The histone variant H2AZ is incorporated preferentially at specific locations in chromatin to modulate chromosome functions. In Saccharomyces cerevisiae, deposition of histone H2AZ is mediated by the multiprotein SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Here, we define interactions between SWR1 components and H2AZ, revealing a link between the ATPase domain of Swr1 and three subunits required for the binding of H2AZ. We discovered that Swc2 binds directly to and is essential for transfer of H2AZ. Swc6 and Arp6 are necessary for the association of Swc2 and for nucleosome binding, whereas other subunits, Swc5 and Yaf9, are required for H2AZ transfer but neither H2AZ nor nucleosome binding. Finally, the C-terminal alpha-helix of H2AZ is crucial for its recognition by SWR1. These findings provide insight on the initial events of histone exchange.

NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF from a Drosophila nuclear extract reduced the level of polymerase that paused in the promoter proximal region of hsp70. Depletion of one NELF subunit in salivary glands using RNA interference also reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborated the cross-linking result and showed that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF were strongly recruited to chromosomal puffs harboring the hsp70 genes. We propose that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation.

NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Here, further characterization of Drosophila NELF is provided. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N- and C-terminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and β1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila.