Positioning the genome within the nucleus

Higher eukaryotic genomes contain both housekeeping genes and genes of which the expression is restricted to a defined time and space. It is well established that a correlation exists between structural organization of the genome and gene expression control. The functional mechanisms underlying this correlation are still poorly understood. Here I describe several observations that are the basis of present concepts of genome organization and nuclear architecture related to functionality. Regarding the relationship between positioning and disturbed cell functionality, I describe observations showing that the proximity of selected gene loci is statistically correlated with their propensity for oncogenic translocations as well as observations of patterns occurring in neurodegenerative disorders where unstable repeats are translated into an expanded polyglutamine tract. Such observations underscore the importance to understand how genetic perturbations lead to the global reorganization of nuclear architecture, chromatin structure and widespread changes in gene expression.     

Map of open and closed chromatin domains in Drosophila genome

Background

Chromatin compactness has been considered a major determinant of gene activity and has been associated with specific chromatin modifications in studies on a few individual genetic loci. At the same time, genome-wide patterns of open and closed chromatin have been understudied, and are at present largely predicted from chromatin modification and gene expression data. However the universal applicability of such predictions is not self-evident, and requires experimental verification.

Results

We developed and implemented a high-throughput analysis for general chromatin sensitivity to DNase I which provides a comprehensive epigenomic assessment in a single assay. Contiguous domains of open and closed chromatin were identified by computational analysis of the data, and correlated to other genome annotations including predicted chromatin “states”, individual chromatin modifications, nuclear lamina interactions, and gene expression. While showing that the widely trusted predictions of chromatin structure are correct in the majority of cases, we detected diverse “exceptions” from the conventional rules. We found a profound paucity of chromatin modifications in a major fraction of closed chromatin, and identified a number of loci where chromatin configuration is opposite to that expected from modification and gene expression patterns. Further, we observed that chromatin of large introns tends to be closed even when the genes are expressed, and that a significant proportion of active genes including their promoters are located in closed chromatin.

Conclusions

These findings reveal limitations of the existing predictive models, indicate novel mechanisms of epigenetic regulation, and provide important insights into genome organization and function.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-988) contains supplementary material, which is available to authorized users.     

Structural insight into recognition of methylated histone tails by retinoblastoma-binding protein 1

Retinoblastoma-binding protein 1 (RBBP1), also named AT-rich interaction domain containing 4A (ARID4A), is a tumor and leukemia suppressor involved in epigenetic regulation in leukemia and Prader-Willi/Angelman syndromes. Although the involvement in epigenetic regulation is proposed to involve its chromobarrel and/or Tudor domains because of their potential binding to methylated histone tails, the structures of these domains and their interactions with methylated histone tails are still uncharacterized. In this work, we first found that RBBP1 contains five domains by bioinformatics analysis. Three of the five domains, i.e. chromobarrel, Tudor, and PWWP domains, are Royal Family domains, which potentially bind to methylated histone tails. We further purified these domains and characterized their interaction with methylated histone tails by NMR titration experiments. Among the three Royal Family domains, only the chromobarrel domain could recognize trimethylated H4K20 (with an affinity of ～3 mm), as well as recognizing trimethylated H3K9, H3K27, and H3K36 (with lower affinities). The affinity could be further enhanced up to 15-fold by the presence of DNA. The structure of the chromobarrel domain of RBBP1 determined by NMR spectroscopy has an aromatic cage. Mutagenesis analysis identified four aromatic residues of the cage as the key residues for methylated lysine recognition. Our studies indicate that the chromobarrel domain of RBBP1 is responsible for recognizing methylated histone tails in chromatin remodeling and epigenetic regulation, which presents a significant advance in our understanding of the mechanism and relationship between RBBP1-related gene suppression and epigenetic regulation.     

Chromatin organization in relation to the nuclear periphery

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.     

Spatial genome organization during T-cell differentiation

The spatial organization of genomes within the mammalian cell nucleus is non-random. The functional relevance of spatial genome organization might be in influencing gene expression programs as cells undergo changes during development and differentiation. To gain insight into the plasticity of genomes in space and time and to correlate the activity of specific genes with their nuclear position, we systematically analyzed the spatial genome organization in differentiating mouse T-cells. We find significant global reorganization of centromeres, chromosomes and gene loci during the differentiation process. Centromeres were repositioned from a preferentially internal distribution in undifferentiated cells to a preferentially peripheral position in differentiated CD4+ and CD8+ cells. Chromosome 6, containing the differentially expressed T-cell markers CD4 and CD8, underwent differential changes in position depending on whether cells differentiated into CD4+ or CD8+ thymocytes. Similarly, the two marker loci CD4 and CD8 showed distinct behavior in their position relative to the chromosome 6 centromere at various stages of differentiation. Our results demonstrate that significant spatial genome reorganization occurs during differentiation and indicate that the relationship between dynamic genome topology and single gene regulation is highly complex.     

