Frank O. Fackelmayer

Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements

We identified four proteins in nuclear extracts from HeLa cells which specifically bind to a scaffold attachment region (SAR) element from the human genome. Of these four proteins, SAF‐A (scaffold attachment factor A), shows the highest affinity for several homologous and heterologous SAR elements from vertebrate cells. SAF‐A is an abundant nuclear protein and a constituent of the nuclear matrix and scaffold. The homogeneously purified protein is a novel double stranded DNA binding protein with an apparent molecular weight of 120 kDa. SAF‐A binds at multiple sites to the human SAR element; competition studies with synthetic polynucleotides indicate that these sites most probably reside in the multitude of A/T‐stretches which are distributed throughout this element. In addition we show by electron microscopy that the protein forms large aggregates and mediates the formation of looped DNA structures.

SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA.

ABSTRACT SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.

The novel SAR-binding domain of scaffold attachment factor A (SAF-A) is a target in apoptotic nuclear breakdown

The scaffold attachment factor A (SAF‐A) is an abundant component of the nuclear scaffold and of chromatin, and also occurs in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. Evidence from previous experiments had suggested that SAF‐A most likely has at least two different functions, being involved both in nuclear architecture and RNA metabolism. We now show that the protein has a novel scaffold‐associated region (SAR)‐specific bipartite DNA‐binding domain which is independent from the previously identified RNA‐binding domain, the RGG box. During apoptosis, but not during necrosis, SAF‐A is cleaved in a caspase‐dependent way. Cleavage occurs within the bipartite DNA‐binding domain, resulting in a loss of DNA‐binding activity and a concomitant detachment of SAF–A from nuclear structural sites. On the other hand, cleavage does not compromise the association of SAF‐A with hnRNP complexes, indicating that the function of SAF‐A in RNA metabolism is not affected in apoptosis. Our results suggest that detachment of SAF–A from SARs, caused by apoptotic proteolysis of its DNA‐binding domain, is linked to the formation of oligonucleosomal‐sized DNA fragments and could therefore contribute to nuclear breakdown in apoptotic cells.

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI‐1, a vector in which a chromosomal scaffold/matrix‐attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T‐antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI‐1 by crosslinking with cis‐diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south‐western analysis, the vector shows exclusive binding to hnRNP‐U/SAF‐A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA–protein complex demonstrates that pEPI‐1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.

Dynamics of Human Protein Arginine Methyltransferase 1(PRMT1) in Vivo

Arginine methylation is a posttranslational protein modification catalyzed by a family of protein arginine methyltransferases (PRMT), the predominant member of which is PRMT1. Despite its major role in arginine methylation of nuclear proteins, surprisingly little is known about the subcellular localization and dynamics of PRMT1. We show here that only a fraction of PRMT1 is located in the nucleus, but the protein is predominantly cytoplasmic. Fluorescence recovery after photobleaching experiments reveal that PRMT1 is highly mobile both in the cytoplasm and the nucleus. However, inhibition of methylation leads to a significant nuclear accumulation of PRMT1, concomitant with the appearance of an immobile fraction of the protein in the nucleus, but not the cytoplasm. Both the accumulation and immobility of PRMT1 is reversed when re-methylation is allowed, suggesting a mechanism where PRMT1 is trapped by unmethylated substrates such as core histones and heterogeneous nuclear ribonucleoprotein proteins until it has executed the methylation reaction.

Human protein arginine methyltransferases in vivo - Distinct properties of eight canonical members of the PRMT family

Methylation of arginine residues is a widespread post-translational modification of proteins catalyzed by a small family of protein arginine methyltransferases (PRMTs). Functionally, the modification appears to regulate protein functions and interactions that affect gene regulation, signalling and subcellular localization of proteins and nucleic acids. All members have been, to different degrees, characterized individually and their implication in cellular processes has been inferred from characterizing substrates and interactions. Here, we report the first comprehensive comparison of all eight canonical members of the human PRMT family with respect to subcellular localization and dynamics in living cells. We show that the individual family members differ significantly in their properties, as well as in their substrate specificities, suggesting that they fulfil distinctive, non-redundant functions in vivo. In addition, certain PRMTs display different subcellular localization in different cell types, implicating cell- and tissue-specific mechanisms for regulating PRMT functions.

