MAR-binding Proteins Research Articles

Chromatin attachment to the nuclear matrix represses hypocotyl elongation in Arabidopsis thaliana

The nuclear matrix is a nuclear compartment that has diverse functions in chromatin regulation and transcription. However, how this structure influences epigenetic modifications and gene expression in plants is largely unknown. In this study, we show that a nuclear matrix binding protein, AHL22, together with the two transcriptional repressors FRS7 and FRS12, regulates hypocotyl elongation by suppressing the expression of a group of genes known as SMALL AUXIN UP RNAs (SAURs) in Arabidopsis thaliana. The transcriptional repression of SAURs depends on their attachment to the nuclear matrix. The AHL22 complex not only brings these SAURs, which contain matrix attachment regions (MARs), to the nuclear matrix, but it also recruits the histone deacetylase HDA15 to the SAUR loci. This leads to the removal of H3 acetylation at the SAUR loci and the suppression of hypocotyl elongation. Taken together, our results indicate that MAR-binding proteins act as a hub for chromatin and epigenetic regulators. Moreover, we present a mechanism by which nuclear matrix attachment to chromatin regulates histone modifications, transcription, and hypocotyl elongation.

Satb2 regulates proliferation and nuclear integrity of pre-osteoblasts.

Special AT-rich sequence binding protein 2 (Satb2) is a matrix attachment region (MAR) binding protein. Satb2 impacts skeletal development by regulating gene transcription required for osteogenic differentiation. Although its role as a high-order transcription factor is well supported, other roles for Satb2 in skeletal development remain unclear. In particular, the impact of dosage sensitivity (heterozygous mutations) and variance on phenotypic severity is still not well understood. To further investigate molecular and cellular mechanisms of Satb2-mediated skeletal defects, we used the CRISPR/Cas9 system to generate Satb2 mutations in MC3T3-E1 cells. Our data suggest that, in addition to its role in differentiation, Satb2 regulates progenitor proliferation. We also find that mutations in Satb2 cause chromatin defects including nuclear blebbing and donut-shaped nuclei. These defects may contribute to a slight increase in apoptosis in mutant cells, but apoptosis is insufficient to explain the proliferation defects. Satb2 expression exhibits population-level variation and is most highly expressed from late G1 to late G2. Based on these data, we hypothesize that Satb2 may regulate proliferation through two separate mechanisms. First, Satb2 may regulate the expression of genes necessary for cell cycle progression in pre-osteoblasts. Second, similar to other MAR-binding proteins, Satb2 may participate in DNA replication. We also hypothesize that variation in the severity or penetrance of Satb2-mediated proliferation defects is due to stochastic variation in Satb2 binding to DNA, which may be buffered in some genetic backgrounds. Further elucidation of the role of Satb2 in proliferation has potential impacts on our understanding of both skeletal defects and cancer.

Expression of nuclear matrix proteins binding matrix attachment regions in prostate cancer. PARP-1: New player in tumor progression.

Prostate cancer (PCa) displays infrequent point mutations, whereas genomic rearrangements are highly prevalent. In eukaryotes, the genome is compartmentalized into chromatin loop domains by the attachment to the nuclear matrix (NM), and it has been demonstrated that several recombination hot spots are situated at the base of loops. Here, we have characterized the binding between NM proteins and matrix attachment regions (MARs) in PCa. Nontumor and 44 PCa tissues were analyzed. More aggressive tumors were characterized by an increase in the complexity of the NM protein patterns that was synchronous with a decrease in the number of proteins binding the MAR sequences. PARP-1 was the protein that showed the most evident changes. The expression of the PARP-1 associated with NM increased and it was dependent on tumor aggressiveness. Immunohistochemical analysis showed that the protein was significantly overexpressed in tumor cells. To explore the role of PARP-1 in PCa progression, PCa cells were treated with the PARP inhibitor, ABT-888. In androgen-independent PC3 cells, PARP inhibition significantly decreased cell viability, migration, invasion, chromatin loop dimensions and histone acetylation. Collectively, our study provides evidence that MAR-binding proteins are involved in the development and progression of PCa. PARP could play a key role in the compartmentalization of chromatin and in the development of the more aggressive phenotype. Thus, PARP can no longer be viewed only as an enzyme involved in DNA repair, but that its role in chromatin modulation could provide the basis for a new therapeutic approach to the treatment of PCa.

