Chicken MAR-binding protein ARBP is homologous to rat methyl-CpG-binding protein MeCP2.

Here, we describe the cloning and further characterization of chicken ARBP, an abundant nuclear protein with a high affinity for MAR/SARs. Surprisingly, ARBP was found to be homologous to the rat protein MeCP2, previously identified as a methyl-CpG-binding protein. A region spanning 125 amino acids in the N-terminal halves is 96.8% identical between chicken ARBP and rat MeCP2. A deletion mutation analysis using Southwestern and band shift assays identified this highly conserved region as the MAR DNA binding domain. Alignment of chicken ARBP with rat and human MeCP2 proteins revealed six trinucleotide amplifications generating up to 34-fold repetitions of a single amino acid. Because MeCP2 was previously localized to pericentromeric heterochromatin in mouse chromosomes, we analyzed the in vitro binding of ARBP to various repetitive sequences. In band shift experiments, ARBP binds to two chicken repetitive sequences as well as to mouse satellite DNA with high affinity similar to that of its binding to chicken lysozyme MAR fragments. In mouse satellite DNA, use of several footprinting techniques characterized two high-affinity binding sites, whose sequences are related to the ARBP binding site consensus in the chicken lysozyme MAR (5'-GGTGT-3'). Band shift experiments indicated that methylation increased in vitro binding of ARBP to mouse satellite DNA two- to fivefold. Our results suggest that ARBP/MeCP2 is a multifunctional protein with roles in loop domain organization of chromatin, the structure of pericentromeric heterochromatin, and DNA methylation.     

The role of nuclear matrix proteins binding to matrix attachment regions (Mars) in prostate cancer cell differentiation

In tumor progression definite alterations in nuclear matrix (NM) protein composition as well as in chromatin structure occur. The NM interacts with chromatin via specialized DNA sequences called matrix attachment regions (MARs). In the present study, using a proteomic approach along with a two-dimensional Southwestern assay and confocal laser microscopy, we show that the differentiation of stabilized human prostate carcinoma cells is marked out by modifications both NM protein composition and bond between NM proteins and MARs. Well-differentiated androgen-responsive and slowly growing LNCaP cells are characterized by a less complex pattern and by a major number of proteins binding MAR sequences in comparison to 22Rv1 cells expressing androgen receptor but androgen-independent. Finally, in the poorly differentiated and strongly aggressive androgen-independent PC3 cells the complexity of NM pattern further increases and a minor number of proteins bind the MARs. Furthermore, in this cell line with respect to LNCaP cells, these changes are synchronous with modifications in both the nuclear distribution of the MAR sequences and in the average loop dimensions that significantly increase. Although the expression of many NM proteins changes during dedifferentiation, only a very limited group of MAR-binding proteins seem to play a key role in this process. Variations in the expression of poly (ADP-ribose) polymerase (PARP) and special AT-rich sequence-binding protein-1 (SATB1) along with an increase in the phosphorylation of lamin B represent changes that might trigger passage towards a more aggressive phenotype. These results suggest that elucidating the MAR-binding proteins that are involved in the differentiation of prostate cancer cells could be an important tool to improve our understanding of this carcinogenesis process, and they could also be novel targets for prostate cancer therapy.     

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.     

The insulator binding protein CTCF associates with the nuclear matrix

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.     

核基质附着区与转基因表达

核基质附着区 (matrixattachmentregions,MARs)是与核基质 (或核骨架 )特异结合的DNA序列 ,属于非编码序列 ,富含AT。通过与核基质的结合 ,它能使染色质形成独立的环状结构 ,调控基因的转录和表达 ,减少由于位置效应引起的转基因沉默。MARs在提高转基因表达水平、消除转基因个体间表达水平的差异、抑制转基因沉默等方面起着重要的作用。就MAR的一些结构功能特征及其在基因工程中的应用等方面进行了阐述。     

Chicken MAR binding protein p120 is identical to human heterogeneous nuclear ribonucleoprotein (hnRNP) U.

We have previously identified two proteins from chicken oviduct nuclei that specifically bind to matrix/scaffold attachment regions (MARs/SARs). Here one of these proteins, named p120 due to its apparent molecular weight, is purified to near homogeneity and shown to be identical to a previously described component of heterogeneous nuclear ribonucleoprotein particles, hnRNP U, on the basis of amino acid sequence analysis of tryptic peptides. p120 binds to multiple MAR fragments provided they have a minimal length of approximately 700 bp. Binding of MAR fragments is specifically competed by homoribopolymers poly(G) and poly(I), which form four-stranded structures. Our results suggest that p120/hnRNP U may serve a dual function, first as a component of hnRNP particles, and second as an element in the higher-order organization of chromatin.     

