Role of Nucleosome Remodeling Factor NURF in Transcriptional Activation of Chromatin

Molecular Cell, Vol. 1, 141–150, December, 1997, Copyright 1997 by Cell Press

Role of Nucleosome Remodeling Factor NURF in Transcriptional Activation of Chromatin Gaku Mizuguchi, Toshio Tsukiyama, Jan Wisniewski,† and Carl Wu* Laboratory of Molecular Cell Biology National Cancer Institute National Institutes of Health Building 37, Room 5E-26 Bethesda, Maryland 20892-4255

Summary The Drosophila nucleosome remodeling factor (NURF) is a protein complex of four subunits that assists transcription factor-mediated perturbation of nucleosomes in an ATP-dependent manner. We have investigated the role of NURF in activating transcription from a preassembled chromatin template and have found that NURF is able to facilitate transcription mediated by a GAL4 derivative carrying both a DNA binding and an activator domain. Interestingly, once nucleosome remodeling by the DNA binding factor is accomplished, a high level of NURF activity is not continuously required for recruitment of the general transcriptional machinery and transcription for at least 100 nucleotides. Our results provide direct evidence that NURF is able to assist gene activation in a chromatin context, and identify a stage of NURF dependence early in the process leading to transcriptional initiation. Introduction Biochemical and genetic findings over the past decade have established that the compression of eukaryotic DNA in chromatin suppresses gene activity in a general manner. This repression extends from the level of the nucleosome, the primary unit of chromatin organization, to the higher order folding of nucleosome arrays and the organization of silent regions of chromatin (for reviews, see Grunstein, 1990; Kornberg and Lorch, 1992; Van Holde et al., 1995; Fletcher and Hansen, 1996; Koshland and Strunnikov, 1996; Ramakrishnan, 1997). How chromatin-mediated repression is overcome to permit transcription is a central problem in the control of gene expression. Current studies reveal that multiple mechanisms are involved in counteracting this repression, including DNA structure, histone modification, and the action of nonhistone regulators of nucleosome structure (for reviews, see Becker, 1994; Owen-Hughes and Workman, 1994; Paranjape et al., 1994; Wolffe, 1994; Kornberg and Lorch, 1995; Brownell and Allis, 1996; Felsenfeld, 1996; Tsukiyama and Wu, 1997). A novel class of chromatin remodeling proteins whose activities are associated with ATP hydrolysis has recently emerged. The prototype of this class, yeast SWI2/ * To whom correspondence should be addressed. † Present address: StressGen Biotechnologies, Victoria, BC V82 4B9, Canada.

SNF2, was identified genetically as a positive transcriptional regulator and contains sequence motifs closely related to those found in DNA-stimulated ATPases (for reviews, see Carlson and Laurent, 1994; Peterson, 1996). The SWI2/SNF2 protein is contained in a very large multisubunit complex that is evolutionarily conserved in yeast, Drosophila, and human cells (Tamkun et al., 1992; Khavari et al., 1993; Cairns et al., 1994; Chiba et al., 1994; Peterson et al., 1994; Dingwall et al., 1995; Muchardt et al., 1995, 1996; Wang et al., 1996a, 1996b). Biochemical studies show that the yeast and human SWI/SNF complex can directly assist site-specific binding of GAL4 derivatives in an ATP-dependent manner and can assist in local reconfiguration within an array of preassembled nucleosomes (Cote et al., 1994; Imbalzano et al., 1994, 1996; Kwon et al., 1994; Brown et al., 1996; OwenHughes et al., 1996; Wang et al., 1996b). RSC is an essential and abundant multisubunit complex that contains a subunit related to SWI2/SNF2 and has DNAdependent ATPase activity and the ability to alter nucleosome structure in vitro (Cairns et al., 1996). CHD-1, another member of the SWI2/SNF2 family, is localized to the decondensed interbands of polytene chromosomes and to a number of active chromosome puffs (Stokes et al., 1996). Another activity has been identified in Drosophila embryo extracts, which increases accessibility of nucleosomal arrays to restriction enzymes and can render mobile an entire nucleosomal array in an ATP-dependent manner (Varga-Weisz et al., 1995). In previous studies, we purified the nucleosome remodeling factor NURF as a four-subunit complex that assists GAGA transcription factor-mediated reconfiguration of nucleosomes in an ATP-dependent fashion (Tsukiyama et al., 1994; Tsukiyama and Wu, 1995). The 140 kDa subunit of NURF is encoded by Drosophila ISWI, related in the ATPase domain to the Drosophila SWI2/SNF2 homolog, brm (Elfring et al., 1994; Tsukiyama et al., 1995). Unlike other members of the SWI2/ SNF2 family, the ATPase activity of NURF is stimulated significantly more by nucleosomes than by free DNA or by core histones, indicating a preferential interaction of NURF with nucleosomes. This interaction involves the flexible, positively charged histone tails that protrude from the nucleosome surface (Georgel et al., 1997). Like the GAGA factor, other sequence-specific factors such as HSF (Tsukiyama et al., 1995; Wall et al., 1995), GAL4 (Pazin et al., 1994), Sp1 and NF-kB (Pazin et al., 1996), NF-E2 (Armstrong and Emerson, 1996), and lac repressor (Pazin et al., 1997) have been shown to remodel nucleosomes reconstituted with chromatin assembly extracts derived from Drosophila embryos that contain active NURF and other remodeling activities (Becker and Wu, 1992; Kamakaka et al., 1993). In several cases, the sequence-specific factors were also found to stimulate transcription many-fold on the reconstituted chromatin templates. Despite these advances, none of the aforementioned chromatin remodeling activities have been shown to facilitate activation of a chromatin template in an in vitro

