Heterogeneous interactions and polymer entropy decide organization and dynamics of chromatin domains

Chromatin is known to be organized into multiple domains of varying sizes and compaction. While these domains are often imagined as static structures, they are highly dynamic and show cell-to-cell variability. Since processes such as gene regulation and DNA replication occur in the context of these domains, it is important to understand their organization, fluctuation, and dynamics. To simulate chromatin domains, one requires knowledge of interaction strengths among chromatin segments. Here, we derive interaction-strength parameters from experimentally known contact maps and use them to predict chromatin organization and dynamics. Taking two domains on the human chromosome as examples, we investigate its three-dimensional organization, size/shape fluctuations, and dynamics of different segments within a domain, accounting for hydrodynamic effects. Considering different cell types, we quantify changes in interaction strengths and chromatin shape fluctuations in different epigenetic states. Perturbing the interaction strengths systematically, we further investigate how epigenetic-like changes can alter the spatio-temporal nature of the domains. Our results show that heterogeneous weak interactions are crucial in determining the organization of the domains. Computing effective stiffness and relaxation times, we investigate how perturbations in interactions affect the solid- and liquid-like nature of chromatin domains. Quantifying dynamics of chromatin segments within a domain, we show how the competition between polymer entropy and interaction energy influence the timescales of loop formation and maintenance of stable loops.     

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.     

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.     

A Lamin-Associated Chromatin Model for Chromosome Organization

We propose a simple model for chromatin organization based on the interaction of the chromatin fibers with lamin proteins along the nuclear membrane. Lamin proteins are known to be a major factor that influences chromatin organization and hence gene expression in the cells. We provide a quantitative understanding of lamin-associated chromatin organization in a crowded macromolecular environment by systematically varying the heteropolymer segment distribution and the strength of the lamin-chromatin attractive interaction. Our minimal polymer model reproduces the formation of lamin-associated-domains and provides an in silico tool for quantifying domain length distributions for different distributions of heteropolymer segments. We show that a Gaussian distribution of heteropolymer segments, coupled with strong lamin-chromatin interactions, can qualitatively reproduce observed length distributions of lamin-associated-domains. Further, lamin-mediated interaction can enhance the formation of chromosome territories as well as the organization of chromatin into tightly packed heterochromatin and the loosely packed gene-rich euchromatin regions.     

Cohesin mediates chromatin interactions that regulate mammalian β-globin expression

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.     

Joining the loops: beta-globin gene regulation

The mammalian beta-globin locus is a multigene locus containing several globin genes and a number of regulatory elements. During development, the expression of the genes changes in a process called "switching." The most important regulatory element in the locus is the locus control region (LCR) upstream of the globin genes that is essential for high-level expression of these genes. The discovery of the LCR initially raised the question how this element could exert its effect on the downstream globin genes. The question was solved by the finding that the LCR and activate globin genes are in physical contact, forming a chromatin structure named the active chromatin hub (ACH). Here we discuss the significance of ACH formation, provide an overview of the proteins implicated in chromatin looping at the beta-globin locus, and evaluate the relationship between nuclear organization and beta-globin gene expression.     

Capturing native interactions: intrinsic methods to study chromatin conformation

The 3D organization of chromatin controls gene expression through spatial interactions between genomic loci. FISH and 3C‐based methods that are commonly used to study chromatin organization utilize chemical crosslinking, a step that may introduce biases in detectable chromatin interactions. In their recent study, Papantonis and colleagues (Brant et al, 2016 ) developed alternative new methods of detecting chromatin contacts without the use of chemical crosslinking agents. These tools increase the resolution and confidence at which interactions can be identified, and may be informative for chromatin interaction dynamics.     

Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei

Despite the major advancements during the last decade with respect to both knowledge of higher order chromatin organization in the cell nucleus and the elucidation of epigenetic mechanisms of gene control, the true three-dimensional (3D) chromatin structure of endogenous active and inactive gene loci is not known. The present study was initiated as an attempt to close this gap. As a model case, we compared the chromatin architecture between the genetically active and inactive domains of the imprinted Prader-Willi syndrome (PWS) locus in human fibroblast and lymphoblastoid cell nuclei by 3D fluorescence in situ hybridization and quantitative confocal laser scanning microscopy. The volumes and 3D compactions of identified maternal and paternal PWS domains were determined in stacks of light optical serial sections using a novel threshold-independent approach. Our failure to detect volume and compaction differences indicates that possible differences are below the limits of light optical resolution. To overcome this limitation, spectral precision distance microscopy, a method of localization microscopy at the nanometer scale, was used to measure 3D distances between differentially labeled probes located both within the PWS region and in its neighborhood. This approach allows the detection of intranuclear differences between 3D distances down to about 70-90 nm, but again did not reveal clearly detectable differences between active and inactive PWS domains. Despite this failure, a comparison of the experimental 3D distance measurements with computer simulations of chromatin folding strongly supports a non-random higher order chromatin configuration of the PWS locus and argues against 3D configurations based on giant chromatin loops. Our results indicate that the search for differences between endogenous active and inactive PWS domains must be continued at still smaller scales than hitherto possible with conventional light microscopic procedures. The possibilities to achieve this goal are discussed.     

