Nuclear Matrix Targeting Sequence Research Articles

Expansion of polyalanine tracts in the QA domain may play a critical role in the clavicular development of cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal-dominant inheritable disease which is secondary to haploinsufficiency of the transcription factor runt-related transcription factor 2 (RUNX2). During the past 10 years, additional cases of RUNX2 mutation have been identified in nearly 500 families with CCD, including familial and sporadic cases (Otto et al. 2002). Some degree of clavicular hypoplasia is the most consistent feature of the disease and is often opposed at the mid line (Tang et al. 2007), while complete absence is rare and complicated to explain the specific mechanism. RUNX2, which is mapped to chromosome 6p21, was identified as the CCD causative gene in 1997 (Mundlos et al. 1997). This gene is the key transcription factor for osteoblastic differentiation, chondrocyte maturation and skeletal morphogenesis (Komori et al. 1997). Moreover, stability of RUNX2 protein, regulated by CBFb, is required for skeletal development by regulating chondrocyte differentiation, proliferation and osteoblast differentiation (Qin et al. 2014). RUNX2-deficient mice displayed impaired bone development manifestation as wide sutures, decreased bone ossification and hypoplastic clavicles (Kundu et al. 2002). RUNX2 consists of eight coding exons and spans a genomic region of 130 kb. According to the analysis of the protein structure, RUNX2 has several functional domains: QA domain, runt domain, nuclear localization signal (NSL), PST domain, VWRPY and nuclear matrix targeting sequence (NMTS). The QA domain, a stretch of 23 polyglutamine and 17 polyalanine tracts, is located in the N-terminus of RUNX2. An in vitro mutagenesis study showed that the QA domain prevented CBFb from binding to RUNX2. Deletionof the QA domain resulted in a four-fold decrease

Characterization of the nuclear matrix targeting sequence (NMTS) of the BPV1 E8/E2 protein — the shortest known NMTS

Technological advantages in sequencing and proteomics have revealed the remarkable diversity of alternative protein isoforms. Typically, the localization and functions of these isoforms are unknown and cannot be predicted. Also the localization signals leading to particular subnuclear compartments have not been identified and thus, predicting alternative functions due to alternative subnuclear localization is limited only to very few subnuclear compartments. Knowledge of the localization and function of alternative protein isoforms allows for a greater understanding of cellular complexity. In this article, we characterize a short and well-defined signal targeting the bovine papillomavirus type 1 E8/E2 protein to the nuclear matrix. The targeting signal comprises the peptide coded by E8 ORF, which is spliced together with part of the E2 ORF to generate the E8/E2 mRNA. Localization to the nuclear matrix correlates well with the transcription repression activities of E8/E2; a single point mutation directs the E8/E2 protein into the nucleoplasm, and transcription repression activity is lost. Our data prove that adding as few as ˜10 amino acids by alternative transcription/alternative splicing drastically alters the function and subnuclear localization of proteins. To our knowledge, E8 is the shortest known nuclear matrix targeting signal.

Nuclear matrix-targeting of the osteogenic factor Runx2 is essential for its recognition and activation of the alkaline phosphatase gene

BackgroundA good understanding of the mechanism of gene regulation that is involved in bone mineralization is critical for the design of anabolic treatments for bone deficiency diseases. Alkaline phosphatase (ALP) expressed by osteoblasts plays an important role in promoting bone mineralization by hydrolyzing pyrophosphate. However, the mechanism by which the expression of ALP is regulated during osteoblast differentiation has not been thoroughly investigated. MethodsChromatin immunoprecipitation, EMSA and mutagenesis were used to identify the Runx2 binding sites on ALP gene and to analyze the role of nuclear matrix-localization of Runx2 on the recognition and activation of ALP gene. ResultsUsing chromatin immunoprecipitation, we determined that both ectopic and endogenous Runx2 bound to ALP intron 1 in a region containing a cluster of five putative core-sites. The third one (I1C3) among those fives was bound most strongly in vitro by Runx2 and acted as a Runx2-dependent transcriptional enhancer. Furthermore, a Runx2 mutant lacking the nuclear matrix-targeting sequence (Runx2ΔNMTS) bound to the ALP gene less efficiently than the wild-type protein and a Runx2 mutant that is deficient in its ability to bind to DNA (Runx2K120A) accumulated largely in the nuclear matrix. ConclusionsNuclear matrix-localization of Runx2 influences its ALP gene recognition. General significanceOur results showed for the first time that ALP is a direct target gene of Runx2 and illustrated that the recognition/binding and activation of the ALP by this transcription factor are dependent on its nuclear matrix-targeting.