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transcription reaction. In this paper, we report that NURF is able to assist activation of a chromatin template by a GAL4 derivative carrying a constitutive activating region from the heat shock transcription factor, HSF. We also show that once nucleosome reconfiguration is accomplished, a high level of NURF is not continuously required for subsequent recruitment of the general transcriptional machinery and transcription initiation. These results provide direct evidence that NURF is able to remodel nucleosomes for gene activation in chromatin and suggest a point in the process of transcription initiation when the nucleosome disordering function of NURF may be especially required.

Results ATP-Dependent Nucleosome Remodeling Is Required for Activation of a Chromatin Template by GAL4-HSF To evaluate the requirements for activation of a preassembled chromatin template, we reconstituted a plasmid carrying five tandemly repeated GAL4 binding sites (UASG) upstream of the TATA box and the adenovirus E4 minimal core promoter (pGIE-0; Pazin et al. 1994) in a transcriptionally inert nucleosome array using a Drosophila chromatin assembly extract. Two related versions of the chromatin assembly extract were effective in reconstituting chromatin for transcriptional activation (S-150, Becker and Wu, 1992; S-190, Kamakaka et al., 1993); we present results obtained with the S-190 extract to facilitate direct comparison with previous transcription studies. After chromatin assembly (6 hr), ATP and proteins not associated with the reconstituted chromatin were removed by Sepharose CL4B gel filtration chromatography. For energy-dependent chromatin remodeling, the partially purified chromatin was incubated with a saturating amount of GAL 4 derivatives in the presence or absence of fresh ATP (30 min). The remodeled chromatin was then directly assayed for transcription by incubation with a Drosophila soluble nuclear fraction to form preinitiation complexes (30 min) (Kamakaka et al., 1991; Kamakaka and Kadonaga, 1994). The soluble nuclear fraction, termed nuclear extract (NE) hereafter, contains a highly active RNA polymerase II transcriptional apparatus, is deficient in histones and other inhibitory proteins, and has weak ATP-dependent chromatin remodeling activity, allowing unambiguous analysis of the earlier remodeling step (Pazin et al., 1994; unpublished data). Transcription was finally enabled by the addition of all four NTPs (10 min), and the RNA products were analyzed by primer extension. The results were highly reproducible; data sets for each figure are presented with the average-fold activation from a minimum of three experiments. As shown in Figure 1A (lanes 4, 7, and 10), no transcription was observed from chromatin templates in reactions lacking a GAL4 derivative. Consistent with previous findings, remodeling in the presence of ATP by GAL4(1–147), carrying essentially the DNA binding domain of GAL4, activated the chromatin template very weakly (2.8-fold; Figure 1A, lane 11) with transcription

