Spatial contacts and nucleosome step movements induced by the NURF chromatin remodeling complex

The nucleosome remodeling factor NURF is a four-subunit, ISWI-containing chromatin remodeling complex that catalyzes nucleosome sliding in an ATP-dependent fashion, thereby modulating the accessibility of the DNA. To elucidate the mechanism of nucleosome sliding, we have investigated by hydroxyl radical footprinting how NURF makes initial contact with a nucleosome positioned at one end of a DNA fragment. NURF binds to two separate locations on the nucleosome: a continuous stretch of linker DNA up to the nucleosome entry site and a region asymmetrically surrounding the nucleosome dyad within the minor grooves, close to residues of the histone H4 tail that have been implicated in the activation of ISWI activity. Kinetic analysis reveals that nucleosome sliding occurs in apparent increments or steps of 10 bp. Furthermore, single nucleoside gaps as well as nicks about two helical turns before the dyad interfere with sliding, indicating that structural stress at this region assists the relative movement of DNA. These findings support a sliding model in which the position-specific tethering of NURF forces a translocating ISWI ATPase to pump a DNA distortion over the histone octamer, thereby changing the translational position of the nucleosome.     

Heterochromatin protein 2 interacts with Nap-1 and NURF: a link between heterochromatin-induced gene silencing and the chromatin remodeling machinery in Drosophila

Heterochromatin protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster that binds to heterochromatin protein 1 (HP1) and has been implicated in heterochromatin-induced gene silencing. Heretofore, HP1 has been the only known binding partner of HP2, a large protein devoid of sequence motifs other than a pair of AT hooks. In an effort to identify proteins that interact with HP2 and assign functions to its various domains, nuclear proteins were fractionated under nondenaturing conditions. On separation of nuclear proteins, nucleosome assembly protein 1 (Nap-1) has an overlapping elution profile with HP2 (assayed by Western blot) and has been identified by mass spectrometry in fractions with HP2. Upon probing fractions in which HP2 and Nap-1 are both present, we find that the nucleosome remodeling factor (NURF), an ISWI-dependent chromatin remodeling complex, is also present. Results from coimmunoprecipitation experiments suggest that HP2 interacts with Nap-1 as well as with NURF; NURF appears to interact directly with both HP2 and Nap-1. Three distinct domains within HP2 mediate the interaction with NURF, allowing us to assign NURF binding domains in addition to the AT hooks and HP1 binding domains already mapped in HP2. Mutations in Nap-1 are shown to suppress position effect variegation, suggesting that Nap-1 functions to help to assemble chromatin into a closed form, as does HP2. On the basis of these interactions, we speculate that HP2 may cooperate with these factors in the remodeling of chromatin for silencing.     

Chromatin remodeling in vivo: evidence for a nucleosome sliding mechanism

Members of the ISWI family of chromatin remodeling factors exhibit ATP-dependent nucleosome sliding, loading, and spacing activities in vitro. However, it is unclear which of these activities are utilized by ISWI complexes to remodel chromatin in vivo. We therefore sought to identify the mechanisms of chromatin remodeling by Saccharomyces cerevisiae Isw2 complex at its known sites of action in vivo. To address this question, we developed a method of identifying intermediates of the Isw2-dependent chromatin remodeling reaction as it proceeded. We show that Isw2 complex catalyzes nucleosome sliding at two different classes of target genes in vivo, in each case sliding nucleosomes closer to the promoter regions. In contrast to its biochemical activities in vitro, nucleosome sliding by Isw2 complex in vivo is unidirectional and localized to a few nucleosomes at each site, suggesting that Isw2 activity is constrained by cellular factors.     

A Novel Pzg-NURF Complex Regulates Notch Target Gene Activity

Drosophila putzig was identified as a member of the TRF2–DREF complex that is involved in core promoter selection. Additionally, putzig regulates Notch signaling, however independently of DREF. Here, we show that Putzig associates with the NURF complex. Loss of any NURF component including the NURF-specific subunit Nurf 301 impedes binding of Putzig to Notch target genes, suggesting that NURF recruits Putzig to these sites. Accordingly, Putzig can be copurified with any NURF member. Moreover, Nurf 301 mutants show reduced Notch target gene activity and enhance Notch mutant phenotypes. These data suggest a novel Putzig–NURF chromatin complex required for epigenetic activation of Notch targets.     

ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF.

Drosophila NURF is an ATP-dependent chromatin remodeling complex that contains ISWI, a member of the SWI2/SNF2 family of ATPases. We demonstrate that NURF catalyzes the bidirectional redistribution of mononucleosomes reconstituted on hsp70 promoter DNA. In the presence of NURF, nucleosomes adopt one predominant position from an ensemble of possible locations within minutes. Movements occur in cis, with no transfer to competing DNA. Migrating intermediates trapped by Exo III digestion reveal progressive nucleosome motion in increments of several base pairs. All four core histones are retained quantitatively during this process, indicating that the general integrity of the histone octamer is maintained. We suggest that NURF remodels nucleosomes by transiently decreasing the activation energy for short-range sliding of the histone octamer.