The Snf-2-related CREB-binding protein activator protein (SRCAP) serves as a coactivator for a number of transcription factors known to interact with CBP. Swr1, the closest Saccharomyces cerevisiae ortholog of SRCAP, is a component of the chromatin remodeling complex SWR-C, which catalyzes exchange of the histone variant H2A.Z into nucleosomes. In this report, we use a combination of conventional chromatography and anti-SRCAP immunoaffinity chromatography to purify a native human SRCAP complex with a polypeptide composition similar to that of SWR-C, and we show for the first time that this SRCAP-containing complex supports ATP-dependent exchange of histone dimers containing H2B and H2A.Z into mononucleosomes reconstituted with recombinant H2A, H2B, H3, and H4. Our findings, together with previous evidence implicating H2A.Z in transcriptional regulation, suggest that SRCAP's coactivator function may depend on its ability to promote incorporation of H2A.Z into chromatin.

We explored the mechanisms of chromatin compaction and transcriptional regulation by poly(ADP-ribose) polymerase 1 (PARP-1), a nucleosome-binding protein with an NAD ؉ -dependent enzymatic activity. By using atomic force microscopy and a complementary set of biochemical assays with reconstituted chromatin, we showed that PARP-1 promotes the localized compaction of chromatin into supranucleosomal structures in a manner independent of the amino-terminal tails of core histones. In addition, we defined the domains of PARP-1 required for nucleosome binding, chromatin compaction, and transcriptional repression. Our results indicate that the DNA binding domain (DBD) of PARP-1 is necessary and sufficient for binding to nucleosomes, yet the DBD alone is unable to promote chromatin compaction and only partially represses RNA polymerase II-dependent transcription in an in vitro assay with chromatin templates (ϳ50% of the repression observed with wild-type PARP-1). Furthermore, our results show that the catalytic domain of PARP-1, which does not bind nucleosomes on its own, cooperates with the DBD to promote chromatin compaction and efficient transcriptional repression in a manner independent of its enzymatic activity. Collectively, our results have revealed a novel function for the catalytic domain in chromatin compaction. In addition, they show that the DBD and catalytic domain cooperate to regulate chromatin structure and chromatin-dependent transcription, providing mechanistic insights into how these domains contribute to the chromatin-dependent functions of PARP-1.Chromatin is the physiological template for nuclear processes involving genomic DNA, including transcription, replication, recombination, and repair. Nucleosomes, the fundamental repeating units of chromatin, are protein-DNA complexes containing 146 bp of DNA wrapped around a protein core containing two copies each of four core histone proteins (H2A, H2B, H3, and H4) (22). Chromatin exists in various conformations, including open conformations where individual nucleosomes are separated, freely accessible, and mobile, as well as compact conformations where nucleosomes are juxtaposed and partially occluded in higher-order structures (6, 11, 37). The extent of nucleosome mobility and chromatin compaction are two determinants of the activity of chromatin-dependent processes, such as transcription by RNA polymerase II (Pol II) (11,25,33).A variety of chromatin "architectural" proteins are known to bind to nucleosomes, promote chromatin compaction, and modulate transcriptional responses (23,28). For example, the linker histone H1 binds at or near the dyad axis of nucleosomes and promotes the formation of a higher-order structure known as the 30-nm fiber (28, 38). In the 30-nm fiber, nucleosomes are tightly packed, and the nucleosomal DNA is less accessible and more refractory to transcription (28). We have recently shown that poly(ADP-ribose) polymerase 1 (PARP-1) is a nucleosome binding protein with linker histone-like properties. Like H1, PARP-1 binds at or near ...