Role of Nuclear Matrix Associated Region (mar) Binding Proteins in the Regulation of T Helper Cell Differentiation

Nuclear matrix is the major hub of nuclear functions that includes transcription, post transcriptional splicing, DNA repair etc. and also majorly involved in the differentiation of T cell programmes. Though plenty of reports illustrate about the role of lineage specific transcription factors involved in the process of T cell differentiation, not much is known about the active participation of nuclear matrix associated proteins in the modulation of chromatin architecture necessary for the T cell differentiation. This review comprehensively explore the necessary role of nuclear matrix associated proteins in the modulation of chromatin architecture relative to gene positioning, necessary to facilitate the T cell differentiation pattern.

Lessons from Anaplasma phagocytophilum: Chromatin Remodeling by Bacterial Effectors

Bacterial pathogens can alter global host gene expression via histone modifications and chromatin remodeling in order to subvert host responses, including those involved with innate immunity, allowing for bacterial survival. Shigella flexneri, Listeria monocytogenes, Chlamydia trachomatis, and Anaplasma phagocytophilum express effector proteins that modify host histones and chromatin structure. A. phagocytophilum modulates granulocyte respiratory burst in part by dampening transcription of several key phagocyte oxidase genes. The A. phagocytophilum protein AnkA localizes to the myeloid cell nucleus where it binds AT-rich regions in the CYBB promoter and decreases its transcription. AT-rich regions of DNA are characteristic of matrix attachment regions (MARs) which are critical for chromatin structure and transcription. MAR-binding proteins, such as SATB1, interact with histone modifying enzymes resulting in altered gene expression. With A. phagocytophilum infection, histone deacetylase 1 (HDAC1) expression is increased and histone H3 acetylation is decreased at the CYBB promoter, suggesting a role for AnkA in altering host epigenetics and modulating gene transcription, at this, and perhaps other loci. This review will focus on how bacterial pathogens alter host epigenetics, by specifically examining A. phagocytophilum AnkA cis-regulation of CYBB transcription and epigenetic changes associated with infection.

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.

Cis -regulation of microRNA expression by scaffold/matrix-attachment regions

microRNAs (miRNAs) spatio-temporally modulate gene expression; however, very little is known about the regulation of their expression. Here, we hypothesized that the well-known cis-regulatory elements of gene expression, scaffold/matrix-attachment regions (MARs) could modulate miRNA expression. Accordingly, we found MARs to be enriched in the upstream regions of miRNA genes. To determine their role in cell type-specific expression of miRNAs, we examined four individual miRNAs (let-7b, miR-17, miR-93 and miR-221) and the miR-17–92 cluster, known to be overexpressed in neuroblastoma. Our results show that MARs indeed define the cell-specific expression of these miRNAs by tethering the chromatin to nuclear matrix. This is brought about by cell type-specific binding of HMG I/Y protein to MARs that then promotes the local acetylation of histones, serving as boundary elements for gene activation. The binding, chromatin tethering and gene activation by HMG I/Y was not observed in fibroblast control cells but were restricted to neuroblastoma cells. This study implies that the association of MAR binding proteins to MARs could dictate the tissue/context specific regulation of miRNA genes by serving as a boundary element signaling the transcriptional activation.

