A physical approach to segregation and folding of the Caulobacter crescentus genome

Bacterial genomes are functionally organized. This organization is dynamic and globally changing throughout the cell cycle. Upon initiation of replication of the chromosome, the two origins segregate and move towards their new location taking along the newly replicated genome. Caulobacter crescentus employs a dedicated active partitioning (Par) system to move one copy of the parS centromere to the distal pole, while the other stays at the stalked pole. In this issue of Molecular Microbiology, Hong and McAdams describe studies on the speed of segregation of parS and regions up to 150 kb away. They show clear differences in segregation rates between parS and 50 kb flanking regions versus regions further away. To assess segregation rates the authors track fluorescent markers during movement using time-lapse microscopy. The relation between genomic and physical distance of pairs of markers reflects how the genome is folded. This relation permits testing experimental data against models from polymer physics. Such models are helpful in understanding principles of genome folding. Although long used in studies on eukaryotes, this approach has rarely been applied to bacteria. Finally, the authors give the first direct evidence for a role of the bacterial chromatin protein HU in folding the genome in vivo.     

Compaction and transport properties of newly replicated Caulobacter crescentus DNA

Upon initiating replication of the Caulobacter chromosome, one copy of the parS centromere remains at the stalked pole; the other moves to the distal pole. We identified the segregation dynamics and compaction characteristics of newly replicated Caulobacter DNA during transport (highly variable from cell to cell) using time-lapse fluorescence microscopy. The parS centromere and a length (also highly variable) of parS proximal DNA on each arm of the chromosome are segregated with the same relatively slow transport pattern as the parS locus. Newly replicated DNA further than about 100 kb from parS segregates with a different and faster pattern, while loci at 48 kb from parS segregate with the slow pattern in some cells and the fast pattern in others. The observed parS-proximal DNA compaction characteristics have scaling properties that suggest the DNA is branched. HU2-deletion strains exhibited a reduced compaction phenotype except near the parS site where only the ΔHU1ΔHU2 double mutant had a compaction phenotype. The chromosome shows speed-dependent extension during translocation suggesting the DNA polymer is under tension. While DNA segregation is highly reliable and succeeds in virtually all wild-type cells, the high degree of cell to cell variation in the segregation process is noteworthy.     

Chromatin globules: a common motif of higher order chromosome structure?

Recent technological advances in the field of chromosome conformation capture are facilitating tremendous progress in the ability to map the three-dimensional (3D) organization of chromosomes at a resolution of several Kb and at the scale of complete genomes. Here we review progress in analyzing chromosome organization in human cells by building 3D models of chromatin based on comprehensive chromatin interaction datasets. We describe recent experiments that suggest that long-range interactions between active functional elements are sufficient to drive folding of local chromatin domains into compact globular states. We propose that chromatin globules are commonly formed along chromosomes, in a cell type specific pattern, as a result of frequent long-range interactions among active genes and nearby regulatory elements. Further, we speculate that increasingly longer range interactions can drive aggregation of groups of globular domains. This process would yield a compartmentalized chromosome conformation, consistent with recent observations obtained with genome-wide chromatin interaction mapping.     

An SMC ATPase mutant disrupts chromosome segregation in Caulobacter

Accurate replication and segregation of the bacterial genome are essential for cell cycle progression. We have identified a single amino acid substitution in the Caulobacter structural maintenance of chromosomes (SMC) protein that disrupts chromosome segregation and cell division. The E1076Q point mutation in the SMC ATPase domain caused a dominant-negative phenotype in which DNA replication was able to proceed, but duplicated parS centromeres, normally found at opposite cell poles, remained at one pole. The cellular positions of other chromosomal loci were in the wild-type order relative to the parS centromere, but chromosomes remained unsegregated and appeared to be stacked upon one another. Purified SMC-E1076Q was deficient in ATP hydrolysis and exhibited abnormally stable binding to DNA. We propose that SMC spuriously links the duplicated chromosome immediately after passage of the replication fork. In wild-type cells, ATP hydrolysis opens the SMC dimer, freeing one chromosome to segregate to the opposite pole. The loss of ATP hydrolysis causes the SMC-E1076Q dimer to remain bound to both chromosomes, inhibiting segregation.     

