BIOLOGICAL OVERVIEW

The compaction of the eukaryotic genome in nucleosomes limits the access of DNA to regulatory proteins and the enzymes that process genetic information. To overcome this constraint, cells employ a variety of strategies to disrupt locally the organization of nucleosomes. Prominent among these are several distinct classes of enzymes that covalently modify specific residues of the N-terminal histone tails, thereby affecting nucleosome stability or higher order nucleosome interactions. A second group of multisubunit protein complexes uses the energy of ATP hydrolysis to alter chromatin structure and mobilize nucleosomes. NURF is an Imitation SWI (ISWI) containing complex of four proteins that uses the energy of ATP hydrolysis to catalyze nucleosome sliding. Three NURF components have been identified previously. cDNAs encoding the largest NURF subunit have been cloned, revealing a 301 kDa polypeptide (NURF301) that shares structural motifs with ACF1. Full and partial NURF complexes have been reconstructed from recombinant proteins and NURF301 and the ISWI ATPase have been shown to be necessary and sufficient for accurate and efficient nucleosome sliding. An HMGA/HMGI(Y)-like domain of NURF301 that facilitates nucleosome sliding indicates the importance of DNA conformational changes in the sliding mechanism. NURF301 also shows interactions with sequence-specific transcription factors, providing a basis for targeted recruitment of the NURF complex to specific genes (Xiao, 2001). NURF301 is identical to the protein coded for by the Drosophila gene Enhancer of bithorax, described by Bhosekar and Babu in 1987 (FlyBase).

To date, four classes of ATP-dependent chromatin remodeling complexes, each containing the SWI2/SNF2 (see Drosophila Brahma), ISWI, CHD/Mi-2, and INO80 ATPases, or their highly related paralogs, have been characterized. Of these, the SWI2/SNF2-containing complexes (SWI/SNF complexes) are most extensively studied and have been demonstrated to play a role in transcription in vivo and in vitro. Similarly, complexes containing ISWI, Mi-2, and INO80 are also implicated in transcription and possibly other DNA transactions. Although the SWI2/SNF2 family protein complexes display similar levels of ATP utilization and generally increase chromatin accessibility, each family member appears to recognize different aspects of the chromatin substrate and can produce different outcomes in the process of chromatin remodeling (Xiao, 2001 and references therein).

For instance, the ATPase activity of SWI/SNF is stimulated by either free or nucleosomal DNA, while the ISWI and Mi-2 proteins or complexes are stimulated only by nucleosomes. ISWI proteins or complexes require the flexible N-terminal histone tails, particularly the H4 tail, for stimulating ATPase activity and inducing nucleosome mobility, but histone tails have no apparent role in activating the Mi-2 proteins. ISWI complexes mediate nucleosome 'sliding,' the movement of histone octamers in cis without permanent diplacement from DNA. By contrast, the SWI/SNF or RSC complexes can mobilize nucleosomes, create a stably remodeled nucleosome intermediate, or transfer a histone octamer from one DNA fragment to another. These differences indicate that there are diverse mechanisms by which nucleosome structure can be altered to increase nucleosome dynamics and DNA accessibility (Xiao, 2001 and references therein).

Biochemical assays for activities that can disrupt or enhance the periodic organization of nucleosome arrays have been used to identify several chromatin remodeling complexes that contain the ISWI ATPase. These include NURF, ACF, CHRAC, RSF, WCRF, ISWI-B, and ISWI-D complexes in metazoa and the Isw1 and Isw2 complexes in budding yeast. Although the native ISWI complexes and the recombinant ISWI protein have provided substantial insights, the mechanism by which they catalyze nucleosome sliding is just beginning to be addressed. In addition, the protein components of the larger ISWI complexes, including NURF, RSF, and the yeast Isw1 complex have not been fully defined (Xiao, 2001 and references therein).

Previous studies of NURF demonstrated a complex of four distinct subunits. Three have been identified previously: NURF140, the ISWI ATPase; NURF55, a WD-40 repeat protein also found in other protein complexes involved in histone metabolism, and NURF38, an inorganic pyrophosphatase. The fourth and largest NURF component (previously designated NURF215) was found to be unusually sensitive to proteolysis during purification, resulting in a heterogeneous set of polypeptides of ~215 kDa that hindered protein sequencing efforts. The largest NURF component, NURF301 has now been cloned and characterized and evidence is provided showing that it has a central role in the assembly, remodeling activity, and biology of the NURF complex (Xiao, 2001).