SOX9 determines RUNX2 transactivity by directing intracellular degradation

Mesenchymal stem cell differentiation is controlled by the cooperative activity of a network of signaling mechanisms. Among these, RUNX2 and SOX9 are the major transcription factors for osteogenesis and chondrogenesis, respectively. Their expression is overlapped both temporally and spatially during embryogenesis. Here we have demonstrated that RUNX2 and SOX9 physically interact in intact cells and have confirmed that SOX9 can inhibit the transactivation of RUNX2. In addition, RUNX2 exerts reciprocal inhibition on SOX9 transactivity. In analyses of the mechanism by which SOX9 regulated RUNX2 function, we demonstrated that SOX9 induced a dose-dependent degradation of RUNX2. Although RUNX2 is normally degraded by the ubiquitin-proteasome pathway, we found that SOX9-mediated degradation was proteasome-independent but phosphorylation-dependent and required the presence of the RUNX2 C-terminal domain, which contains a nuclear matrix targeting sequence (NMTS). Furthermore, SOX9 was able to decrease the level of ubiquitinated RUNX2 and direct RUNX2 to the lysosome for degradation. SOX9 also preferentially directed β-catenin, an intracellular mediator of canonical Wnt signaling, for lysosomal breakdown. Consequently, the mechanisms by which SOX9 regulates RUNX2 function may underlie broader signaling pathways that can influence osteochondrogenesis and mesenchymal fate.

Nuclear matrix binding is critical for progesterone receptor movement into nuclear foci

The ovarian hormone progesterone is essential for normal breast development, and progesterone analogues are implicated in increasing breast cancer risk. The progesterone receptor (PR) is a transcription factor that, when ligand activated, moves rapidly into nuclear foci associated with transcriptional activity. However, the role of intranuclear trafficking signals in the focal location of PR is unknown. We have identified a mutation in PR that ablates its binding to the nuclear matrix and prevents PR movement into nuclear foci. Nuclear matrix binding mutants lack transcriptional activity and inhibit dimerization, demonstrating the critical role of matrix binding for PR dynamics and activity. DNA binding of PR is required for fidelity of location in foci, as DNA binding domain (DBD) mutants form aberrant foci with reduced mobility and altered tethering to the nucleus. Mutations in either the nuclear matrix targeting sequence or DBD domains were dominant in preventing wild-type receptor from moving to appropriate nuclear locations, demonstrating that both partner proteins in a PR dimer must have intact intranuclear trafficking signals for correct receptor positioning within the nucleus. This study has demonstrated that positioning of PR in foci within the nucleus is critically regulated by intranuclear trafficking signals, which play a key role in transcriptional activity and are relevant to its action in normal and malignant breast cells.