Characterization and heterologous expression of a new matrix attachment region binding protein from the unicellular green alga Dunaliella salina

Although interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) are implicated in various nuclear functions, the understanding of the regulatory mechanism of MARs is still poor. A few MAR-binding proteins (MARBP) have been isolated from some plants and animals, but not from the unicellular algae. Here, we identify a novel MAR-binding protein, namely DMBP-1, from the halotolerant alga Dunaliella salina. The cDNA of DMBP-1 is 2322-bp long and contains a 1626 bp of an open reading frame encoding a polypeptide of 542 amino acids (59 kDa). The DMBP-1 expressed in Escherichia coli specifically binds A/T-rich MAR DNA. The DMBP-1 fused to green fluorescent protein appears only inside the nuclei of Chinese hamster ovarian cells transfected with the pEGFP-MBP, indicating that the protein is located in the nuclei. The findings mentioned above may contribute to better understanding of the nuclear matrix-MAR interactions.

The L2a element is a mouse CD8 silencer that interacts with MAR-binding proteins SATB1 and CDP

Previous transgenic-reporter and targeted-deletion studies indicate that the subset-specific expression of CD8αβ heterodimers is controlled by multiple enhancer activities, since no silencer elements had been found within the locus. We have identified such a silencer as L2a, a previously characterized ∼220 bp nuclear matrix associating region (MAR) located ∼4.5 kb upstream of CD8α. L2a transgenes driven by the E8 I enhancer showed no reporter expression in thymic subsets or T cells in splenic, inguinal and mesenteric lymph node peripheral T cells. Deletion of L2a resulted in significant reporter de-repression, even in the CD4 +CD8 + double positive (DP) thymocyte population. L2a contains binding sites for two MAR-interacting proteins, SATB1 and CDP. We found that that binding of these factors was markedly influenced by the content and spacing of L2a sub-motifs (L and S) and that SATB1 binds preferentially to the L motif both in vitro and in vivo. A small fraction of the transgenic CD8 single positive (SP) thymocytes and peripheral CD8 + T cells bypassed L2a-silencing to give rise to variegated expression of the transgenic reporter. Crossing the L2a-containing transgene onto a SATB1 knockdown background enhanced variegated expression, suggesting that SATB1 is critical in overcoming L2a-silenced transcription.

Modulation of chromatin by MARs and MAR binding oncogenic transcription factor SMAR1

The orchestration of the events in the cell during the progression of the cell cycle is modulated by various phenomenon which are regulated by structural modules of the cell. The nucleus is a major hub for all these regulatory units which harbour the nuclear matrix, matrix proteins and chromatin. The histone modifications etch a complex code on the chromatin and the matrix proteins in consort with the histone code regulate the gene expression. SMAR1 is a matrix attachment region binding protein that interacts with chromatin modulators like HDAC1, Sin3A and causes chromatin condensation. SMAR1 modulates the chromatin at the Vbeta locus and plays a prominent role in V(D)J recombination. Such indispensable function of SMAR1 by the modulation of chromatin in the context of malignancy and V(D)J recombination emphasizes that MAR binding proteins regulate the complex events of the cell and perturbed expression causes disease conditions.

SATB1 binds an intronic MAR sequence in human PI3kγ in vitro

In our previous study, an intronic MAR sequence in human PI3Kgamma gene (PIMAR) was identified using bioinformatics and biochemical methods. We used MatInspector software to identify potential binding sites for MAR-binding proteins in PIMAR. In this study, a tissue-specific MAR-binding protein (SATB1) was used to characterize the potential binding sites. Southwestern blot analysis indicates that recombinant SATB1 directly binds PIMAR sequence in vitro. Reporter gene assay showed that overexpression of SATB1 downregulates the luciferase reporter linked with reversed PIMAR by approximately threefold in the NIH-3T3 cell line. These results indicate that SATB1 may play antagonistic roles in PI3Kgamma transcriptional regulation.

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7–7.0 and 6.2–7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.