AHM1, a novel type of nuclear matrix-localized, MAR binding protein with a single AT hook and a J domain-homologous region

Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook-containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain-homologous region and a Zn finger-like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger-like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones.     

Possible role of H1 histone in replication timing

AT‐rich repetitive DNA sequences become late replicating during cell differentiation. Replication timing is not correlated with LINE density in human cells (Ryba et al. 2010). However, short and properly spaced runs of oligo dA or dT present in nuclear matrix attachment regions (MARs) of the genome are good candidates for elements of AT‐rich repetitive late replicating DNA. MAR attachment to the nuclear matrix is negatively regulated by chromatin binding of H1 histone, but this is counteracted by H1 phosphorylation, high mobility group proteins or, indirectly, core histone acetylation. Fewer MAR attachments correlates positively with longer average DNA loop size, longer replicons and an increase of late replicating DNA.     

杜氏盐藻中的核基质与核基质结合区

真核生物细胞核DNA通过核基质结合区（Matrix attachment region,MAR）附着到核基质上。为了进一步探索染色体DNA与核基质之间的相互作用,从单细胞真核藻类-杜氏盐藻中克隆出了MAR片段。首先构建了杜氏盐藻的随机MAR文库,通过体外结合实验分离出能与核基质结合的MAR序列。从构建的MAR文库中,筛选出3个能与核基质结合的MAR,其中两个片段与核基质具有较强的结合力,测序分析表明具有MAR片段的一些典型特征性基序。     

Suppression of transgene silencing by matrix attachment regions in maize: a dual role for the maize 5' ADH1 matrix attachment region

Matrix attachment regions (MARs) are DNA sequences that bind an internal nuclear network of nonhistone proteins called the nuclear matrix. Thus, they may define discrete gene-containing chromatin loops in vivo. We have studied the effects of flanking transgenes with MARs on transgene expression levels in maize callus and in transformed maize plants. Three MAR elements, two from maize (Adh1 5' MAR and Mha1 5' MAR) and one from yeast (ARS1), had very different effects on transgene expression that bore no relation to their affinity for the nuclear matrix in vitro. In callus, two of the MAR elements (Adh1 5' MAR and ARS1) reduced transgene silencing but had no effect on the variability of expression. In transgenic plants, Adh1 5' MAR had the effect of localizing beta-glucuronidase expression to lateral root initiation sites. A possible model accounting for the function of Adh1 5' MAR is discussed.     

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Genomic DNA in higher eucaryotic cells is organized into a series of loops, each of which may be affixed at its base to the nuclear matrix via a specific matrix attachment region (MAR). In this report, we describe the distribution of MARs within the amplified dihydrofolate reductase (DHFR) domain (amplicon) in the methotrexate-resistant CHO cell line CHOC 400. In one experimental protocol, matrix-attached and loop DNA fractions were prepared from matrix-halo structures by restriction digestion and were analyzed for the distribution of amplicon sequences between the two fractions. A second, in vitro method involved the specific binding to the matrix of cloned DNA fragments from the amplicon. Both methods of analysis detected a MAR in the replication initiation locus that we have previously defined in the DHFR amplicon, as well as in the 5'-flanking region of the DHFR gene. The first of these methods also suggests the presence of a MAR in a region mapping approximately 120 kilobases upstream from the DHFR gene. Each of these MARs was detected regardless of whether the matrix-halo structures were prepared by the high-salt or the lithium 3,5-diiodosalicylate extraction protocols, arguing against their artifactual association with the proteinaceous scaffolding of the nucleus during isolation procedures. However, the in vitro binding assay did not detect the MAR located 120 kilobases upstream from the DHFR gene but did detect specific matrix attachment of a sequence near the junction between amplicons. The results of these experiments suggest that (i) MARs can occur next to different functional elements in the genome, with the result that a DNA loop formed between two MARs can be smaller than a replicon; and (ii) different methods of analysis detect a somewhat different spectrum of matrix-attached DNA fragments.     

Use of matrix attachment regions (MARs) to minimize transgene silencing

Matrix attachment regions (MARs) are operationally defined as DNA elements that bind specifically to the nuclear matrix in vitro. It is possible, although unproven, that they also mediate binding of chromatin to the nuclear matrix in vivo and alter the topology of the genome in interphase nuclei. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable expression in transgenic plants or cell lines, most likely by minimizing gene silencing. Our review explores current data and presents several plausible models to explain MAR effects on transgene expression.