NURF301 shares four protein motifs with ACF1, a subunit of the ISWI-containing chromatin assembly and remodeling complexes ACF and CHRAC. Sequences comprising a third of the NURF301 protein -- a glutamine-rich domain of ~450 residues and the N-terminal region of ~500 residues -- are unique, indicating that NURF301 has functions in addition to those exhibited by ACF1. NURF301 also contains four LXXLL motifs that define sites for interaction with nuclear hormone receptors. Together with the observed interactions between NURF301 and GAGA (Trithorax-like) factor, Heat shock factor, and VP16, NURF301 is likely to present a broad target for interactions with a wide variety of sequence-specific transcriptional regulators (Xiao, 2001).

The identification of GAGA factor in a general search for NURF-interacting proteins is striking. This finding provides a plausible explanation for the original discovery that NURF, rather than another chromatin remodeling complex, was the key ATP-dependent activity required with GAGA factor for proper reconstitution of DNase hypersensitivity of chromatin at the Drosophila hsp70 promoter (Tsukiyama, 1994; Tsukiyama, 1995). The binding of NURF301 to GAGA factor suggests that NURF may be recruited to facilitate the local mobilization of nucleosomes for transcriptional activation or repression, depending on gene context. In fact, there is strong evidence for recruitment of SWI/SNF complexes by specific transcription factors and for recruitment of the Isw2 complex by the Ume6p repressor. Given the observed coimmunoprecipitation of NURF and GAGA factor and the in vitro binding of GAGA factor to NURF301 and, to a lesser extent, ISWI, it is interesting that the immunostaining patterns for GAGA factor and ISWI do not overlap extensively on Drosophila polytene chromosomes. It will be of interest to further investigate the in vivo and in vitro interactions of GAGA factor with ISWI complexes (ACF, CHRAC, NURF) (Xiao, 2001).

With the cloning of all four NURF subunits, an active NURF complex could be reconstructed from purified recombinant proteins. NURF301 is important for the structural integrity of NURF. Interestingly, only two NURF subunits, NURF301 and ISWI, appear to be necessary and sufficient to reconstitute accurate and efficient nucleosome sliding. These results are in concurrence with the dual requirement for ACF1 and ISWI for full reconstitution of chromatin assembly and nucleosome sliding activity of the ACF complex (Xiao, 2001).

How might NURF301 contribute to the induction of nucleosome sliding by NURF? The presence of four protein motifs common to ACF1 and NURF301 suggests that these two proteins may share common functions that contribute to nucleosome destabilization when coupled to the activity of the ISWI engine. However, the presence of a unique N-terminal region including an HMGA-like domain in NURF301 suggests additional mechanisms. HMGA proteins bind four-way DNA junctions, induce structural changes in nucleosomes, and also facilitate formation of stereospecific multiprotein-DNA enhanceosome complexes by changing DNA conformation. A surprisingly high number of chromatin remodeling proteins, including ISWI, brm, SWI2/SNF2, Rsc1, and Rsc2 contain the AT-hook motif. Indeed, the single AT hook of yeast Rsc1 and Rsc2 proteins is essential for in vivo function, and deletion of a segment of mammalian brm/SNF2alpha containing an AT hook affects function in cultured cells and affinity for chromatin (Xiao, 2001).

In the HMGA-like domain of NURF301, the motif corresponding to the second of three AT hooks in HMGA is missing. This AT hook is critical for sequence-specific DNA binding, indicating that the domain of NURF301 is adapted for binding to general DNA sequences. Moreover, the motif corresponding to the third AT hook of HMGA lacks a conserved proline in NURF301; mutagenesis studies indicate that such a variant may bind to DNA nonselectively. Hence, the interaction of the HMGA-like domain of NURF301 may be largely independent of the AT content of nucleosomal DNA. The demonstration that the HMGA domain of NURF301 is functionally linked to nucleosome binding, nucleosome-dependent stimulation of ATPase activity, and, importantly, nucleosome sliding, underscores the use of an architectural DNA binding domain as an integral part of a nucleosome remodeling mechanism. These findings are consistent with the results that also reveal an important contribution of the HMGA protein in TBP-induced nucleosome sliding on the interferon-ß promoter (Xiao, 2001).

