Thermodynamic Pathways to Genome Spatial Organization in the Cell Nucleus

The architecture of the eukaryotic genome is characterized by a high degree of spatial organization. Chromosomes occupy preferred territories correlated to their state of activity and, yet, displace their genes to interact with remote sites in complex patterns requiring the orchestration of a huge number of DNA loci and molecular regulators. Far from random, this organization serves crucial functional purposes, but its governing principles remain elusive. By computer simulations of a statistical mechanics model, we show how architectural patterns spontaneously arise from the physical interaction between soluble binding molecules and chromosomes via collective thermodynamics mechanisms. Chromosomes colocalize, loops and territories form, and find their relative positions as stable thermodynamic states. These are selected by thermodynamic switches, which are regulated by concentrations/affinity of soluble mediators and by number/location of their attachment sites along chromosomes. Our thermodynamic switch model of nuclear architecture, thus, explains on quantitative grounds how well-known cell strategies of upregulation of DNA binding proteins or modification of chromatin structure can dynamically shape the organization of the nucleus.     

Review: nuclear lamins--structural proteins with fundamental functions

The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed of both peripheral and integral membrane proteins, including lamins and lamina-associated proteins. Lamins can interact with one another, with lamina-associated proteins, with nuclear scaffold proteins, and with chromatin. Likewise, most of the lamina-associated proteins are likely to interact directly with chromatin. The nuclear lamina is required for proper cell cycle regulation, chromatin organization, DNA replication, cell differentiation, and apoptosis. Mutations in proteins of the nuclear lamina can disrupt these activities and cause genetic diseases. The structure and assembly of the nuclear lamina proteins and their roles in chromatin organization and cell cycle regulation were recently reviewed. In this review, we discuss the roles of the nuclear lamina in DNA replication and apoptosis and analyze how mutations in nuclear lamina proteins might cause genetic diseases.     

The specification and maintenance of renal cell types by epigenetic factors

The specification of cell lineages and patterning in the embryo occurs sequentially as specific regions are increasingly restricted in their developmental fates. When and how this occurs is still not entirely clear. Nevertheless, the roles of epigenetic regulatory genes in partitioning the genome into active and inactive domains is evident in a variety of organisms and is highly conserved through evolution. The function of Pax2 in the kidney has been inferred by the phenotypic analysis of loss-of-function mutants in mice, fish, and humans. Although Pax2 and the related gene, Pax8, are essential for early intermediate mesoderm specification and are found in the epithelial lineage arising from that mesoderm, how these proteins regulate cell lineage restriction and gene expression patterns has remained obscure. Our recent data, suggests that Pax proteins help establish chromatin domains within cell lineages by providing the locus and tissue specificity for epigenetic imprinting complexes that modify histones. The novel protein PTIP is a key adaptor that links Pax proteins and possibly many other types of DNA binding proteins to a histone H3K4 methyltransferase complex. Given the prevalence of Pax2 expression in kidney development and in kidney disease, we now need to address the effects of epigenetics on renal disease states, on the stability of the terminal epithelial phenotype, and in the aging cell.     

Local geometry and elasticity in compact chromatin structure

The hierarchical packaging of DNA into chromatin within a eukaryotic nucleus plays a pivotal role in both the accessibility of genomic information and the dynamics of replication. Our work addresses the role of nanoscale physical and geometric properties in determining the structure of chromatin at the mesoscale level. We study the packaging of DNA in chromatin fibers by optimization of regular helical morphologies, considering the elasticity of the linker DNA as well as steric packing of the nucleosomes and linkers. Our model predicts a broad range of preferred helix structures for a fixed linker length of DNA; changing the linker length alters the predicted ensemble. Specifically, we find that the twist registry of the nucleosomes, as set by the internucleosome repeat length, determines the preferred angle between the nucleosomes and the fiber axis. For moderate to long linker lengths, we find a number of energetically comparable configurations with different nucleosome-nucleosome interaction patterns, indicating a potential role for kinetic trapping in chromatin fiber formation. Our results highlight the key role played by DNA elasticity and local geometry in regulating the hierarchical packaging of the genome.     

