SMARDependent Interactions at the IgH Enhancer

Section 4.2 has shown that most of the ubiquitous proteins at the scaffold comply with BUR-binding characteristics (review: Galande 2002). While these proteins differ widely regarding their DNA-binding domains, it is noteworthy that most of them comprise a structural motif that might confer high affinity towards BURs. Recently, the SAF box could be localized in several DNA-binding proteins and can now be considered as one of the prototype domains. Another motif, the 11-residue "AT hook", was first detected in the HMG-I(Y) protein and associates with multiple A/T tracts that are separated by 6-8 bp, resembling the architecture of BURs. HMG1/2 and related proteins with multiple HMG boxes recognize irregular DNA structures in a sequence-nonspecific manner. One preferred substrate is the cruciform structure that arises by intrastrand pairing at inverted repeats that are common in S/MARs (Mielke et al. 1996). As base unpairing is a prerequisite for cruciform formation, the latter process directly depends on SIDD properties. While the DNA binding features of PARP-1 and YY1 are governed by Zn-finger domains, the PARP-1/Ku70/86 complex recognizes S/MARs, probably as a consequence of its ss-DNA recognition potential (Galande and Kohwi-Shigematsu 1999). Our own laboratory has detected that YY1 consensus sequences with regulatory potential are consistently found adjacent to UEs, probably in order to utilize the factor's bending potential (see Sect. 5.1.3 and Klar and Bode 2005).

The following section will extend these aspects to cell-specific BUR binders in lymphocytes and discuss the regulatory networks of which they are part. Here, the immunoglobulin heavy-chain (IgH) locus has become a paradigm. Within the locus, transcription is controlled by promoters, located 5' of the individual variable (VH) gene segments, and by a composite downstream enhancer (E||). The E|| region can be subdivided into an enhancer core (220 bp) and two 310-350-bp flanking S/MARs that were first defined by matrix-binding assays in vitro. According to our convention, these S/MARs are classified as being "facultative elements" (Fig. 1) as the associating factors, in striking contrast to the ubiquitous core-enhancer binding counterparts, are cell-type restricted. They have, therefore, the potential to cause dynamic changes of nuclear structures in accord with their suggested function.

4.2.1 SATB1

A factor binding to the 3' E^-associated S/MAR in T-cells, special AT-rich DNA binding protein 1 (SATB1), became the founding father of all BUR binders when it was identified and cloned by virtue of its ability to bind to the core-unpairing element (CUE) located within the 3' S/MAR. SATB1 does not associate with mutated CUEs that lack the unwinding property (Bode et al. 1992). Since it neither binds to nor is competed off by ssDNA, the CUE must be recognized indirectly through an altered sugar phosphate backbone. Thereby it differs from other S/MAR-binding proteins such as mutant p53, which trigger a strand separation. We have anticipated, therefore, that its association would inhibit transcription, and our early data were in accord with this (somewhat simplistic) expectation (Kohwi-Shigematsu et al. 1997).

SATB1 comprises an unusual combination of a S/MAR-binding domain and a homeodomain, both of which are necessary for the recognition of the CUE. In addition, a dimerization motif is needed for BUR binding (Cai et al. 2003). This motif is homologous to PDZ domains, modular protein-binding structures with at least three distinct types of binding:

• Association with specific recognition sequences at the carboxy-termini of proteins

• Association with other PDZ domains to form heterodimers

• Homodimerization, which for SATB1 is one prerequisite for the recognition of BURs

The fact that a BUR-binding protein contains a putative PDZ domain has important biological implications as it is to be expected that SATB1 functions can be strengthened or modulated by recruiting other PDZ-containing proteins to the S/MARs. This is the likely mechanism by which SATB1 builds up dynamic, cage-like structures within the nucleus (see below).

SATB1 was among the first cell-type-restricted S/MAR binders. It is expressed predominantly (but not exclusively: see Wen et al. 2005) in thymocytes where it represents one of the few gene products that are induced early upon peripheral T cell activation. A biological function emerged from the phenotype of SATB1 knockout mice, where SATB1 was found essential for orchestrating the spatial and temporal expression of a large number of T cell- and stage-specific genes: in the absence of SATB1, T cell development was severely impaired, and immature CD3-/CD4-/CD8- triple negative thymocytes were largely reduced in number. At the same time, SATB1-deficient thymocytes and T-cells in lymph nodes became prone to apoptosis.

The comparison of SATB1 knockout and wild-type mice indicated a role for SATB1 in the dysregulation of about 2% of the genes (Alvarez et al. 2000), also evidenced by the fact that the respective S/MARs were found to be detached from the nuclear matrix in vivo. These observations support the hypothesis that, in their normal context, specific genes are actively anchored to the nuclear matrix and that this association enables an appropriate regulation. Together these data show that there are factors acting as BUR-dependent regulators of cell function. Although the field is still in its infancy, other proteins that are readily detected on Southwestern blots or that are retained on BUR affinity columns may serve related functions at the bases of chromatin loop domains (Galande 2002).

How do SATB1 functions relate to details of the nuclear architecture? Cai et al. (2003) have demonstrated a then unknown nuclear distribution, i.e. a "cage-like" SATB1-containing structure circumscribing heterochromatic areas. They showed that this cage shares properties with a nuclear matrix as it resists the conventional extraction steps. The localization of the SATB1 network outside heterochromatic regions agrees with a model in which the attachment points (BURs) in the network provide landing platforms for chromatin-remodeling complexes (CHRAC, NURD), which constantly rearrange nucleosomes to support both positive and negative tran-scriptional actions. These pilot findings have widened our view into how a single protein can link the expression of hundreds of genes to nuclear organization.

4.2.2 SATB2

Dobreva et al. (2003) characterized a novel cell type-specific S/MAR-binding protein, SATB2, which is abundantly expressed in pre-B- and B-cells where it serves certain SATBl-like functions. SATB2 differs from its closely related thymocyte-specific relative by sumoylation-dependent modifications. Sumoylation is a recently detected post-translational modification system, biochemically analogous to, but functionally distinct from, ubiquitinylation as it involves the covalent attachment of a SUMO (small ubiquitin-related modifier) sequence to substrate proteins. Mutation of two internal conjugation sites (lysines) clearly enhances its activation potential in parallel to the association with endogenous S/MARs in vivo, whereas N-terminal fusions with SUMO decrease SATB2-mediated gene activation. Since sumoylation is involved in targeting SATB2 to the nuclear periphery, this may cause modulation of SATB2 activities.

BRIGHT (B cell regulator of IgH transcription) is yet another factor transactivating the intronic IgH enhancer by binding to the S/MARs as a tetramer (Kaplan et al. 2001). BRIGHT contains regions homologous to the Drosophila SWI complex, suggesting that it might be involved in chromatin remodelling. A number of experiments suggest that the cell-type specificity of the E^ enhancer is governed by negative regulatory mechanisms that are dominant to this and other B-cell specific transcriptional activators and that these actions can be ascribed to interference with nuclear matrix attachment. A responsible negative regulatory factor, first called NF-^NR and later found related to Cux/CDP, binds to multiple sites flanking IgH enhancer. Interestingly, the expression of NF-^NR displays a unique developmental pattern, as it is present in most cell lines outside of the B-cell lineage (T cells, macrophages and fibroblasts), but also in B-cells early in development. In contrast, it is absent from more mature cells that express high levels of IgH chains (Wang et al.1999).

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