Modulation of Runx2 Activity by Estrogen Receptor-α: Implications for Osteoporosis and Breast Cancer

The transcription factors Runx2 and estrogen receptor-alpha (ERalpha) are involved in numerous normal and disease processes, including postmenopausal osteoporosis and breast cancer. Using indirect immunofluorescence microscopy and pull-down techniques, we found them to colocalize and form complexes in a ligand-dependent manner. Estradiol-bound ERalpha strongly interacted with Runx2 directly through its DNA-binding domain and only indirectly through its N-terminal and ligand-binding domains. Runx2's amino acids 417-514, encompassing activation domain 3 and the nuclear matrix targeting sequence, were sufficient for interaction with ERalpha's DNA-binding domain. As a consequence of the interaction, Runx2's transcriptional activation activity was strongly repressed, as shown by reporter assays in COS7 cells, breast cancer cells, and late-stage MC3T3-E1 osteoblast cultures. Metaanalysis of gene expression in 779 breast cancer biopsies indicated negative correlation between the expression of ERalpha and Runx2 target genes. Selective ER modulators (SERM) induced ERalpha-Runx2 interactions but led to various functional outcomes. The regulation of Runx2 by ERalpha may play key roles in osteoblast and breast epithelial cell growth and differentiation; hence, modulation of Runx2 by native and synthetic ERalpha ligands offers new avenues in selective ER modulator evaluation and development.

SUMO Conjugation to the Matrix Attachment Region-binding Protein, Special AT-rich Sequence-binding Protein-1 (SATB1), Targets SATB1 to Promyelocytic Nuclear Bodies Where It Undergoes Caspase Cleavage

SATB1 (special AT-rich sequence-binding protein-1) provides a key link between DNA loop organization, chromatin modification/remodeling, and association of transcription factors at matrix attachment regions (MARs). To investigate the role of SATB1 in cellular events, we performed a yeast two-hybrid screen that identified SUMO-1, Ubc9, and protein inhibitor of activated STAT (PIAS) family members as SATB1 interaction partners. These proteins, working in concert, enhanced SUMO conjugation to lysine-744 of SATB1. Overexpression of SUMO or PIAS in Jurkat cells, which express high levels of endogenous SATB1, exhibited enhanced caspase cleavage of this MAR-associating protein. Sumoylation-deficient SATB1 (SATB1(K744R)) failed to display the characteristic caspase cleavage pattern; however, fusion of SUMO in-frame to SATB1(K744R) restored cleavage. A SUMO-independent interaction of inactive caspase-6 and SATB1 was noted. A subset of total cellular SATB1 localized into promyelocytic leukemia nuclear bodies where enhanced SATB1 cleavage was detected subsequent to caspase activation. These results reveal a novel sumoylation-directed caspase cleavage of this key regulatory molecule. The role of regulated proteolysis of SATB1 may be to control transcription in immune cells during normal cell functions or to assist in efficient and rapid clearance of nonfunctional or potentially damaging immune cells.

Sp2 Localizes to Subnuclear Foci Associated with the Nuclear Matrix

We have reported that extracts prepared from many human and mouse cell lines show little or no Sp2 DNA-binding activity and that Sp2 has little or no capacity to stimulate transcription of promoters that are activated by Sp1, Sp3, and Sp4. Using an array of chimeric Sp1/Sp2 proteins we showed further that Sp2 DNA-binding activity and trans-activation are each negatively regulated in mammalian cells. As part of an ongoing effort to study Sp2 function and regulation we characterized its subcellular localization in comparison with other Sp-family members in fixed and live cells. We report that 1) Sp2 localizes largely within subnuclear foci associated with the nuclear matrix, and 2) these foci are distinct from promyelocytic oncogenic domains and appear to be stable during an 18-h time course of observation. Deletion analyses identified a 37 amino acid sequence spanning the first zinc-"finger" that is sufficient to direct nuclear matrix association, and this region also encodes a bipartite nuclear localization sequence. A second nuclear matrix targeting sequence is encoded within the Sp2 trans-activation domain. We conclude that Sp2 preferentially associates with the nuclear matrix and speculate that this subcellular localization plays an important role in the regulation of Sp2 function.

Nuclear Matrix Binding Regulates SATB1-mediated Transcriptional Repression

Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.

Distinct LIM domains of Hic-5/ARA55 are required for nuclear matrix targeting and glucocorticoid receptor binding and coactivation.