Advances in higher-order chromatin architecture: the move towards 4D genome

In eukaryotes, the genome is hierarchically packed inside the nucleus, which facilitates physical contact between cis-regulatory elements (CREs), such as enhancers and promoters. Accumulating evidence highlights the critical role of higher-order chromatin structure in precise regulation of spatiotemporal gene expression under diverse biological contexts including lineage commitment and cell activation by external stimulus. Genomics and imaging-based technologies, such as Hi-C and DNA fluorescence in situ hybridization (FISH), have revealed the key principles of genome folding, while newly developed tools focus on improvement in resolution, throughput and modality at single-cell and population levels, and challenge the knowledge obtained through conventional approaches. In this review, we discuss recent advances in our understanding of principles of higher-order chromosome conformation and technologies to investigate 4D chromatin interactions.     

三维基因组学与精准生物学

三维基因组学是以研究真核生物核内基因组空间构象,及其对不同基因转录调控的生物学效应为主要研究内容的一个新的学科方向;也是后基因组学时代研究的一个热门领域。它的研究重点是空间构象与基因转录调控间的关系。通过三维基因组学技术,科学家将能对基因组的折叠和空间构象、转录调控机制、复杂生物学性状、信号传导通路和基因组的运行机制等一系列重要问题进行更深入的探讨和研究,为系统解读生命百科全书和精准生物学的实施奠定坚实基础。本文综述了目前三维基因组学研究领域中的主要技术、研究现状、科研进展、存在问题、未来及与精准生物学的关系等内容。以期能较系统地展示三维基因组学取得的一系列成果,解读从三维空间构象信息到不同基因功能研究的路径,精准决定在转录调控网络中不同基因表达的时空特异性的可能模式。     

Genome modeling: From chromatin fibers to genes

The intricacies of the 3D hierarchical organization of the genome have been approached by many creative modeling studies. The specific model/simulation technique combination defines and restricts the system and phenomena that can be investigated. We present the latest modeling developments and studies of the genome, involving models ranging from nucleosome systems and small polynucleosome arrays to chromatin fibers in the kb-range, chromosomes, and whole genomes, while emphasizing gene folding from first principles. Clever combinations allow the exploration of many interesting phenomena involved in gene regulation, such as nucleosome structure and dynamics, nucleosome-nucleosome stacking, polynucleosome array folding, protein regulation of chromatin architecture, mechanisms of gene folding, loop formation, compartmentalization, and structural transitions at the chromosome and genome levels. Gene-level modeling with full details on nucleosome positions, epigenetic factors, and protein binding, in particular, can in principle be scaled up to model chromosomes and cells to study fundamental biological regulation.     

CTCF mediates the cell-type specific spatial organization of the Kcnq5 locus and the local gene regulation

Chromatin loops play important roles in the dynamic spatial organization of genes in the nucleus. Growing evidence has revealed that the multivalent functional zinc finger protein CCCTC-binding factor (CTCF) is a master regulator of genome spatial organization, and mediates the ubiquitous chromatin loops within the genome. Using circular chromosome conformation capture (4C) methodology, we discovered that CTCF may be a master organizer in mediating the spatial organization of the kcnq5 gene locus. We characterized the cell-type specific spatial organization of the kcnq5 gene locus mediated by CTCF in detail using chromosome conformation capture (3C) and 3C-derived techniques. Cohesion also participated in mediating the organization of this locus. RNAi-mediated knockdown of CTCF sharply diminished the interaction frequencies between the chromatin loops of the kcnq5 gene locus and down-regulated local gene expression. Functional analysis showed that the interacting chromatin loops of the kcnq5 gene locus can repress the gene expression in a luciferase reporter assay. These interacting chromatin fragments were a series of repressing elements whose contacts were mediated by CTCF. Therefore, these findings suggested that the dynamical spatial organization of the kcnq5 locus regulates local gene expression.     

Analysis of the terminus region of the Caulobacter crescentus chromosome and identification of the dif site

The terminus region of the Caulobacter crescentus chromosome and the dif chromosome dimer resolution site were characterized. The Caulobacter genome contains skewed sequences that abruptly switch strands at dif and may have roles in chromosome maintenance and segregation. Absence of dif or the XerCD recombinase results in a chromosome segregation defect. The Caulobacter terminus region is unusual, since it contains many essential or highly expressed genes.     

