Role In Chromatin Organization Research Articles

SAFB1's multiple functions in biological control—lots still to be done!

The examination of scaffold attachment factor B1 (SAFB1) and its multiple functions and tasks in cellular processes provides insight into its role in diseases, such as cancer. SAFB1 is a large multi-domain protein with well-described functions in transcriptional repression, and RNA splicing. It is ubiquitously expressed, and has been shown to be important in numerous cellular processes including cell growth, stress response, and apoptosis. SAFB1 is part of a protein family with at least two other family members, SAFB2 and the SAFB-like transcriptional modulator SLTM. The goal of this prospect article is to summarize known functions of SAFB1, and its roles in cellular processes, but also to speculate on less well described, novel attributes of SAFB1, such as a potential role in chromatin organization. This timely review shows aspects of SAFB1, which are proving to have a complexity far greater than was previously thought.

Investigating repetitively matching short sequencing reads: The enigmatic nature of H3K9me3

Most histone modifications can easily be characterized as either activating or repressive. For example, histone3, lysine 4 trimethylation (H3K4me3) is generally considered a distinct sign of actively transcribed promoters while H3K27me3 is generally found at repressed genes. This is not the case for H3K9me3, the subject of this communication, which is a modification that has traditionally been considered a mark of constitutive heterochromatin, but has also been found in significant levels in expressed genes. We therefore sought to use new high-throughput genome-wide maps of H3K9me3 localization to investigate the conflicting hypotheses concerning the nature of this modification. Before we could accurately analyze the locations of H3K9me3 along the genome, and especially in repetitive locations, we developed a method for accurately utilizing short sequencing reads that do not map uniquely to a location in the genome. Investigating the locations of H3K9me3 along the genome allowed us to determine that, while there are high levels of H3K9me3 outside of genes, this modification is not absent from genes. Therefore, we suggest that H3K9me3 may have a role in chromatin organization rather than being directly related to gene expression. In addition, we have found that there is a need to include repetitively matching reads in any high-throughput sequencing experiment.

Laminopathies and the long strange trip from basic cell biology to therapy

The main function of the nuclear lamina, an intermediate filament meshwork lying primarily beneath the inner nuclear membrane, is to provide structural scaffolding for the cell nucleus. However, the lamina also serves other functions, such as having a role in chromatin organization, connecting the nucleus to the cytoplasm, gene transcription, and mitosis. In somatic cells, the main protein constituents of the nuclear lamina are lamins A, C, B1, and B2. Interest in the nuclear lamins increased dramatically in recent years with the realization that mutations in LMNA, the gene encoding lamins A and C, cause a panoply of human diseases ("laminopathies"), including muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. Here, we review the laminopathies and the long strange trip from basic cell biology to therapeutic approaches for these diseases.

The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

BackgroundHistone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks.ResultsTo gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin.ConclusionFRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1-independent fashion.

Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions

Insulators are protein-bound DNA elements that are thought to play a role in chromatin organization and the regulation of gene expression by mediating intra- and interchromosomal interactions. Suppressor of Hair-wing [Su(Hw)] and Drosophila CTCF (dCTCF) insulators are found at distinct loci throughout the Drosophila melanogaster genome and function by recruiting an additional protein, Centrosomal Protein 190 (CP190). We performed chromatin immunoprecipitation (ChIP) and microarray analysis (ChIP-chip) experiments with whole-genome tiling arrays to compare Su(Hw), dCTCF, boundary element-associated factor (BEAF), and CP190 localization on DNA in two different cell lines and found evidence that BEAF is a third subclass of CP190-containing insulators. The DNA-binding proteins Su(Hw), dCTCF, and BEAF show unique distribution patterns with respect to the location and expression level of genes, suggesting diverse roles for these three subclasses of insulators in genome organization. Notably, cell line-specific localization sites for all three DNA-binding proteins as well as CP190 indicate multiple levels at which insulators can be regulated to affect gene expression. These findings suggest a model in which insulator subclasses may have distinct functions that together organize the genome in a cell type-specific manner, resulting in differential regulation of gene expression.

