. Our results uncover an unexpected plasticity of the meiotic programme and show how chromosome signalling orchestrates nuclear organisation and meiotic progression.

Sequential role of RAD51 paralog complexes in replication fork remodeling and restart.

Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart.

Chromosome organization by a conserved condensin-ParB system in the actinobacterium Corynebacterium glutamicum.

Higher-order chromosome folding and segregation are tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remain poorly characterized, especially in non-model species. Here, we investigate the role of DNA-partitioning protein ParB and SMC condensin complexes in the actinobacterium Corynebacterium glutamicum. Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin (oriC). At least one oriC-proximal parS site is necessary for reliable chromosome segregation. We use chromatin immunoprecipitation and photoactivated single-molecule localization microscopy to show the formation of distinct, parS-dependent ParB-nucleoprotein subclusters. We further show that SMC/ScpAB complexes, loaded via ParB at parS sites, mediate chromosomal inter-arm contacts (as previously shown in Bacillus subtilis). However, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding; instead, this complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. Our results complement current models of ParB-SMC/ScpAB crosstalk and show that some condensin complexes evolved functions that are apparently uncoupled from chromosome folding.

Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.

Meiotic sex chromosome cohesion and autosomal synapsis are supported by Esco2.

In mitotic cells, establishment of sister chromatid cohesion requires acetylation of the cohesin subunit SMC3 (acSMC3) by ESCO1 and/or ESCO2. Meiotic cohesin plays additional but poorly understood roles in the formation of chromosome axial elements (AEs) and synaptonemal complexes. Here, we show that levels of ESCO2, acSMC3, and the pro-cohesion factor sororin increase on meiotic chromosomes as homologs synapse. These proteins are less abundant on the largely unsynapsed sex chromosomes, whose sister chromatid cohesion appears weaker throughout the meiotic prophase. Using three distinct conditional Esco2 knockout mouse strains, we demonstrate that ESCO2 is essential for male gametogenesis. Partial depletion of ESCO2 in prophase I spermatocytes delays chromosome synapsis and further weakens cohesion along sex chromosomes, which show extensive separation of AEs into single chromatids. Unsynapsed regions of autosomes are associated with the sex chromatin and also display split AEs. This study provides the first evidence for a specific role of ESCO2 in mammalian meiosis, identifies a particular ESCO2 dependence of sex chromosome cohesion and suggests support of autosomal synapsis by acSMC3-stabilized cohesion.

Chromosomal Organization and Regulation of Genetic Function in Escherichia coli Integrates the DNA Analog and Digital Information.

In this article, we summarize our current understanding of the bacterial genetic regulation brought about by decades of studies using the Escherichia coli model. It became increasingly evident that the cellular genetic regulation system is organizationally closed, and a major challenge is to describe its circular operation in quantitative terms. We argue that integration of the DNA analog information (i.e., the probability distribution of the thermodynamic stability of base steps) and digital information (i.e., the probability distribution of unique triplets) in the genome provides a key to understanding the organizational logic of genetic control. During bacterial growth and adaptation, this integration is mediated by changes of DNA supercoiling contingent on environmentally induced shifts in intracellular ionic strength and energy charge. More specifically, coupling of dynamic alterations of the local intrinsic helical repeat in the structurally heterogeneous DNA polymer with structural-compositional changes of RNA polymerase holoenzyme emerges as a fundamental organizational principle of the genetic regulation system. We present a model of genetic regulation integrating the genomic pattern of DNA thermodynamic stability with the gene order and function along the chromosomal OriC-Ter axis, which acts as a principal coordinate system organizing the regulatory interactions in the genome.

Inhibition of spindle extension through the yeast S phase checkpoint is coupled to replication fork stability and the integrity of centromeric DNA.

Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.

Disruption of nuclear speckles reduces chromatin interactions in active compartments.

BACKGROUND: Nuclei of eukaryotes contain various higher-order chromatin architectures and nuclear bodies (NBs), which are critical for proper nuclear functions. Recent studies showed that active chromatin regions are associated with nuclear speckles (NSs), a type of NBs involved in RNA processing. However, the functional roles of NSs in 3D genome organization remain unclear. RESULTS: Using mouse hepatocytes as the model, we knocked down SRRM2, a core protein component scaffolding NSs, and performed Hi-C experiments to examine genome-wide chromatin interactions. We found that Srrm2 depletion disrupted the NSs and changed the expression of 1282 genes. The intra-chromosomal interactions were decreased in type A (active) compartments and increased in type B (repressive) compartments. Furthermore, upon Srrm2 knockdown, the insulation of TADs was decreased specifically in active compartments, and the most significant reduction occurred in A1 sub-compartments. Interestingly, the change of intra-TAD chromatin interactions upon Srrm2 depletion was not associated with the alteration of gene expression. CONCLUSIONS: We show that disruption of NSs by Srrm2 knockdown causes a global decrease in chromatin interactions in active compartments, indicating critical functions of NSs in the organization of the 3D genome.

The Nuclear Pore Complex in Cell Type-Specific Chromatin Structure and Gene Regulation.