CRISPR Double Cutting through the Labyrinthine Architecture of 3D Genomes

The genomes are organized into ordered and hierarchical topological structures in interphase nuclei.Within discrete territories of each chromosome,topologically associated domains(TADs) play important roles in various nuclear processes such as gene regulation.Inside TADs separated by relatively constitutive boundaries,distal elements regulate their gene targets through specific chromatin-looping contacts such as long-distance enhancer-promoter interactions.High-throughput sequencing studies have revealed millions of potential regulatory DNA elements,which are much more abundant than the mere ~ 20,000 genes they control.The recently emerged CRISPRCas9 genome editing technologies have enabled efficient and precise genetic and epigenetic manipulations of genomes.The multiplexed and high-throughput CRISPR capabilities facilitate the discovery and dissection of gene regulatory elements.Here,we describe the applications of CRISPR for genome,epigenome,and 3D genome editing,focusing on CRISPR DNA-fragment editing with Cas9 and a pair of sgRNAs to investigate topological folding of chromatin TADs and developmental gene regulation.     

Large-scale reconstruction of 3D structures of human chromosomes from chromosomal contact data

Chromosomes are not positioned randomly within a nucleus, but instead, they adopt preferred spatial conformations to facilitate necessary long-range gene–gene interactions and regulations. Thus, obtaining the 3D shape of chromosomes of a genome is critical for understanding how the genome folds, functions and how its genes interact and are regulated. Here, we describe a method to reconstruct preferred 3D structures of individual chromosomes of the human genome from chromosomal contact data generated by the Hi-C chromosome conformation capturing technique. A novel parameterized objective function was designed for modeling chromosome structures, which was optimized by a gradient descent method to generate chromosomal structural models that could satisfy as many intra-chromosomal contacts as possible. We applied the objective function and the corresponding optimization method to two Hi-C chromosomal data sets of both a healthy and a cancerous human B-cell to construct 3D models of individual chromosomes at resolutions of 1 MB and 200 KB, respectively. The parameters used with the method were calibrated according to an independent fluorescence in situ hybridization experimental data. The structural models generated by our method could satisfy a high percentage of contacts (pairs of loci in interaction) and non-contacts (pairs of loci not in interaction) and were compatible with the known two-compartment organization of human chromatin structures. Furthermore, structural models generated at different resolutions and from randomly permuted data sets were consistent.     

CRISPR/Cas9基因编辑在三维基因组研究中的应用

CRISPR/Cas9系统在基因编辑方面具有巨大优势,能够低成本、可编程、方便快捷地用于动物、植物以及微生物的基因组靶向编辑和功能改造。三维基因组学是近年来兴起的一门研究染色质高级结构动态调控及基因组生物学功能的交叉学科。在三维基因组研究中,通常采用对DNA片段进行基因编辑以模拟基因组结构性变异,标记特定DNA片段,进而研究调控元件对于基因调控、细胞分化、组织发生、器官形成、个体发育的影响,最终阐明三维基因组的组装调控机制和生物学功能。因此,CRISPR及其衍生技术为研究三维基因组提供了极好的遗传学工具。本文主要综述了CRISPR片段编辑及其衍生技术在三维基因组调控与功能研究中的应用,以期为后续研究工作提供理论参考以及新的研究思路。     

核基质附着区广泛参与基因组三维结构折叠的生物信息学分析

在细胞分裂间期,每条染色质都占据着特定的染色质领域（chromosome territory,CT）。每个CT领域内进一步分成不同的拓扑学相关区域（topological associated domain,TAD）,每个TAD又由若干子TAD（sub-TAD）构成。不同的TAD相互聚集,形成基因活跃表达和不表达的A、B两种组份或区室（compartment）。然而,目前对于染色质折叠方式及维持机制的研究尚无定论。核基质附着区（matrix attachment regions,MARs）是在不同物种基因组中广泛存在的一类富含AT序列的与核基质结合的DNA元件,能够通过与CTCF、SATB1等调控蛋白质相互作用,对远距离的基因表达进行调控。本研究以染色质三维结构为背景,通过整合染色质三维结构及组蛋白修饰等组学数据,对MARs元件与染色质三维结构的关系进行研究,对MARs元件参与形成的相互作用网络的结构及功能进行探索。结果发现,MARs元件与染色质三维结构高度相关,而且在高强度相互作用中占据较大的比例,提示MARs元件在染色质折叠方面发挥作用。此外,通过拓扑结构聚类分析还首次揭示,MARs元件分为不同类型,包括维持染色质领域及空间构象等的结构单元部分,以及调控基因表达等的功能单元部分。这表明,MARs元件在基因组三维高级结构的建立、维持以及功能等方面发挥重要作用。     

Techniques for and challenges in reconstructing 3D genome structures from 2D chromosome conformation capture data

Chromosome conformation capture technologies that provide frequency information for contacts between genomic regions have been crucial for increasing our understanding of genome folding and regulation. However, such data do not provide direct evidence of the spatial 3D organization of chromatin. In this opinion article, we discuss the development and application of computational methods to reconstruct chromatin 3D structures from experimental 2D contact data, highlighting how such modeling provides biological insights and can suggest mechanisms anchored to experimental data. By applying different reconstruction methods to the same contact data, we illustrate some state-of-the-art of these techniques and discuss our gene resolution approach based on Brownian dynamics and Monte Carlo sampling.     