Hydrogen peroxide-inducible clone-5 (Hic-5), belongs to the group III LIM domain protein family and contains four carboxyl-terminal LIM domains (LIM1-LIM4). In addition to its role in focal adhesion signaling, Hic-5 acts in the nucleus as a coactivator for some steroid hormone receptors such as the glucocorticoid receptor (GR) and androgen receptor (AR). Based upon its effect on AR transactivation, Hic-5 has also been designated as ARA55. Here, we report mapping studies of Hic-5/ARA55 functional domains and establish that LIM3 and LIM4 are necessary for maximal effects on GR transactivation. However, results from yeast two-hybrid assays demonstrated that these two LIM domains together, while necessary, are not sufficient to interact with the tau2 transactivation domain of GR. LIM4 also functions as a nuclear matrix targeting sequence (NMTS) for Hic-5/ARA55, as it is both necessary and sufficient to target a heterologous protein to the nuclear matrix. Thus, as suggested from previous analysis of LIM domain-containing proteins, separate but highly related LIM domains serve distinct functions.

Functional interaction between nuclear matrix-associated HBXAP and NF-κB

Hepatitis B virus X-associated protein (HBXAP) is a plant homeodomain (PHD) finger-containing protein implicated in transcription regulation. However, the underlying molecular mechanism remains to be defined. Here, we show that HBXAP represses NF-κB-mediated gene activation in a dose-dependent manner. Our results showed that HBXAP and NF-κB colocalize to the nuclear matrix with specific physical interaction between them. HBXAP may depend on its nuclear matrix localization for its repression of NF-κB-mediated gene repression. A specific nuclear matrix targeting sequence of HBXAP was identified. The sequence is included in a region encompassing amino acids 688–722 that could form a coiled-coil structure. The 18-amino acid stretch lies at the core of that structure. The present results showed that either the coiled-coil conformation or the PHD finger domain is crucial for the transcription repression activity of HBXAP on NF-κB-mediated gene activation. Taken together, our results suggest that HBXAP may function as a negative regulator for TNF-α-induced, NF-κB-mediated gene activation.

Functional interaction between nuclear matrix-associated HBXAP and NF-$kappa;B

Hepatitis B virus X-associated protein (HBXAP) is a plant homeodomain (PHD) finger-containing protein implicated in transcription regulation. However, the underlying molecular mechanism remains to be defined. Here, we show that HBXAP represses NF-κB-mediated gene activation in a dose-dependent manner. Our results showed that HBXAP and NF-κB colocalize to the nuclear matrix with specific physical interaction between them. HBXAP may depend on its nuclear matrix localization for its repression of NF-κB-mediated gene repression. A specific nuclear matrix targeting sequence of HBXAP was identified. The sequence is included in a region encompassing amino acids 688–722 that could form a coiled-coil structure. The 18-amino acid stretch lies at the core of that structure. The present results showed that either the coiled-coil conformation or the PHD finger domain is crucial for the transcription repression activity of HBXAP on NF-κB-mediated gene activation. Taken together, our results suggest that HBXAP may function as a negative regulator for TNF-α-induced, NF-κB-mediated gene activation.

Localization of the domains in Runx transcription factors required for the repression of CD4 in thymocytes.

The runt family transcription factors Runx1 and Runx3 are expressed in developing murine thymocytes. We show that enforced expression of full-length Runx1 in CD4(-)CD8(-) thymocytes results in a profound suppression of immature CD4/CD8 double-positive thymocytes and mature CD4 single-positive thymocytes compared with controls. This effect arises from Runx1- or Runx3-mediated repression of CD4 expression, and is independent of positively selecting signals. Runx1 is able to repress CD4 in CD4/CD8 double-positive thymocytes, but not in mature splenic T cells. Runx-mediated CD4 repression is independent of association with the corepressors Groucho/TLE or Sin3. Two domains are required for complete Runx-mediated CD4 repression. These are contained within Runx1 aa 212-262 and 263-360. The latter region contains the nuclear matrix targeting sequence, which is highly conserved among runt family transcription factors across species. The presence of the nuclear matrix targeting sequence is required for Runx-mediated CD4 repression, suggesting that Runx transcription factors are stabilized on the CD4 silencer via association with the nuclear matrix.

