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.     

NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization

Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.     

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.     

Two-step synergism between the progesterone receptor and the DNA-binding domain of nuclear factor 1 on MMTV minichromosomes.

In contrast to its behavior as naked DNA, the MMTV promoter assembled in minichromosomes can be activated synergistically by the progesterone receptor and NF1 in a process involving ATP-dependent chromatin remodeling. The DNA-binding domain of NF1 is required and sufficient for stable occupancy of all receptor-binding sites and for functional synergism. Activation of purified minichromosomes is observed in the absence of SWI/SNF and can be enhanced by recombinant ISWI. Receptor binding to minichromosomes recruits ISWI and NURF38, but not brahma. We propose a two-step synergism in which the receptor triggers a chromatin remodeling event that facilitates access of NF1, which in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules and full transactivation.     

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.     

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI/SNF or NURF

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.