Distinct centromere-like parS sites on the two chromosomes of Vibrio spp

Vibrio cholerae, the cause of cholera, has two circular chromosomes. The parAB genes on each V. cholerae chromosome act to control chromosome segregation in a replicon-specific fashion. The chromosome I (ChrI) parAB genes (parAB1) govern the localization of the origin region of ChrI, while the chromosome II (ChrII) parAB genes (parAB2) control the segregation of ChrII. In addition to ParA and ParB proteins, Par systems require ParB binding sites (parS). Here we identified the parS sites on both V. cholerae chromosomes. We found three clustered origin-proximal ParB1 binding parS1 sites on ChrI. Deletion of these three parS1 sites abrogated yellow fluorescent protein (YFP)-ParB1 focus formation in vivo and resulted in mislocalization of the ChrI origin region. However, as observed in a parA1 mutant, mislocalization of the ChrI origin region in the parS1 mutant did not compromise V. cholerae growth, suggesting that additional (non-Par-related) mechanisms may mediate the partitioning of ChrI. We also identified 10 ParB2 binding parS2 sites, which differed in sequence from parS1. Fluorescent derivatives of ParB1 and ParB2 formed foci only with the cognate parS sequence. parABS2 appears to form a functional partitioning system, as we found that parABS2 was sufficient to stabilize an ordinarily unstable plasmid in Escherichia coli. Most parS2 sites were located within 70 kb of the ChrII origin of replication, but one parS2 site was found in the terminus region of ChrI. In contrast, in other sequenced vibrio species, the distribution of parS1 and parS2 sites was entirely chromosome specific.     

三维基因组学研究进展

三维基因组学是一门研究基因组三维空间结构与功能的新兴学科,主要研究基因组序列在细胞核内的三维空间构象,及其对DNA复制、DNA重组、基因表达调控等生物过程的生物学效应。自染色质构象捕获技术 (3C)出现后,三维基因组学相关研究领域飞速发展。借助于3C及其衍生技术、Hi-C和ChIA-PET等技术,科学家能对各类物种的三维基因组进行更为深入的研究,从而揭示微生物、植物和动物基因组的空间构象、染色质的相互作用模式、转录调控以及不同生物学性状的形成机制;挖掘与生命活动和疾病相关的关键基因和信号通路;推动农业科学、生命科学和医学等领域的快速发展。文中就三维基因组学研究进展作一综述,主要阐述三维基因组学的概念和研究技术的发展及其在农业科学、生命科学和医学等领域的应用,尤其是肿瘤领域所取得的阶段性研究成果。     

The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin-proximal region of the linear chromosome

The mycelial prokaryote Streptomyces coelicolor A3(2) possesses a large linear chromosome (8.67 Mb) with a centrally located origin of replication (oriC). Recently, chromosome partitioning genes (parA and parB) and putative ParB binding sites (parS sequences) were identified in its genome. The S. coelicolor chromosome contains more parS sequences than any other bacterial chromosome characterized so far. Twenty of the 24 parS sequences are densely packed within a relatively short distance (approximately 200 kb) around oriC. A series of in vitro and in vivo experiments showed that S. coelicolor ParB protein interacts specifically with the parS sequences, albeit with a rather low affinity. Our results suggested that the binding of ParB is not only determined by the parS sequence, but also by the location of target DNA close to oriC. The unusually high number and close proximity to each other of the parS sites, together with in vivo and in vitro evidence that multiple ParB molecules may assemble along the DNA from an initial ParB-parS complex, suggest that a large DNA segment around the replication origin may form a massive nucleoprotein complex as part of the replication-partitioning cycle.     

染色体构象俘获技术及其研究进展

真核生物中远距离的调控元件往往通过相互作用形成复杂的染色体相互作用网络,对基因的表达进行三维调节,染色体构象俘获是研究染色体相互作用的有力工具。简要综述了染色体构象俘获技术的基本原理及其研究进展,并对相关技术存在的问题进行了分析,对发展趋势进行了展望。