Showing papers on "Scaffold/matrix attachment region published in 2014"

A comparative encyclopedia of DNA elements in the mouse genome

Abstract: The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases

Chromatin accessibility: a window into the genome

Abstract: Transcriptional activation throughout the eukaryotic lineage has been tightly linked with disruption of nucleosome organization at promoters, enhancers, silencers, insulators and locus control regions due to transcription factor binding. Regulatory DNA thus coincides with open or accessible genomic sites of remodeled chromatin. Current chromatin accessibility assays are used to separate the genome by enzymatic or chemical means and isolate either the accessible or protected locations. The isolated DNA is then quantified using a next-generation sequencing platform. Wide application of these assays has recently focused on the identification of the instrumental epigenetic changes responsible for differential gene expression, cell proliferation, functional diversification and disease development. Here we discuss the limitations and advantages of current genome-wide chromatin accessibility assays with especial attention on experimental precautions and sequence data analysis. We conclude with our perspective on future improvements necessary for moving the field of chromatin profiling forward.

Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

Abstract: To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3,995 proteins. The replication machinery and 485 chromatin factors such as CAF-1, DNMT1 and SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, whereas H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins, and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance.

The Functional Topography of the Arabidopsis Genome Is Organized in a Reduced Number of Linear Motifs of Chromatin States

Abstract: Chromatin is of major relevance for gene expression, cell division, and differentiation. Here, we determined the landscape of Arabidopsis thaliana chromatin states using 16 features, including DNA sequence, CG methylation, histone variants, and modifications. The combinatorial complexity of chromatin can be reduced to nine states that describe chromatin with high resolution and robustness. Each chromatin state has a strong propensity to associate with a subset of other states defining a discrete number of chromatin motifs. These topographical relationships revealed that an intergenic state, characterized by H3K27me3 and slightly enriched in activation marks, physically separates the canonical Polycomb chromatin and two heterochromatin states from the rest of the euchromatin domains. Genomic elements are distinguished by specific chromatin states: four states span genes from transcriptional start sites (TSS) to termination sites and two contain regulatory regions upstream of TSS. Polycomb regions and the rest of the euchromatin can be connected by two major chromatin paths. Sequential chromatin immunoprecipitation experiments demonstrated the occurrence of H3K27me3 and H3K4me3 in the same chromatin fiber, within a two to three nucleosome size range. Our data provide insight into the Arabidopsis genome topography and the establishment of gene expression patterns, specification of DNA replication origins, and definition of chromatin domains.

Chromatin as dynamic 10-nm fibers

Abstract: Since Flemming described a nuclear substance in the nineteenth century and named it “chromatin,” this substance has fascinated biologists. What is the structure of chromatin? DNA is wrapped around core histones, forming a nucleosome fiber (10-nm fiber). This fiber has long been assumed to fold into a 30-nm chromatin fiber and subsequently into helically folded larger fibers or radial loops. However, several recent studies, including our cryo-EM and X-ray scattering analyses, demonstrated that chromatin is composed of irregularly folded 10-nm fibers, without 30-nm chromatin fibers, in interphase chromatin and mitotic chromosomes. This irregular folding implies a chromatin state that is physically less constrained, which could be more dynamic compared with classical regular helical folding structures. Consistent with this, recently, we uncovered by single nucleosome imaging large nucleosome fluctuations in living mammalian cells (∼50 nm/30 ms). Subsequent computational modeling suggested that nucleosome fluctuation increases chromatin accessibility, which is advantageous for many “target searching” biological processes such as transcriptional regulation. Therefore, this review provides a novel view on chromatin structure in which chromatin consists of dynamic and disordered 10-nm fibers.

Regulating the chromatin landscape: structural and mechanistic perspectives.

Abstract: A large family of chromatin remodelers that noncovalently modify chromatin is crucial in cell development and differentiation. They are often the targets of cancer, neurological disorders, and other human diseases. These complexes alter nucleosome positioning, higher-order chromatin structure, and nuclear organization. They also assemble chromatin, exchange out histone variants, and disassemble chromatin at defined locations. We review aspects of the structural organization of these complexes, the functional properties of their protein domains, and variation between complexes. We also address the mechanistic details of these complexes in mobilizing nucleosomes and altering chromatin structure. A better understanding of these issues will be vital for further analyses of subunits of these chromatin remodelers, which are being identified as targets in human diseases by NGS (next-generation sequencing).

Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions

Abstract: ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes.

Diverse lamin-dependent mechanisms interact to control chromatin dynamics: Focus on laminopathies

Abstract: Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals. Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation.