initiating from the E4 promoter at two positions separated by 6 nucleotides (Pazin et al., 1994). Weak activation (2.9-fold) by GAL4(1–147) was also observed when chromatin was not purified by chromatography, thereby allowing remodeling to occur with ATP carried over from the assembly step (Figure 1A, lane 5). When preassembled chromatin templates were remodeled with GAL4(1– 147) fused to a constitutive activating region of the heat shock transcription factor HSF (GAL4-HSF; Wisniewski et al., 1996), activation was 58-fold with freshly added ATP in the remodeling reaction and 63-fold with ATP carried over from the chromatin assembly step (Figure 1A, lanes 12 and 6). This level of activation was z16% of the absolute level of transcription observed on naked DNA (Figure 1A, lane 3). The activation was ATP-dependent, since substantially reduced activation by GAL4HSF was observed when ATP was omitted during the remodeling step (3.3-fold; Figure 1A, lane 9). The results indicate that the repression of basal transcription imposed by nucleosomes is significantly relieved by the joint action of GAL4-HSF and an ATP-dependent nucleosome remodeling activity associated with reconstituted chromatin. To confirm the extent of chromatin remodeling by GAL4-derivatives, we monitored nucleosome structure after the remodeling step (6.5 hr) by digestion with micrococcal nuclease (MNase) and Southern blot hybridization using probes specific for the adenovirus E4 promoter (TATA box) or a distal region 900 bp upstream. As shown in Figure 1B, a regularly spaced array of nucleosomes of z180 bp repeat length was observed on the promoter region when no GAL4 derivatives were introduced in the remodeling step. However, when either GAL4(1–147) or GAL4-HSF was introduced with fresh ATP, or with ATP carried over from the chromatin assembly step, substantial remodeling occurred over the E4 promoter as revealed by the smearing of the oligonucleosomal DNA ladder at intermediate stages of MNase digestion and by the decreased abundance of the 146 bp mononucleosome fragment. No nucleosome remodeling was observed at the distal region. Similar results were obtained using chromatin reconstituted with purified Drosophila histones by salt gradient dialysis (data not shown). The elimination of ATP during the remodeling step significantly decreased nucleosome reconfiguration by the GAL4 derivatives (Figure 1B), although it should be noted that a saturating amount of GAL4 derivative nonetheless suffices to confer factor binding, as indicated by DNase I footprinting (Pazin et al., 1994) and by an increased dinucleosome population during MNase digestion that is attributable to a subpopulation of nucleosome linkers protected by GAL4 binding to the 80 bp UASG element (Figure 1B, promoter region). Our results, which concur with a similar study using GAL4VP16 (Pazin et al., 1994), clearly demonstrate that ATPdependent nucleosome remodeling is associated with transcriptional activation. Activation of Chromatin by GAL4-HSF Is Dependent on NURF In order to identify the ATP-dependent nucleosome remodeling activity associated with activation of chromatin, we briefly treated the reconstituted chromatin with

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Figure 1. ATP-Dependent Nucleosome Remodeling Is Required for Activation of a Chromatin Template by GAL4-HSF (A) Experimental scheme is diagrammed at the top. Transcription of free DNA and chromatin templates were analyzed by primer extension. Left panel (2CL4B column, continuous presence of 3 mM ATP), 100 ng (DNA equivalents) chromatin reconstituted for 6 hr was incubated with GAL4(1–147) or GAL4HSF for 30 min to allow nucleosome remodeling. Samples were then incubated with nuclear extract (NE) to form preinitiation complexes for 30 min before addition of NTPs for 10 min of transcription. Free DNA samples (100 ng) were transcribed in the same way. Right panel (1CL4B column, ATP was removed before addition of GAL4 derivatives), chromatin reconstituted for 6 hr was purified on a Sepharose CL4B spin column and incubated 1/20.5 mM ATP with GAL4 derivatives for 30 min to allow nucleosome remodeling. GAL4(1–147) or GAL4-HSF was added to 100 ng of free plasmid template and incubated for 10 min. The plasmid was then added to S-190 extract or nuclear extract (NE) (each 80 mg protein), incubated for 30 min, and transcribed for 10 min with NTPs. Samples were analyzed for transcription as in the left panel. (B) MNase digestion assay. A portion (20 ml) of the chromatin samples processed for transcription (6.5 hr) was analyzed by MNase digestion (three digestion points) and sequential Southern blot hybridization using oligonucleotide probes specific for the adenovirus E4 TATA box (promoter region), and 2900/2874 bp (distal region). The level of chromatin perturbation and transcription with GAL4-HSF added at 6 hr is comparable to that observed when the DNA binding factor is added at time zero of nucleosome assembly (not shown).