RGS12TS-S localizes at nuclear matrix-associated subnuclear structures and represses transcription: structural requirements for subnuclear targeting and transcriptional repression.

RGS12TS-S, an 1,157-amino-acid RGS protein (regulator of G protein signaling), is a nuclear protein that exhibits a unique pattern of subnuclear organization into nuclear foci or dots when expressed endogenously or ectopically. We now report that RGS12TS-S is a nuclear matrix protein and identify structural determinants that target this protein to the nuclear matrix and to discrete subnuclear sites. We also determine the relationship between RGS12TS-S-decorated nuclear dots and known subnuclear domains involved in control of gene expression and provide the first evidence that RGS12TS-S is functionally involved in the regulation of transcription and cell cycle events. A novel nuclear matrix-targeting sequence was identified that is distinct from a second novel motif needed for targeting RGS12TS-S to nuclear dots. RGS12TS-S nuclear dots were distinct from Cajal bodies, SC-35 domains, promyelocytic leukemia protein nuclear bodies, Polycomb group domains, and DNA replication sites. However, RGS12TS-S inhibited S-phase DNA synthesis in various tumor cell lines independently of Rb and p53 proteins, and its prolonged expression promoted formation of multinucleated cells. Expression of RGS12TS-S dramatically reduced bromo-UTP incorporation into sites of transcription. RGS12TS-S, when tethered to a Gal4 DNA binding domain, dramatically inhibited basal transcription from a Gal4-E1b TATA promoter in a histone deacetylase-independent manner. Structural analysis revealed a role for the unique N-terminal domain of RGS12TS-S in its transcriptional repressor and cell cycle-regulating activities and showed that the RGS domain was dispensable for these functions. These results provide novel insights into the structure and function of RGS12TS-S in the nucleus and demonstrate that RGS12TS-S possesses biological activities distinct from those of other members of the RGS protein family.

The Homeodomain Coordinates Nuclear Entry of the Lhx3 Neuroendocrine Transcription Factor and Association with the Nuclear Matrix

LIM homeodomain transcription factors regulate development in complex organisms. To characterize the molecular signals required for the nuclear localization of these proteins, we examined the Lhx3 factor. Lhx3 is essential for pituitary organogenesis and motor neuron specification. By using functional fluorescent derivatives, we demonstrate that Lhx3 is found in both the nucleoplasm and nuclear matrix. Three nuclear localization signals were mapped within the homeodomain, and one was located in the carboxyl terminus. The homeodomain also serves as the nuclear matrix targeting sequence. No individual signal is alone required for nuclear localization of Lhx3; the signals work in combinatorial fashion. Specific combinations of these signals transferred nuclear localization to cytoplasmic proteins. Mutation of nuclear localization signals within the homeodomain inhibited Lhx3 transcriptional function. By contrast, mutation of the carboxyl-terminal signal activated Lhx3, indicating that this region is critical to transcriptional activity and may be a target of regulatory pathways. The pattern of conservation of the nuclear localization and nuclear matrix targeting signals suggests that the LIM homeodomain factors use similar mechanisms for subcellular localization. Furthermore, upon nuclear entry, association of Lhx3 with the nuclear matrix may contribute to LIM homeodomain factor interaction with other classes of transcription factors.

Direct Interaction of the KRAB/Cys2-His2Zinc Finger Protein ZNF74 with a Hyperphosphorylated Form of the RNA Polymerase II Largest Subunit

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys2-His2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with pre- mRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.