Differential Nuclease Sensitivity Profiling of Chromatin Reveals Biochemical Footprints Coupled to Gene Expression and Functional DNA Elements in Maize

Abstract: The eukaryotic genome is organized into nucleosomes, the fundamental units of chromatin The positions of nucleosomes on DNA regulate protein-DNA interactions and in turn influence DNA-templated events Despite the increasing number of genome-wide maps of nucleosome position, how global changes in gene expression relate to changes in nucleosome position is poorly understood We show that in nucleosome occupancy mapping experiments in maize (Zea mays), particular genomic regions are highly susceptible to variation introduced by differences in the extent to which chromatin is digested with micrococcal nuclease (MNase) We exploited this digestion-linked variation to identify protein footprints that are hypersensitive to MNase digestion, an approach we term differential nuclease sensitivity profiling (DNS-chip) Hypersensitive footprints were enriched at the 5′ and 3′ ends of genes, associated with gene expression levels, and significantly overlapped with conserved noncoding sequences and the binding sites of the transcription factor KNOTTED1 We also found that the tissue-specific regulation of gene expression was linked to tissue-specific hypersensitive footprints These results reveal biochemical features of nucleosome organization that correlate with gene expression levels and colocalize with functional DNA elements This approach to chromatin profiling should be broadly applicable to other species and should shed light on the relationships among chromatin organization, protein-DNA interactions, and genome regulation

DNA bridging and looping by HMO1 provides a mechanism for stabilizing nucleosome-free chromatin.

Abstract: The regulation of chromatin structure in eukaryotic cells involves abundant architectural factors such as high mobility group B (HMGB) proteins. It is not understood how these factors control the interplay between genome accessibility and compaction. In vivo, HMO1 binds the promoter and coding regions of most ribosomal RNA genes, facilitating transcription and possibly stabilizing chromatin in the absence of histones. To understand how HMO1 performs these functions, we combine single molecule stretching and atomic force microscopy (AFM). By stretching HMO1-bound DNA, we demonstrate a hierarchical organization of interactions, in which HMO1 initially compacts DNA on a timescale of seconds, followed by bridge formation and stabilization of DNA loops on a timescale of minutes. AFM experiments demonstrate DNA bridging between strands as well as looping by HMO1. Our results support a model in which HMO1 maintains the stability of nucleosome-free chromatin regions by forming complex and dynamic DNA structures mediated by protein–protein interactions.

Closing the (nuclear) envelope on the genome: how nuclear lamins interact with promoters and modulate gene expression.

Abstract: The nuclear envelope shapes the functional organization of the nucleus. Increasing evidence indicates that one of its main components, the nuclear lamina, dynamically interacts with the genome, including the promoter region of specific genes. This seems to occur in a manner that accords developmental significance to these interactions. This essay addresses key issues raised by recent data on the association of nuclear lamins with the genome. We discuss how lamins interact with large chromatin domains and with spatially restricted regions on gene promoters. We address the relationship between these interactions, chromatin modifications and gene expression outcomes. Lamin-genome contacts are redistributed after cell division and during stem cell differentiation, with evidence of lineage specificity. Thus, we also speculate on a developmental role of lamin interactions with specific genes. Finally, we highlight how concepts arising from this recent work lay the foundations of future challenges and investigations.

A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis

Abstract: Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function.

DNA topology and transcription.

Abstract: Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions.

A Smad3 and TTF-1/NKX2-1 complex regulates Smad4-independent gene expression

Abstract: Thyroid transcription factor-1 (TTF-1, also known as NKX2-1) is a tissue-specific transcription factor in lung epithelial cells. Although TTF-1 inhibits the epithelial-to-mesenchymal transition induced by transforming growth factor-β (TGF-β) in lung adenocarcinoma cells, the mechanism through which TTF-1 inhibits the functions of TGF-β is unknown. Here we show that TTF-1 disrupts the nuclear Smad3-Smad4 complex without affecting the nuclear localization of phospho-Smad3. Genome-wide analysis by chromatin immunoprecipitation followed by sequencing revealed that TTF-1 colocalizes with Smad3 on chromatin and alters Smad3-binding patterns throughout the genome, while TTF-1 generally inhibits Smad4 binding to chromatin. Moreover, Smad3 binds to chromatin together with TTF-1, but not with Smad4, at some Smad3-binding regions when TGF-β signaling is absent, and knockdown of Smad4 expression does not attenuate Smad3 binding in these regions. Thus, TTF-1 may compete with Smad4 for interaction with Smad3, and in the presence of TTF-1, Smad3 regulates the transcription of certain genes independently of Smad4. These findings provide a new model of regulation of TGF-β-Smad signaling by TTF-1.

Chromatin without the 30-nm fiber: constrained disorder instead of hierarchical folding.