BAT3 and SET1A Form a Complex with CTCFL/BORIS To Modulate H3K4 Histone Dimethylation and Gene Expression

Chromatin status is characterized in part by covalent posttranslational modifications of histones that regulate chromatin dynamics and direct gene expression. BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein that is thought to play a role in chromatin organization and gene expression. BORIS is a cancer-germ line gene; these are genes normally present in male germ cells (testis) that are also expressed in cancer cell lines as well as primary tumors. This work identifies SET1A, an H3K4 methyltransferase, and BAT3, a cochaperone recruiter, as binding partners for BORIS, and these proteins bind to the upstream promoter regions of two well-characterized procarcinogenic genes, Myc and BRCA1. RNA interference (RNAi) knockdown of BAT3, as well as SET1A, decreased Myc and BRCA1 gene expression but did not affect the binding properties of BORIS, but RNAi knockdown of BORIS prevented the assembly of BAT3 and SET1A at the Myc and BRCA1 promoters. Finally, chromatin analysis suggested that BORIS and BAT3 exert their effects on gene expression by recruiting proteins such as SET1A that are linked to changes in H3K4 dimethylation. Thus, we propose that BORIS acts as a platform upon which BAT3 and SET1A assemble and exert effects upon chromatin structure and gene expression.

Characterization, expression and subcellular distribution of a novel MFP1 (matrix attachment region-binding filament-like protein 1) in onion

MFP1 (matrix attachment region-binding filament-like protein 1) is a conserved nuclear and chloroplast DNA-binding protein encoded by a nuclear gene, well characterized in dicot species. In monocots, only a 90 kDa MFP1-related protein had been characterized in the nucleus and nuclear matrix of Allium cepa proliferating cells. We report here a novel MFP1-related nuclear protein of 80 kDa in A. cepa roots, with M(r) and pI values similar to those of MFP1 proteins in dicot species, and which also displays a dual location, in the nucleus and chloroplasts of leaf cells. However, this novel protein is not a nuclear matrix component. It shows a spotted intranuclear distribution in small foci differing from the nuclear bodies containing the 90 kDa protein. In electron microscopy analysis, the intranuclear foci containing the 80 kDa MFP1 appeared as small loose structures at the periphery of condensed chromatin patches. This protein was also located in the nucleolus. It was abundant in meristematic cells, but its level fell when proliferation stopped. This different expression and distribution, and its preferential location at the boundaries between heterochromatin and euchromatin, suggest that the novel 80 kDa protein might be associated with decondensed DNA and could play a role in chromatin organization.

CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region

The CTCF paralog BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein thought to play a role in chromatin organization and gene expression. Under normal physiologic conditions, BORIS is predominantly expressed during embryonic male germ cell development; however, it is also expressed in tumors and tumor cell lines and, as such, has been classified as a cancer-germline or cancer-testis gene. It has been suggested that BORIS may be a pro-proliferative factor, whereas CTCF favors antiproliferation. BORIS and CTCF share similar zinc finger DNA-binding domains and seem to bind to identical target sequences. Thus, one critical question is the mechanism governing the DNA-binding specificity of these two proteins when both are present in tumor cells. Chromatin immunoprecipitation (ChIP) in HCT116 cells and their hypermethylated variant showed that BORIS binds to methylated DNA sequences, whereas CTCF binds to unmethylated DNA. Electromobility shift assays, using both whole-cell extracts and in vitro translated CTCF and BORIS protein, and methylation-specific ChIP PCR showed that BORIS is a methylation-independent DNA-binding protein. Finally, experiments in murine hybrid cells containing either the maternal or paternal human chromosome 11 showed that BORIS preferentially binds to the methylated paternal H19 differentially methylated region, suggesting a mechanism in which the affinity of CTCF for the unmethylated maternal allele directs the DNA binding of BORIS toward the paternal allele.

Otefin, a Nuclear Membrane Protein, Determines the Fate of Germline Stem Cells in Drosophila via Interaction with Smad Complexes

Nuclear envelope proteins play important roles in chromatin organization, gene regulation, and signal transduction; however, the physiological role of these proteins remains elusive. We found that otefin (ote), which encodes a nuclear lamin-binding protein [corrected], is essential for germline stem cell (GSC) maintenance. We show that Ote, as an intrinsic factor, is both necessary and sufficient to regulate GSC fate. Furthermore, we demonstrate that ote is required for the Dpp/BMP signaling pathway to silence bam transcription. By structure-function analysis, we demonstrate that the nuclear membrane localization of Ote is essential for its role in GSC maintenance. Finally, we show that Ote physically interacts with Medea/Smad4 at the bam silencer element to regulate GSC fate. Thus, we demonstrate that specific nuclear membrane components mediate signal-dependent transcriptional effects to control stem cell behavior.

DNA methylation: The nuts and bolts of repression

DNA methylation is an epigenetic modification which plays an important role in chromatin organization and gene expression. DNA methylation can silence genes and repetitive elements through a process which leads to the alteration of chromatin structure. The mechanisms which target DNA methylation to specific sites in the genome are not fully understood. In this review, we will discuss the mechanisms which lead to the long-term silencing of genes and will survey the progression that has been made in determining the targeted mechanisms for de novo DNA methylation.