Protein Arginine Methyltransferase 1 and 8 Interact with FUS to Modify Its Sub-Cellular Distribution and Toxicity In Vitro and In Vivo

Amyotrophic lateral sclerosis (ALS) is a late onset and progressive motor neuron disease. Mutations in the gene coding for fused in sarcoma/translocated in liposarcoma (FUS) are responsible for some cases of both familial and sporadic forms of ALS. The mechanism through which mutations of FUS result in motor neuron degeneration and loss is not known. FUS belongs to the family of TET proteins, which are regulated at the post-translational level by arginine methylation. Here, we investigated the impact of arginine methylation in the pathogenesis of FUS-related ALS. We found that wild type FUS (FUS-WT) specifically interacts with protein arginine methyltransferases 1 and 8 (PRMT1 and PRMT8) and undergoes asymmetric dimethylation in cultured cells. ALS-causing FUS mutants retained the ability to interact with both PRMT1 and PRMT8 and undergo asymmetric dimethylation similar to FUS-WT. Importantly, PRMT1 and PRMT8 localized to mutant FUS-positive inclusion bodies. Pharmacologic inhibition of PRMT1 and PRMT8 activity reduced both the nuclear and cytoplasmic accumulation of FUS-WT and ALS-associated FUS mutants in motor neuron-derived cells and in cells obtained from an ALS patient carrying the R518G mutation. Genetic ablation of the fly homologue of human PRMT1 (DART1) exacerbated the neurodegeneration induced by overexpression of FUS-WT and R521H FUS mutant in a Drosophila model of FUS-related ALS. These results support a role for arginine methylation in the pathogenesis of FUS-related ALS.

Protein arginine methyltransferase 6 enhances polyglutamine-expanded androgen receptor function and toxicity in spinal and bulbar muscular atrophy.

Summary Polyglutamine expansion in androgen receptor (AR) is responsible for spinobulbar muscular atrophy (SBMA) that leads to selective loss of lower motor neurons. Using SBMA as a model, we explored the relationship between protein structure/function and neurodegeneration in polyglutamine diseases. We show here that protein arginine methyltransferase 6 (PRMT6) is a specific co-activator of normal and mutant AR and that the interaction of PRMT6 with AR is significantly enhanced in the AR mutant. AR and PRMT6 interaction occurs through the PRMT6 steroid receptor interaction motif, LXXLL, and the AR activating function 2 surface. AR transactivation requires PRMT6 catalytic activity and involves methylation of arginine residues at Akt consensus site motifs, which is mutually exclusive with serine phosphorylation by Akt. The enhanced interaction of PRMT6 and mutant AR leads to neurodegeneration in cell and fly models of SBMA. These findings demonstrate a direct role of arginine methylation in polyglutamine disease pathogenesis.

Effects of the heterogeneous nuclear ribonucleoprotein U (hnRNP U/SAF-A) on glucocorticoid-dependent transcription in vivo.

The glucocorticoid receptor (GR) is a ligand dependent transcription factor, which regulates the transcription of multiple hormone-dependent genes. The transcriptional regulation by GR takes place by interaction of GR with the basal transcription machinery and by recruiting glucocorticoid receptor interacting proteins (GRIPs). Previously we identified hnRNP U/SAF-A as a factor interfering with GR-dependent transcription by repressing glucocorticoid induced activation. To gain insight into the mechanisms that govern this interference, we have now investigated the transcription of GR-dependent reporter genes in Ltk(-) cells transiently transfected with a variety of hnRNP U constructs. We demonstrate that a hnRNP U construct lacking the GR-binding domain acts as a dominant negative factor that now enhances GR-driven transcription. In addition, hnRNP U repression of glucocorticoid induced transcription was found to be dependent on the amount of cotransfected GR, where a high amount of GR leads to ligand-inducible repression of GR-dependent reporter gene activity by hnRNP U, whereas low amounts of GR showed nearly no effect. The relative concentrations of GR, hnRNP U and DNA-binding sites for GR are important for the effect of hnRNP U on transcription, suggesting a model where hnRNP-U acts as a storage site for intranuclear GR.

Nucleo‐cytoplasmic shuttling of protein arginine methyltransferase 1 (PRMT1) requires enzymatic activity

Methylation of arginine residues is a widespread post‐translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMT), of which PRMT1 is the predominant member in human cells. We have previously described the localization and mobility of PRMT1 in live cells, and found that it shuttles between the nucleus and the cytoplasm depending on the methylation status of substrate proteins. Recently, amino‐terminal splicing isoforms of PRMT1 were shown to differ significantly in intracellular localization, the most interesting being splice variant 2 that carries a nuclear export signal in its amino terminus, and is expressed in increased levels in breast cancer cells. We show here that enzymatic activity is required for nucleo‐cytoplasmic shuttling of PRMT1v2, as a catalytically inactive mutant highly accumulates in the nucleus and displays altered intranuclear mobility as determined by fluorescence recovery after photobleaching experiments. Our results indicate that nuclear export of PRMT1v2 is dominant over activity‐independent nuclear import, but can only occur after activity‐dependent release of the enzyme from substrates, suggesting that shuttling of the enzyme provides a dynamic mechanism for the regulation of substrate methylation.