Nuclear pore complex (NPC)-mediated nucleocytoplasmic trafficking is essential for key cellular processes, such as cell growth, cell differentiation, and gene regulation. The NPC has also been viewed as a nuclear architectural platform that impacts genome function and gene expression by mediating spatial and temporal coordination between transcription factors, chromatin regulatory proteins, and transcription machinery. Recent findings have uncovered differential and cell type-specific expression and distinct chromatin-binding patterns of individual NPC components known as nucleoporins (Nups). Here, we examine recent studies that investigate the functional roles of NPCs and Nups in transcription, chromatin organization, and epigenetic gene regulation in the context of development and disease.

A subtelomeric region affects telomerase-negative replicative senescence in Saccharomyces cerevisiae.

In eukaryotes, telomeres determine cell proliferation potential by triggering replicative senescence in the absence of telomerase. In Saccharomyces cerevisiae, senescence is mainly dictated by the first telomere that reaches a critically short length, activating a DNA-damage-like response. How the corresponding signaling is modulated by the telomeric structure and context is largely unknown. Here we investigated how subtelomeric elements of the shortest telomere in a telomerase-negative cell influence the onset of senescence. We found that a 15 kb truncation of the 7L subtelomere widely used in studies of telomere biology affects cell growth when combined with telomerase inactivation. This effect is likely not explained by (i) elimination of sequence homology at chromosome ends that would compromise homology-directed DNA repair mechanisms; (ii) elimination of the conserved subtelomeric X-element; (iii) elimination of a gene that would become essential in the absence of telomerase; and (iv) heterochromatinization of inner genes, causing the silencing of an essential gene in replicative senescent cells. This works contributes to better delineate subtelomere functions and their impact on telomere biology.

C. elegans ZHP-4 is required at multiple distinct steps in the formation of crossovers and their transition to segregation competent chiasmata.

Correct segregation of meiotic chromosomes depends on DNA crossovers (COs) between homologs that culminate into visible physical linkages called chiasmata. COs emerge from a larger population of joint molecules (JM), the remainder of which are repaired as noncrossovers (NCOs) to restore genomic integrity. We present evidence that the RNF212-like C. elegans protein ZHP-4 cooperates with its paralog ZHP-3 to enforce crossover formation at distinct steps during meiotic prophase: in the formation of early JMs and in transition of late CO intermediates into chiasmata. ZHP-3/4 localize to the synaptonemal complex (SC) co-dependently followed by their restriction to sites of designated COs. RING domain mutants revealed a critical function for ZHP-4 in localization of both proteins to the SC and for CO formation. While recombination initiates in zhp-4 mutants, they fail to appropriately acquire pro-crossover factors at abundant early JMs, indicating a function for ZHP-4 in an early step of the CO/NCO decision. At late pachytene stages, hypomorphic mutants exhibit significant levels of crossing over that are accompanied by defects in localization of pro-crossover RMH-1, MSH-5 and COSA-1 to designated crossover sites, and by the appearance of bivalents defective in chromosome remodelling required for segregation. These results reveal a ZHP-4 function at designated CO sites where it is required to stabilize pro-crossover factors at the late crossover intermediate, which in turn are required for the transition to a chiasma that is required for bivalent remodelling. Our study reveals an essential requirement for ZHP-4 in negotiating both the formation of COs and their ability to transition to structures capable of directing accurate chromosome segregation. We propose that ZHP-4 acts in concert with ZHP-3 to propel interhomolog JMs along the crossover pathway by stabilizing pro-CO factors that associate with early and late intermediates, thereby protecting designated crossovers as they transition into the chiasmata required for disjunction.

Dynamic changes in ORC localization and replication fork progression during tissue differentiation.

BACKGROUND: Genomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role. RESULTS: We exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, we found that sites of ORC binding show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited. CONCLUSIONS: These studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation.

Transcription factors orchestrate dynamic interplay between genome topology and gene regulation during cell reprogramming.

Chromosomal architecture is known to influence gene expression, yet its role in controlling cell fate remains poorly understood. Reprogramming of somatic cells into pluripotent stem cells (PSCs) by the transcription factors (TFs) OCT4, SOX2, KLF4 and MYC offers an opportunity to address this question but is severely limited by the low proportion of responding cells. We have recently developed a highly efficient reprogramming protocol that synchronously converts somatic into pluripotent stem cells. Here, we used this system to integrate time-resolved changes in genome topology with gene expression, TF binding and chromatin-state dynamics. The results showed that TFs drive topological genome reorganization at multiple architectural levels, often before changes in gene expression. Removal of locus-specific topological barriers can explain why pluripotency genes are activated sequentially, instead of simultaneously, during reprogramming. Together, our results implicate genome topology as an instructive force for implementing transcriptional programs and cell fate in mammals.

Destabilization of the replication fork protection complex disrupts meiotic chromosome segregation.

The replication fork protection complex (FPC) coordinates multiple processes that are crucial for unimpeded passage of the replisome through various barriers and difficult to replicate areas of the genome. We examine the function of Swi1 and Swi3, fission yeast's primary FPC components, to elucidate how replication fork stability contributes to DNA integrity in meiosis. We report that destabilization of the FPC results in reduced spore viability, delayed replication, changes in recombination, and chromosome missegregation in meiosis I and meiosis II. These phenotypes are linked to accumulation and persistence of DNA damage markers in meiosis and to problems with cohesion stability at the centromere. These findings reveal an important connection between meiotic replication fork stability and chromosome segregation, two processes with major implications to human reproductive health.