Advances in imaging the interphase nucleus using thin cryosections

The mammalian genome is partitioned amongst various chromosomes and encodes for approximately 30,000 protein-coding genes. Gene expression occurs after exit from mitosis, when chromosomes partially decondense within the cell nucleus to allow the enzymatic activities that work on chromatin to access each gene in a regulated fashion. Differential patterns of gene expression evolve during cell differentiation to give rise to the over 200 cell types in higher eukaryotes. The architectural organisation of the genome inside the interphase cell nucleus, and associated enzymatic activities, reveals dynamic and functional compartmentalization of the genome. In this review, I highlight the advantages of Tokuyasu cryosectioning on the investigation of nuclear structure and function. Robert Feulgen Prize 2007 Winner lecture presented at the 49th Symposium of the Society for Histochemistry in Freiburg i.Br., Germany, 26–29 September 2007.     

"On the move"ments of nuclear components in living cells

The use of GFP fusion proteins has dramatically changed our view of how the cell nucleus is organized and how functions are carried out. In this review we focus on recent advances related to the dynamics of chromatin domains, as well as the dynamics of nuclear proteins and several nuclear organelles.     

核骨架——细胞核内生命活动的重要结构体系

综合分析了国际国内近年来有关核骨架研究的新进展,从几个方面的研究事实,包括核骨架对染色质ＤＮＡ的有序组织,核骨架参与ＤＮＡ复制和基因的表达与调控以及核骨架的起源进化等,阐明核骨架是细胞核内染色质结构的有序组织者和功能活动的参与者,核内纷繁复杂的生命活动能有条不紊地进行,核骨架在其中扮演了重要角色。     

Distribution of the partitioning protein KorB on the genome of IncP-1 plasmid RK2

The ParB family partitioning protein, KorB, of plasmid RK2 is central to a regulatory network coordinating replication, maintenance and transfer genes. Previous immunofluorescence microscopy indicated that the majority of KorB is localized in plasmid foci. The 12 identified KorB binding sites on RK2 are differentiated by: position relative to promoters; binding strength; and cooperativity with other repressors and so the distribution of KorB may be sequestered around a sub-set of sites. However, chromatin immunoprecipitation analysis showed that while RK2 DNA molecules appear to sequester KorB to create a higher local concentration, cooperativity between DNA binding proteins does not result in major differences in binding site occupancy. Thus under steady state conditions all operators are close to fully occupied and this correlates with gene expression on the plasmid being highly repressed.     

ADAM19 在小鼠睾丸发育中的时空表达

目的 阐明含有去整合素和金属蛋白酶结构域的跨膜蛋白19(ADAM19)在小鼠睾丸发育中的作用.方法 采用半定量RT-PCR和免疫组化两种实验方法,分别检测ADAM19 mRNA和蛋白质在小鼠睾丸发育中的时空表达.结果 ①最早在胚胎发育的15.5 d才能检测到ADAM19 mRNA的表达,后其表达随着胚胎发育天数的增加而逐渐升高,到围产期表达水平达到最高.出生后,ADAM19 mRNA的表达呈现显著下降的趋势,到成体睾丸中就几乎检测不到ADAM19的表达.②和其mRNA表达变化趋势一样,ADAM19蛋白也是首次在胚胎发育的15.5 d被检测到,一直持续存在到出生后一周,一周后则几乎检测不到;阳性表达信号主要定位在睾丸的曲细精管(睾索)中.结论 ADAM19 在小鼠睾丸中的表达具有显著的发育依赖性.     

NSort/DB:An Intranuclear Compartment Protein Database

Distinct substructures within the nucleus are associated with a wide variety of important nuclear processes.Structures such as chromatin and nuclear pores have specific roles,while others such as Cajal bodies are more functionally varied.Understanding the roles of these membraneless intra-nuclear compartments requires extensive data sets covering nuclear and compartment-associated proteins.NSort/DB is a database providing access to intra-or sub-nuclear compartment associations for the mouse nuclear proteome.Based on resources ranging from large-scale curated data sets to detailed experiments,this data set provides a high-quality set of annotations of non-exclusive association of nuclear proteins with structures such as promyelocytic leukaemia bodies and chromatin.The database is searchable by protein identifier or compartment,and has a documented web service API.The search interface,web service and data download are all freely available online at http://www.nsort.org/db/.Availability of this data set will enable systematic analyses of the protein complements of nuclear compartments,improving our understanding of the diverse functional repertoire of these structures.