HMGA-like binding of NURF301 to DNA at the entrance and/or exit of the nucleosome might contribute to a local change in DNA conformation (e.g., a twist or bulge). ATP-dependent propagation of the local distortion over the surface of the histone octamer by the ISWI engine, perhaps facilitated by other nucleosome binding regions of NURF301 and the single AT hook of ISWI, would result in a net change in the translational position of the nucleosome core particle. The ability to routinely purify wild-type and mutant NURF complexes should greatly facilitate biochemical and biophysical studies of the mechanism of nucleosome sliding (Xiao, 2001).

GENE STRUCTURE

cDNA clone length - 8800

Bases in 5' UTR - 600

Bases in 3' UTR - 198

PROTEIN STRUCTURE

Amino Acids - 2669

Structural Domains

Sequence matches were found throughout the NURF301 ORF for all 11 peptides derived from the largest NURF subunit, and one peptide derived from a NURF215 preparation previously obtained by conventional, multistep chromatography and SDS-PAGE. The results provide rigorous evidence that the ORF identified encodes the largest NURF subunit. In addition, Northern blot analysis shows a single ~9 kb mRNA species, consistent with the size of full-length cDNA. The identity of NURF301 was confirmed by Western blot analysis, which shows that polyclonal antibodies raised against a peptide in the NURF301 ORF reacted specifically with the largest NURF subunit. In addition, antibodies against the ISWI subunit of NURF could immunoprecipitate NURF301 from a conventionally purified NURF preparation, indicating that NURF301 is an integral part of the NURF complex rather than a cofractionating species (Xiao, 2001).

Sequence analysis of the NURF301 ORF using SMART and MacVector has revealed that NURF301 contains many protein sequence motifs conserved in transcription factors and chromatin proteins. These include an N-terminal region with sequence homology to HMGI/Y, a DDT domain, three PHD fingers, a WAC and WAKZ motif, a glutamine-rich region, and a bromodomain. The relative arrangement of WAC, WAKZ, PHD finger, and bromodomain motifs in NURF301 is similar to that found in Drosophila ACF1 and human ACF1/WCRF180, the large subunits of ACF, CHRAC, and WCRF chromatin remodeling and assembly complexes. However, the glutamine-rich and HMGI/Y-like protein motifs are not present in ACF1 (Xiao, 2001).

HMGI/Y proteins (revised nomenclature HMGA) are small polypeptides that contain three separate AT hooks, short peptide motifs that bind to the minor groove of AT-rich stretches and other sequences, followed by a C-terminal acidic tail. Although a wide variety of DNA binding proteins and chromatin remodeling factors (e.g., ISWI, brm, Rsc1, Rsc2) carry the AT-hook motif, apparently none are as similar to HMGA as the N-terminal region of NURF301. This domain conserves two of the three AT hooks as well as flanking residues. In addition, a C-terminal acidic region is found, although its location is 18 amino acids further C-terminal from the acidic tail of HMGA. Several residues that undergo posttranslational modification in HMGA are also conserved in NURF301 (Xiao, 2001).

A search of genome databases revealed human cDNAs showing sequence similarity to NURF301. By sequencing overlapping human cDNA clones, an ORF of 8967 nucleotides, predicting a 322,948 Da polypeptide (p323) was reconstructed. The nucleotide sequence of human p323 is basically identical to BPTF, a human bromodomain transcription factor (Jones, 2000); p323 has two additional exons and may be a product of alternative splicing. The N-terminal 2200 nucleotides of p323 and BPTF are also essentially identical to the cDNA for human FAC1, first identified as a protein reactive to a monoclonal antibody to Alzheimer's disease brain homogenates and implicated in transcription (Bowser, 1995; Jordan-Sciutto, 2000). The protein sequence of human p323/BPTF is ~35% identical (~50% similar) to NURF301 over the entire coding region, suggesting that it is the human ortholog of NURF301 (Xiao, 2001).

Currently, NURF301 (GenBank Accession number AF417921) corresponds to two predicted genes in the BDGP, CG17135 and CG7022, located adjacent to one another on the genomic sequence AE003467. The gene assignment in this region will require revision.

date revised: 15 November 2001

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