Influence of Chromatin Structure on Induction of Double-Strand Breaks in Mammalian Cells Irradiated with DNA-Incorporated125I

In this study the induction of double-strand breaks (DSBs) was investigated in Chinese hamster V79-379A cells irradiated with the Auger-electron emitter (125)I incorporated into DNA. The role of chromatin organization was studied by pulse-labeling synchronized cells with (125)IdU before decay accumulation in early or late S phase. Pulsed-field gel electrophoresis and fragment-size analysis were used to quantify the distribution of DNA fragments in irradiated intact cells and naked DNA as well as in DNA from asynchronously labeled cultures in a different scavenging environment. The results show that in intact cells, after accumulation of decays at -70 degrees C in the presence of 10% DMSO, almost four times more DSBs were induced in late S phase compared with early S phase and the fragment distribution was clearly non-random with an excess of fragments <0.2 Mbp. The DSB yield was 0.6 DSB/cell and decay for cells irradiated in early S phase and 2.3 DSBs/cell and decay for cells irradiated in late S phase. When similar experiments were performed on naked genomic DNA or intact cells irradiated with gamma rays, the difference in yield was not as prominent. These data imply a role of chromatin organization in the induction of DSBs by DNA-incorporated (125)I. In summary, the results presented here suggest that the yield of DSBs as well as the fragment distribution induced by (125)IdU decay may vary significantly depending on the chromatin organization during S phase and the labeling procedure used.

Dynamic Regulation of Histone H3 Methylation at Lysine 4 in Mammalian Spermatogenesis1

Spermatogenesis is a highly complex cell differentiation process that is governed by unique transcriptional regulation and massive chromatin alterations, which are required for meiosis and postmeiotic maturation. The underlying mechanisms involve alterations to the epigenetic layer, including histone modifications and incorporation of testis-specific nuclear proteins, such as histone variants and protamines. Histones can undergo methylation, acetylation, and phosphorylation among other modifications at their N-terminus, and these modifications can signal changes in chromatin structure. We have identified the temporal and spatial distributions of histone H3 mono-, di-, and trimethylation at lysine 4 (K4), and the lysine-specific histone demethylase AOF2 (amine oxidase flavin-containing domain 2, previously known as LSD1) during mammalian spermatogenesis. Our results reveal tightly regulated distributions of H3-K4 methylation and AOF2, and that H3-K4 methylation is very similar between the mouse and the marmoset. The AOF2 protein levels were found to be higher in the testes than in the somatic tissues. The distribution of AOF2 matched the cell- and stage-specific patterns of H3-K4 methylation. Interaction studies revealed unique epigenetic regulatory complexes associated with H3-K4 methylation in the testis, including the association of AOF2 and methyl-CpG-binding domain protein 2 (MBD2a/b) in a complex with histone deacetylase 1 (HDAC1). These studies enhance our understanding of epigenetic modifications and their roles in chromatin organization during male germ cell differentiation in both normal and pathologic states.

141st ENMC International Workshop Inaugural Meeting of the EURO-Laminopathies Project Nuclear Envelope-linked Rare Human Diseases: From Molecular Pathophysiology towards Clinical Applications 10–12 March 2006, Naarden, The Netherlands

EURO-Laminopathies is a European Commissionfunded research project, including 13 scientific group leaders in the fields of human genetics, clinical research, structural biology, molecular cell biology, and pharmaceutical industry, and one administrative manager from eight countries (Austria, France, Germany, Italy Israel, Spain, Switzerland and the United Kingdom). The consortium held its kick-off meeting as an International ENMC Workshop in Naarden on the weekend of March 10–12, 2006. The EURO-Laminopathies project aims at understanding the molecular mechanisms of laminopathies, which are rare human diseases linked tomutations in genes encoding nuclear envelope proteins, such as A-type lamins (LMNA), proteins involved in the post-translational processing of A-type lamins (ZMPSTE24), and lamin-binding proteins (EMD, LBR, LAP2). Laminopathies are clinically manifested after birth, can affect different tissues andprogressively develop during childhood or adolescence, often leading to early death. Efficient therapies have been hampered by the lack

Chromatin Immunoprecipitation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications

INTRODUCTIONIn cells and tissues, the histone proteins that constitute the nucleosomes can present multiple post-translational modifications, such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and lysine ubiquitination. On their own, or in combination, these covalent modifications on the core histones are thought to play essential roles in chromatin organization and gene expression in eukaryotes. Importantly, patterns of histone modifications may be somatically conserved and can, thereby, maintain locus-specific repression/activity in defined lineages, or throughout development. Indirect immunofluorescence studies on cultured cells have been pivotal in unraveling the roles of histone modifications. However, to address in detail what happens at specific sites in vivo, chromatin immunoprecipitation (ChIP) is the method of choice. Here, we describe how ChIP can be performed on non-fixed chromatin from animal cells or tissues (fresh or frozen) to analyze histone modifications at specific chromosomal sites. These protocols are suitable only for analyzing histones and their modifications. For other applications, chromatin immunoprecipitation should be performed on cross-linked chromatin.

