SAF-Box, a conserved protein domain name that specifically recognizes scaffold attachment region DNA. nonhistone proteins and that serves as a scaffold for loops of chromatin (reviewed in reference 5). This matrix has been associated with the regulation of transcription, DNA replication, and RNA processing (44). The DNA regions anchoring the chromosomal DNA to the nuclear matrix (called matrix-associated regions [MARs] or scaffold-attachment regions [SARs] [8]) are composed of AT-rich sequences. They have a unique structure characterized by a narrow minor groove, a high unwinding potential and the (S)-GNE-140 formation of hairpin structures (5). S/MARs are located in the introns of several large genes (32, 53) and also at borders of transcription models (23, 47). S/MAR elements have often been implicated in the regulation of gene expression. They are frequently found close to enhancers (37, 48), and they can stimulate gene expression of heterologous reporter genes when integrated into the genome (56) and can regulate chromatin accessibility (21, 30). Several S/MAR binding proteins have been identified and characterized over recent years. One of the first was the scaffold attachment factor-A (SAF-A), initially identified as a component of RNP particles (35) but later also characterized as a S/MAR-binding protein (17, 18, 24, 36, 52). Other S/MAR binding proteins include ubiquitous, abundant proteins such as SAF-B (50), topoisomerase II (1), histone H1 (28), lamin B1 (42), HMGI/Y (66), and nucleolin (11), but also proteins that are expressed in a more cell-type-specific fashion, such as SatB1 (10) and p114 (63). Even though many of these proteins have been thoroughly characterized biochemically, it is still relatively unclear how they are involved in the gene regulating processes of S/MAR elements. In recent years much progress has been made in the characterization of the events at the site of transcription initiation. Of the factors involved the transcriptional coactivators p300/CBP were originally described as conversation modules that form a (S)-GNE-140 bridge between the basal transcription machinery and upstream transcription factors (25). CBP was cloned by virtue of its binding to CREB (7) and p300 as an adenoviral-E1A associating protein (15). Viral oncogenes such as the adenovirus E1A and simian computer virus 40 (SV40) large T can interact with p300/CBP and disrupt the conversation of p300/CBP with other factors. The homologous proteins p300 and CBP contain three cysteine-histidine (CH)-rich regions, of which CH3 constitutes the major conversation site for adenovirus E1A. An additional role of p300/CBP became clear when it was found that p300/CBP have intrinsic acetyltransferase activity (4, 45) and associate with other proteins with acetyltransferase activity such as p/CAF (64). The acetylation of histone tails is generally related to transcription activation (57). In addition, acetylation of transcription factors by p300/CBP can lead to either activation or Rabbit polyclonal to NF-kappaB p105-p50.NFkB-p105 a transcription factor of the nuclear factor-kappaB ( NFkB) group.Undergoes cotranslational processing by the 26S proteasome to produce a 50 kD protein. repression of transcription (55). Here we report a strong conversation between p300 and SAF-A. We show that p300 and SAF-A bind to S/MAR elements and that their binding is usually disrupted when the viral protein E1A or SV40 large T is present. The binding of p300 and SAF-A to S/MAR elements seems to be restricted to the transcriptional inactive state. However, while no transcription occurs, these S/MAR elements are bound by acetylated histones, suggesting that the presence of p300 at S/MAR elements might realize a localized chromatin state ready for transcription. MATERIALS (S)-GNE-140 AND METHODS Yeast two-hybrid assay. Yeast two-hybrid screening was performed as described previously (3) with a random-primed human cDNA testis library fused to a VP16 transactivating domain name by using the vector pVP16 (61) and the yeast strain Y190 (59). The bait plasmid pMD4-p300 (amino acids [aa] 1570 to 1848) was made by cloning an 834-bp p300 cDNA fragment into pMD4. pMD4 was a gift from M. van Dijk and R. Bernards. Y190, which contains LacZ and HIS3 reporter genes, was transformed with plasmid pMD4-p300 and the pVP16 cDNA library. In the screen 5 106 transformants were tested, and the HIS+ positive colonies were subsequently tested for -galactosidase activity by colony filter assay. Of the double-positive clones (HIS+, LacZ+) one contained the coding sequence for aa 633 to 806 of SAF-A. Cell.