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.     

史氏鲟免疫球蛋白重链可变区序列及多样性

为了研究史氏鲟免疫球蛋白重链可变区基因的组织结构和多样性,采用RTPCR技术从史氏鲟(Acipenserschrenckii)脾脏总RNA中获得了免疫球蛋白重链可变区cDNA克隆,随机挑取31个阳性克隆进行测序。结果表明:所有序列相同率高于75%,前导肽相同率高于90%,应属于同1个VH家族。其变异主要存在于互补性决定区,特别是CDR3区。在D片段序列中发现大量保守的基因序列(motif)。并发现多个VH基因片段可以共用一个J片段的现象。在基因组DNA重排过程中,VH片段可以与任意的D和J片段结合。此外,史氏鲟免疫球蛋白重链可变区的VH,D和J片段的随机重排外,外切核酸酶作用,以及在重排位点大量N,P片段的插入现象,都大大增加了鲟鱼免疫球蛋白的多样性。     

Changes in histone acetylation are associated with differences in accessibility of V(H) gene segments to V-DJ recombination during B-cell ontogeny and development

Although V(D)J recombination is thought to be regulated by changes in the accessibility of chromatin to the recombinase machinery, the mechanisms responsible for establishing "open" chromatin are poorly understood. We performed a detailed study of the acetylation status of histones associated with 11 V(H) gene segments, their flanking regions, and various intergenic elements during B-cell development and ontogeny, when V(D)J recombination is highly regulated. Histone H4 shows higher and more-regulated acetylation than does histone H3 in the V(H) locus. In adult pro-B cells, V(H) gene segments are acetylated prior to V(D)J rearrangement, with higher acetylation associated with J(H)-distal V(H) gene segments. While large regions of the V(H) locus have similar patterns of histone acetylation, acetylation is narrowly confined to the gene segments, their flanking promoters, and recombinase signal sequence elements. Thus, histone acetylation in the V(H) locus is both locally and globally regulated. Increased histone acetylation accompanies preferential recombination of J(H)-proximal V(H) gene segments in early B-cell ontogeny, and decreased histone acetylation accompanies inhibition of V-DJ recombination in a transgenic model of immunoglobulin heavy-chain allelic exclusion. Thus, changes in histone acetylation appear to be important for both promotion and inhibition of V-DJ rearrangement during B-cell ontogeny and development.     

Inter-chromosomal gene regulation in the mammalian cell nucleus

Cellular phenotypes can critically rely on mono-allelic gene expression. Recent studies suggest that in mammalian cells inter-chromosomal DNA interactions may mediate the decision which allele to activate and which to silence. Here, these findings are discussed in the context of knowledge on gene competition, chromatin dynamics, and nuclear organization. We argue that data obtained by 4C technology strongly support the idea that chromatin folds according to self-organizing principles. In this concept, the nuclear positioning of a given locus is probabilistic as it also depends on the properties of neighbouring DNA segments and, by extrapolation, the whole chromosome. The linear distribution of repetitive DNA sequences and of active and inactive DNA regions is important for the folding and relative positioning of chromosomes. This stochastic concept of nuclear organization predicts that tissue-specific interactions between two selected loci present on different chromosomes will be rare.     

Chromatin looping and the probability of transcription

Recent studies of several multigene clusters have shown that gene activation by a remote enhancer is associated with chromatin loop formation. It is not fully understood how a chromatin loop forms in a nucleus or how it is involved in gene regulation. In this article, we propose that the major feature that determines loop formation is the flexibility of chromatin, and that this flexibility is modulated by histone acetylation (and other modifications). Thus, histone modifications will modulate distribution of the preferential looping site in chromatin, which, in turn, determines the probability of interaction between a remote enhancer and the cognate genes. This model can explain gene expression changes in the Hoxd gene cluster and the beta-globin locus.     

Tn5-FISH,a novel cytogenetic method to image chromatin interactions with sub-kilobase resolution

There is an increasing interest in understanding how three-dimensional (3D) organization of the genome is regulated. Different strategies have been employed to identify genome-wide chromatin interactions. However, due to current limitations in resolving genomic contacts, visualization and validation of these genomic loci with sub-kilobase resolution remain unsolved to date. Here, we describe Tn5 transposase-based Fluorescencein situhybridization (Tn5-FISH), a PCR-based, cost-effective imaging method, which can co-localize the genomic loci with sub-kilobase resolution, dissect genome architecture, and verify chromatin interactions detected by chromatin configuration capture (3C)-derived methods. To validate this method, short-range interactions in keratin-encoding gene (KRT) locus in topologically associated domain (TAD) were imaged by triple-color Tn5-FISH, indicating that Tn5-FISH is very useful to verify short-range chromatin interactions inside the contact domain and TAD. Therefore, Tn5-FISH can be a powerful molecular tool for the clinical detection of cytogenetic changes in numerous genetic diseases such as cancers.