Transcriptional Coactivator PC4, a Chromatin-Associated Protein, Induces Chromatin Condensation

Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G2/M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization.

Scaffold/matrix attachment regions and intrinsic DNA curvature

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs.

Glucocorticoid sensitivity: pathology, mutations and clinical implications

Glucocorticoids exert diverse effects on virtually all cell types and tissues. Subtle changes in sensitivity may be generalized and congenital or acquired in a tissue-specific manner. Such changes may lead to altered susceptibility to metabolic diseases, such as ischemic heart disease, or to insensitivity to the therapeutic actions of synthetic glucocorticoids such as in inflammatory disease. This review will cover current theories of how glucocorticoids exert genetic and other congenital effects on glucocorticoid sensitivity, and acquired changes in glucocorticoid sensitivity seen principally in inflammatory and malignant disease. Recent important developments in the field include the impact of genetic variation within the glucocorticoid receptor gene, the effects of early life experience on long-term glucocorticoid sensitivity, studies identifying the role of nuclear factor κB in modulating glucocorticoid sensitivity in vitro and in vivo, and the action of macrophage migration inhibitory factor in modulating the anti-inflammatory effects of glucocorticoids. The role of chromatin organization in regulating glucocorticoid action on proinflammatory genes is discussed, as is the regulation of glucocorticoid sensitivity in human malignancy in the context of pathogenesis and treatment response.

Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region

In the yeast Saccharomyces cerevisiae that lacks lamins, the nuclear pore complex (NPC) has been proposed to serve a role in chromatin organization. Here, using fluorescence microscopy in living cells, we show that nuclear pore proteins of the Nup84 core complex, Nup84p, Nup145Cp, Nup120p, and Nup133p, serve to anchor telomere XI-L at the nuclear periphery. The integrity of this complex is shown to be required for repression of a URA3 gene inserted in the subtelomeric region of this chromosome end. Furthermore, altering the integrity of this complex decreases the efficiency of repair of a DNA double-strand break (DSB) only when it is generated in the subtelomeric region, even though the repair machinery is functional. These effects are specific to the Nup84 complex. Our observations thus confirm and extend the role played by the NPC, through the Nup84 complex, in the functional organization of chromatin. They also indicate that anchoring of telomeres is essential for efficient repair of DSBs occurring therein and is important for preserving genome integrity.

The mobile nucleoporin Nup2p and chromatin-bound Prp20p function in endogenous NPC-mediated transcriptional control

Nuclear pore complexes (NPCs) govern macromolecular transport between the nucleus and cytoplasm and serve as key positional markers within the nucleus. Several protein components of yeast NPCs have been implicated in the epigenetic control of gene expression. Among these, Nup2p is unique as it transiently associates with NPCs and, when artificially tethered to DNA, can prevent the spread of transcriptional activation or repression between flanking genes, a function termed boundary activity. To understand this function of Nup2p, we investigated the interactions of Nup2p with other proteins and with DNA using immunopurifications coupled with mass spectrometry and microarray analyses. These data combined with functional assays of boundary activity and epigenetic variegation suggest that Nup2p and the Ran guanylyl-nucleotide exchange factor, Prp20p, interact at specific chromatin regions and enable the NPC to play an active role in chromatin organization by facilitating the transition of chromatin between activity states.

The nuclear lamina comes of age

Many nuclear proteins form lamin-dependent complexes, including LEM-domain proteins, nesprins and SUN-domain proteins. These complexes have roles in chromatin organization, gene regulation and signal transduction. Some link the nucleoskeleton to cytoskeletal structures, ensuring that the nucleus and centrosome assume appropriate intracellular positions. These complexes provide new insights into cell architecture, as well as a foundation for the understanding of the molecular mechanisms that underlie the human laminopathies - clinical disorders that range from Emery-Dreifuss muscular dystrophy to the accelerated ageing seen in Hutchinson-Gilford progeria syndrome.