HnRNPs SAFA

Mattern et al. (1997 and references therein) have identified the most abundant proteins that are exclusively present in the internal nuclear network. In line with earlier reports (Nakayasu and Berezney 1991), many of these belong to the group of heterogeneous nuclear ribonucleoproteins (hnRNPs), the sites of nascent transcripts and RNA maturation. These findings support a model in which major matrix protein constituents are involved in RNA metabolism, packaging and transport.

The most abundant component of this group, called "scaffold-attachment factor A" (SAF-A/ hnRNP-U), was first characterized in LIS-extracted scaffolds (Kipp et al. 2000 and references therein). SAF-A associates with multiple S/MARs, and UV-cross-linking experiments show that this established RNA binder is also associated with DNA in vivo. HeLa-cells contain about 2 million molecules per nucleus, half of which associate with the nuclear matrix in a salt-resistant manner. The other half is either bound to hnRNP particles or resides in a DNAseI extractable fraction.

The primary structure of SAF-A reflects its dual function as there are two independent nucleic acid-binding domains, (1) a C-terminal RNA/ssDNA binding domain (RGG box) and (2) a S/MAR-specific 45-amino acid N-terminal domain, called "SAF box", which is split and inactivated during apoptosis (Kipp et al. 2000). The SAF box is reminiscent of a homeobox lacking the DNA recognition helix and was the first characterized protein domain specifically recognizing S/MARs. SAF boxes are present in organisms as distant as yeast, plants and mammals, but not in prokaryotes, compatible with their specific binding to S/MAR-DNA. Originally unexpected, SAF-box-containing proteins from evolutionarily distant eukaryotes are not orthologs since homologies outside the SAF box are barely detectable. For most of these proteins, the function has remained unknown, with the exception of SAF-B and E1B-AP5, which serve related functions in the nuclear architecture and/or RNA metabolism. Interestingly, a poly(ADP-ribosyl)ase (PARP) from A. thaliana contains two SAF boxes in tandem, suggesting that these can substitute for certain functions of, for instance, Zn-finger domains (Kipp et al. 2000). Being a ubiquitous S/MAR-binding component of the nuclear matrix, a separate chapter Sect. 5.1) will be devoted to PARP-1, the major representative of this class in mammals.

In vitro, SAF-A shows a pronounced propensity to self-polymerize, and this state is required to recognize S/MAR-DNA. Binding follows a cooperative mode, which is also typical for scaffold-S/MAR interactions (Kay and Bode 1994): each individual domain interacts only weakly with a DNA element, i.e. an UE according to Fig. 1. Only the simultaneous binding of SAF boxes to multiple UEs confers a strong and at the same time specific interaction in accord with the "mass-binding mode." This model explains the well-known phenomenon that there are hardly any naturally occurring S/MARs below a critical length of 250 bp. The failure of ssDNA to compete for the interaction of S/MARs with SAF boxes shows that SAF-box proteins alone cannot explain all criteria of scaffold-S/MAR interactions. However, a superimposition of SAF- and lamin-binding characteristics could well account for it.

Fackelmayer and colleagues (2004) have reported results from a defined model system to elucidate the role of scaffolds in DNA replication. They used extracts from Xenopus laevis eggs that contain all necessary components to assemble replication-competent nuclei, but do not support interfering reactions such as transcription or RNA maturation. They show, for the first time, that SAF-A provides an architectural framework on which active replication factories are assembled. Even when DNA is removed, SAF-A continues to form a nuclear reticulum. A dominant-negative approach indicates that the same is true in cultured cells. Consistent with its proposed role as a structural component, SAF-A/GFP fusions subjected to "fluorescence recovery after photobleaching" (FRAP) studies showed the protein to be rather immobile. Taken together, the data point to a structuring role of a SAF-A scaffold for DNA replication (congress report by Jackson 2005 and in preparation).

Meanwhile, SAF-A has been implicated in several more functions. Its association with histoneacetyltransferases (Martens et al. 2002) may be taken as an indication that S/MAR effects on histone hyperacetylation (Schlake et al.1994) are actually mediated by SAF-A implementing the protein in transcriptional potentiation (Sect. 3). Its involvement in the maintenance of nonviral episomes (Jencke et al. 2001) emphasizes its function in DNA replication.

3.2.1 SAF-A and Nuclear Hormone Receptor Functions

Nuclear hormone receptors are paradigms of regulated transcription factor systems that integrate signal transduction into nuclear architecture. The family includes receptors for steroid and thyroid hormones, for vitamin A and vitamin D. These receptors have become important pharmacological targets in a wide variety of clinical disorders, such as fertility issues and autoimmune diseases, which are now amenable to treatment with agonists or antagonists. It is anticipated that blocking the scaffold association of hormone receptors should have comparable effects to such an approach, but on a different level of regulation. Synergistic effects may arise when these treatments are used in combination (review: Fackelmayer 2004).

In the absence of a ligand most members of this receptor class localize to the cytoplasm. After hormone binding, they are translocated to the nucleus where they establish a punctuate pattern that is resistant to enzymatic removal of chromatin and thereby suggestive for nuclear matrix binding. It has been hypothesized that matrix-acceptor protein interactions occur in a cell-type-specific fashion. Identifying the acceptor proteins may therefore guide the development of specialized agents to modulate hormone-dependent gene expression patterns.

For the glucocorticoid receptor (GR), a minimal NMTS has been identified. If it was applied to screen for relevant acceptor proteins, SAF-A was recovered. Subsequent assays suggest that SAF-A might serve as the docking site for ligand-bound GR on the matrix. Comparable results are now available, demonstrating the specific interaction of a steroid hormone receptor with SAF-B, the second-most ubiquitous member of the SAF-box family in mammalia. When overexpressed, SAF-B exerts growth inhibition in breast tumor cells. In this context, it is noted that 20% of all breast tumors lack detectable levels of SAF-B, owing to aberrations at its gene locus (Fackelmayer 2004).

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