Abstract: Several hierarchical levels of DNA packaging are believed to exist in chromatin, starting from a 10-nm chromatin fiber that is further packed into a 30-nm fiber. Transitions between the 30-nm and 10-nm fibers are thought to be essential for the control of chromatin transcriptional status. However, recent studies demonstrate that in the nuclei, DNA is packed in tightly associated 10-nm fibers that are not compacted into 30-nm fibers. Additionally, the accessibility of DNA in chromatin depends on the local mobility of nucleosomes rather than on decompaction of chromosome regions. These findings argue for reconsidering the hierarchical model of chromatin packaging and some of the basic definitions of chromatin. In particular, chromatin domains should be considered as three-dimensional objects, which may include genomic regions that do not necessarily constitute a continuous domain on the DNA chain.

The role of chromatin dynamics in immune cell development.

Abstract: In immune cells, as in all mammalian cells, nuclear DNA is wrapped around histones to form nucleosomes. The positioning and modifications of nucleosomes throughout the genome defines the chromatin state of the cell and has a large impact on gene regulation. Chromatin state is dynamic throughout immune cell development and activation. High-throughput open chromatin assays, such as DNase-seq, can be used to find regulatory element across the genome and, when combined with histone modifications, can specify their function. During hematopoiesis, distal regulatory elements, known as enhancers, are established by pioneer factors that alter chromatin state. Some of these enhancers are lost, some are gained, and some are maintained as a memory of the cell's developmental origin. The enhancer landscape is unique to the cell lineage-with different enhancers regulating the same promoter-and determines the mechanism of cell type-specific activation after exposure to stimuli. Histone modification and promoter architecture govern the diverse responses to stimulation. Furthermore, chromatin dynamics may explain the high plasticity of certain tissue-resident immune cell types. Future epigenomic research will depend on the development of more efficient experiments and better methods to associate enhancers with genes. The ultimate goal of mapping genome-wide chromatin state throughout the hematopoietic tree will help illuminate the mechanisms behind immune cell development and function.

Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes

Abstract: In eukaryotic cells, transgene expression levels may be limited by an unfavourable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which may protect transgenes from such position effect. We evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres, which are commonly associated to low or inconsistent expression because of their repressive chromatin environment. Although to variable extents, matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE) and the chicken cHS4 insulator acted as barrier elements, protecting a telomeric-distal transgene from silencing. MARs also increased the probability of silent gene reactivation in time-course experiments. Additionally, all MARs improved the level of expression in non-silenced cells, unlike other elements. MARs were associated to histone marks usually linked to actively expressed genes, especially acetylation of histone H3 and H4, suggesting that they may prevent the spread of silencing chromatin by imposing acetylation marks on nearby nucleosomes. Alternatively, an UCOE was found to act by preventing deposition of repressive chromatin marks. We conclude that epigenetic DNA elements used to enhance and stabilize transgene expression all have specific epigenetic signature that might be at the basis of their mode of action.

S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection

Abstract: Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human s-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

A chimeric HS4-SAR insulator (IS2) that prevents silencing and enhances expression of lentiviral vectors in pluripotent stem cells.

Abstract: Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells.

Functional organization and dynamics of the cell nucleus

Abstract: The eukaryotic cell nucleus enclosed within the nuclear envelope harbors organized chromatin territories and various nuclear bodies as sub-nuclear compartments. This higher-order nuclear organization provides a unique environment to regulate the genome during replication, transcription, maintenance, and other processes. In this review, we focus on the plant four-dimensional nuclear organization, its dynamics and function in response to signals during development or stress.

The Eμ Enhancer Region Influences H Chain Expression and B Cell Fate without Impacting IgVH Repertoire and Immune Response In Vivo

Abstract: The IgH intronic enhancer region Eμ is a combination of both a 220-bp core enhancer element and two 310-350-bp flanking scaffold/matrix attachment regions named MARsEμ. In the mouse, deletion of the core-enhancer Eμ element mainly affects VDJ recombination with minor effects on class switch recombination. We carried out endogenous deletion of the full-length Eμ region (core plus MARsEμ) in the mouse genome to study VH gene repertoire and IgH expression in developing B-lineage cells. Despite a severe defect in VDJ recombination with partial blockade at the pro-B cell stage, Eμ deletion (core or full length) did not affect VH gene usage. Deletion of this regulatory region induced both a decrease of pre-B cell and newly formed B cell compartments and a strong orientation toward the marginal zone B cell subset. Because Igμ H chain expression was decreased in Eμ-deficient pre-B cells, we propose that modification of B cell homeostasis in deficient animals was caused by "weak" pre-B cell and BCR expression. Besides imbalances in B cell compartments, Ag-specific Ab responses were not impaired in animals carrying the Eμ deletion. In addition to its role in VDJ recombination, our study points out that the full-length Eμ region does not influence VH segment usage but ensures efficient Igμ-chain expression required for strong signaling through pre-B cells and newly formed BCRs and thus participates in B cell inflow and fate.