The behavior of SATB1, a MAR‐binding protein, in response to apoptosis stimulation

As a MAR-binding protein, SATB1 regulates genes by folding chromatin into a loop domain. Apoptosis is known to be accompanied by a collapse of nuclear architecture and cleavage of condensing chromatin into oligonucleosomal fragments. To further understand the functional role of MAR-binding proteins during apoptosis we investigated the relationship of the behavior of SATB1 and the collapse of nuclear architecture in Jurkat cells with immunostaining and Western blot analysis. We demonstrated that SATB1 formed special three-dimensional network distributions during early apoptosis. The distribution change of SATB1 was associated with cleavage of the protein and accompanied by the nuclear architecture collapse. Cleavage of SATB1 was mediated by caspase-3 and was apoptosis specific. Our observations further support the notion that early proteolysis of MAR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.

Specificity of SAF-A and lamin B binding in vitro correlates with the satellite DNA bending state.

There is evidence that Matrix Attachment Region (MAR)-binding proteins also bind satellite DNA (satDNA). The aim of the current work was to determine whether the major nuclear matrix (NM) MAR-binding proteins are able to recognize satDNAs of different locations and what DNA structural features are important for the recognition. In nuclei and NM, a number of the same polypeptides were recognized on a southwestern blot when MAR of immunoglobulin kappa gene (Ig kappa MAR) and pericentromeric (periCEN) satDNA fragments were used. However, the binding decreased dramatically when human and mouse CEN satDNA were used for the probes. After an NM extract was subjected to ion exchange chromatography, the main DNA-binding proteins were identified as SAF-A (scaffold attachment factor A) and lamin B. It was not possible to test the binding of lamin B by gel mobility shift assay (GMSA), but SAF-A showed an ability to distinguish CEN and periCEN satDNA fragments in GMSA. While periCEN fragments have an abnormally slow mobility on electrophoresis, which is a hallmark of bent DNA, CEN satDNA fragments have a normal mobility. A computer analysis was done using the wedge model (Ulanovsky and Trifonov [1987] Nature 326:720-722), which describes how the curved state depends on particular nucleotide sequences. The curved states of the fragments predicted by the model are in good agreement with their ability to be recognized by NM proteins. Thus SAF-A and lamin B are able to recognize conserved structural features of satDNA in the same way that MAR-binding proteins recognize MARs in spite of a lack of a consensus sequence. CEN and periCEN satDNAs are distinguished by proteins in correlation with the helical curvature of these fragments.

SMAR1, a Novel, Alternatively Spliced Gene Product, Binds the Scaffold/Matrix-Associated Region at the T Cell Receptor β Locus

Rearrangement and expression of the T cell receptor β gene are critical events for early T lymphocyte development. To characterize cis-regulatory elements and their associated trans-factors that mediate these events, we have previously identified a nuclear matrix/scaffold-associated region, referred to as MARβ, 400 bp upstream of the Eβ enhancer. Electrophoretic mobility shift assay showed that two known MAR-binding proteins, SATB1 and Cux, bind MARβ. In this article, we report the identification of a novel MAR-binding protein, named SMAR1, that also binds MARβ. SMAR1 shares homology with SATB1 and Cux in the MAR-binding domain/Cut repeat and also with the tetramerization domain of a B cell-specific MAR-binding protein, Bright. The binding of GST–SMAR1 fusion protein to MARβ is inhibited by the presence of an excess amount of MAR-containing DNA from the immunoglobulin κ locus. Smar1 transcripts are most abundant in the thymus and are alternatively spliced. The smar1 gene maps to the distal portion of mouse chromosome 8 at a distance of 111.8 cM.