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。     

Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times without nuclear or cellular division. The Mcm2-7 protein complex is required for licensing of DNA replication. In an effort to understand whether genome reduplication occurs due to absence or failure of the DNA replication licensing system, we analysed the function of Mcm2-3-5 proteins in E. histolytica. In this study, we have cloned E. histolytica (Eh) MCM2 and Eh MCM5 genes, while Eh MCM3 was cloned earlier. The sequence of Eh MCM2-3-5 genes is well conserved with other eukaryotic homologues. We have shown that Eh Mcm2,3 proteins are functional in Saccharomyces cerevisiae. Our studies in E. histolytica showed that Eh Mcm2-3-5 proteins are associated with chromatin constitutively in cycling cells and during arrest of DNA synthesis induced by serum starvation. Alternation of genome duplication with mitosis is regulated by association-dissociation of Mcm2-7 proteins with chromatin in other eukaryotes. Our results suggest that constitutive association of Mcm proteins with chromatin could be one of the reasons why genome reduplication occurs in E. histolytica.     

Introduction of a long synthetic repetitive DNA sequence into cultured tobacco cells

Genome information has been accumulated for many species, and these genes and regulatory sequences are expected to be applied in plants by enhancing or creating new metabolic pathways. We hypothesized that manipulating a long array of repetitive sequences using tethered chromatin modulators would be effective for robust regulation of gene expression in close proximity to the arrays. This approach is based on a human artificial chromosome made of long synthetic repetitive DNA sequences in which we manipulated the chromatin by tethering the modifiers. However, a method for introducing long repetitive DNA sequences into plants has not yet been established. Therefore, we constructed a bacterial artificial chromosome-based binary vector in Escherichia coli cells to generate a construct in which a cassette of marker genes was inserted into 60-kb synthetic human centromeric repetitive DNA. The binary vector was then transferred to Agrobacterium cells and its stable maintenance confirmed. Next, using Agrobacterium-mediated genetic transformation, this construct was successfully introduced into the genome of cultured tobacco BY-2 cells to obtain a large number of stable one-copy strains. ChIP analysis of obtained BY-2 cell lines revealed that the introduced synthetic repetitive DNA has moderate chromatin modification levels with lower heterochromatin (H3K9me2) or euchromatin (H3K4me3) modifications compared to the host centromeric repetitive DNA or an active Tub6 gene, respectively. Such a synthetic DNA sequence with moderate chromatin modification levels is expected to facilitate manipulation of the chromatin structure to either open or closed.     

The specification and maintenance of renal cell types by epigenetic factors

The specification of cell lineages and patterning in the embryo occurs sequentially as specific regions are increasingly restricted in their developmental fates. When and how this occurs is still not entirely clear. Nevertheless, the roles of epigenetic regulatory genes in partitioning the genome into active and inactive domains is evident in a variety of organisms and is highly conserved through evolution. The function of Pax2 in the kidney has been inferred by the phenotypic analysis of loss-of-function mutants in mice, fish and humans. Although Pax2 and the related gene, Pax8, are essential for early intermediate mesoderm specification and are found in the epithelial lineage arising from that mesoderm, how these proteins regulate cell lineage restriction and gene expression patterns has remained obscure. Our recent data, suggests that Pax proteins help establish chromatin domains within cell lineages by providing the locus and tissue specificity for epigenetic imprinting complexes that modify histones. The novel protein PTIP is a key adaptor that links Pax proteins and possibly many other types of DNA binding proteins to a histone H3K4 methyltransferase complex. Given the prevalence of Pax2 expression in kidney development and in kidney disease, we now need to address the effects of epigenetics on renal disease states, on the stability of the terminal epithelial phenotype, and in the aging cell.Key words: kidney development, Pax2, PTIP, histone methyltransferase