The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during T-cell development

SATB1 is expressed primarily in thymocytes and can act as a transcriptional repressor. SATB1 binds in vivo to the matrix attachment regions (MARs) of DNA, which are implicated in the loop domain organization of chromatin. The role of MAR-binding proteins in specific cell lineages is unknown. We generated SATB1-null mice to determine how SATB1 functions in the T-cell lineage. SATB1-null mice are small in size, have disproportionately small thymi and spleens, and die at 3 weeks of age. At the cellular level, multiple defects in T-cell development were observed. Immature CD3(-)CD4(-)CD8(-) triple negative (TN) thymocytes were greatly reduced in number, and thymocyte development was blocked mainly at the DP stage. The few peripheral CD4(+) single positive (SP) cells underwent apoptosis and failed to proliferate in response to activating stimuli. At the molecular level, among 589 genes examined, at least 2% of genes including a proto-oncogene, cytokine receptor genes, and apoptosis-related genes were derepressed at inappropriate stages of T-cell development in SATB1-null mice. For example, IL-2Ralpha and IL-7Ralpha genes were ectopically transcribed in CD4(+)CD8(+) double positive (DP) thymocytes. SATB1 appears to orchestrate the temporal and spatial expression of genes during T-cell development, thereby ensuring the proper development of this lineage. Our data provide the first evidence that MAR-binding proteins can act as global regulators of cell function in specific cell lineages.

Two MAR DNA-binding proteins of the pea nuclear matrix identify a new class of DNA-binding proteins.

Four MAR-binding proteins of 60, 65, 70 and 72 kDa have been detected by South-Western blotting and isolated from pea nuclear matrices. Two cDNAs encoding the 60 and 65 kDa proteins (MARBP-1 and MARBP-2) were isolated from a pea leaf cDNA library by screening with a PCR product obtained using degenerate primers based on an amino acid sequence from the 60 kDa protein. The proteins of 560 and 550 amino acids are 86% identical and contain several KKD/E repeats near the C-terminus. Escherichia coli-expressed MARBP-1 specifically binds A/T-rich MAR DNA. The interaction of MARBP-1/MARBP-2 with MAR DNA involves novel DNA-binding motifs. The MARBP-1 and MARBP-2 genes are expressed in a range of pea tissues and are encoded by genes at different loci. MARBP-1 and MARBP-2 are homologous to yeast nucleolar proteins Nop56p and Nop58p, which are involved in ribosome biogenesis, and to similar highly conserved proteins in other eukaryotes and in archaebacteria. MARBP-1 and MARBP-2 may have multifunctional roles in chromatin organisation and ribosome biogenesis.

The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression.

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.

Nucleolin is a matrix attachment region DNA-binding protein that specifically recognizes a region with high base-unpairing potential.

A DNA affinity column containing a synthetic double-stranded nuclear matrix attachment region (MAR) was used to purify a 100-kDa protein from human erythroleukemia K562 cells. This protein was identified as nucleolin, the key nucleolar protein of dividing cells, which is thought to control rRNA gene transcription and ribosome assembly. Nucleolin is known to bind RNA and single-stranded DNA. We report here that nucleolin is also a MAR-binding protein. It binds double-stranded MARs from different species with high affinity. Nucleolin effectively distinguishes between a double-stranded wild-type synthetic MAR sequence with a high base-unpairing potential and its mutated version that has lost the unpairing capability but is still A+T rich. Thus, nucleolin is not merely an A+T-rich sequence-binding protein but specifically binds the base-unpairing region of MARs. This binding specificity is similar to that of the previously cloned tissue-specific MAR-binding protein SATB1. Unlike SATB1, which binds only double-stranded MARs, nucleolin binds the single-stranded T-rich strand of the synthetic MAR probe approximately 45-fold more efficiently than its complementary A-rich strand, which has an affinity comparable to that of the double-stranded form of the MAR. In contrast to the high selectivity of binding to double-stranded MARs, nucleolin shows only a small but distinct sequence preference for the T-rich strand of the wild-type synthetic MAR over the T-rich strand of its mutated version. The affinity to the T-rich synthetic MAR is severalfold higher than to its corresponding RNA and human telomere DNA. Quantitative cellular fractionation and extraction experiments indicate that nucleolin is present both as a soluble protein and tightly bound to the matrix, similar to other known MAR-binding proteins.

Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.