the anionic detergent Sarkosyl (0.05%) to inactivate NURF (Tsukiyama and Wu, 1995). After chromatography to remove both Sarkosyl and ATP, chromatin was tested for remodeling by incubation with GAL4 derivatives in the presence of fresh ATP, followed by the transcription assay (Figure 2A, top). When GAL4 derivatives were omitted from the remodeling reaction, no detectable transcription was observed for Sarkosyl-inactivated chromatin (Figure 2A, lane 4), and nucleosome organization was not perturbed (Figure 2B, lane 1). Introduction of GAL4 derivatives to Sarkosyl-treated chromatin still gave poor activation, even in the presence of ATP (Figure 2A, lanes 5 and 6), and nucleosome remodeling at the E4 promoter region was undetectable (Figure 2B, lanes 2 and 3), while control, untreated chromatin showed strong activation by GAL4-HSF (Figure 2A, lane 3). To investigate whether NURF could facilitate activation, we introduced in the remodeling reaction a NURF preparation purified up to the penultimate step (P-11 fraction; Tsukiyama et al., 1995). This resulted in a significant restoration of transcription mediated by GAL4-HSF (29-fold activation; Figure 2A, lane 9), about 70% of that observed for the (2Sarkosyl) control (41-fold activation; Figure 2A, lane 3). There was also restoration of nucleosome remodeling at the promoter region (Figure 2B,

lanes 5 and 6), and both remodeling and transcriptional activation assisted by NURF was ATP dependent. To confirm these results, we introduced our most highly purified NURF preparation to the remodeling reaction at a substoichiometric ratio of 1 NURF to 16 nucleosomes (NURF/glycerol gradient fraction, Tsukiyama et al., 1995). This NURF preparation showed a similar rescue of transcriptional activation (33-fold; Figure 2A, lane 10). Since the total ISWI to nucleosome ratio in vivo is 1:20 (Tsukiyama et al., 1995; Tsukiyama and Wu, 1997), the 1:16 ratio of NURF to nucleosomes employed in our assay is unlikely to be an order of magnitude greater than the physiological ratio, assuming that a significant fraction of ISWI is contained within the NURF complex. Taken together, the data suggest that a substantial portion of the Sarkosyl-sensitive requirement for activation can be satisfied by NURF. We further addressed whether other remodeling activities besides NURF could activate chromatin in our assay. Sarkosyl-treated pGIE-0 chromatin was tested with two ATP-dependent chromatin remodeling activities: the Sarkosyl-sensitive Drosophila CHRAC complex (Varga-Weisz et al., 1997) and the yeast SWI/SNF complex (Cote et al., 1994). As shown in Figure 3A, neither CHRAC nor SWI/SNF introduced at levels comparable

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results raise the interesting possibility that the two other chromatin remodeling activities we have assayed may be specialized for different configurations of promoters and enhancers, different stages of transcription, or for chromosomal functions separate from transcription.

Figure 2. NURF Is Able to Activate Chromatin (A) Experimental scheme is diagrammed at the top. Primer extension analysis showing transcriptional activation of chromatin templates by GAL4 derivatives. Chromatin assembled for 6 hr was either not treated (left panel) or treated (right panel) with 0.05% Sarkosyl, purified by gel filtration, and incubated with GAL4 derivatives, 1/20.5 ml NURF (100 ng/ml P11 fraction or 50 ng/ml glycerol gradient fraction) and 10.5 mM ATP for 30 min to allow nucleosome remodeling. Chromatin templates were then incubated for 30 min with nuclear extract (NE) to form preinitiation complexes, followed by addition of NTPs for 10 min of transcription. The concentration of NURF employed was estimated by silver staining of NURF-140 and NURF-38 (Tsukiyama and Wu, 1995), which are equimolar in the complex, and by an assumption of equimolarity for the NURF-55 and NURF-215. (B) MNase digestion assay. Chromatin samples processed as for transcription (6.5 hr) were analyzed by MNase digestion and sequential Southern blot hybridization as in Figure 1.

to NURF in ATPase units for SWI/SNF, or ISWI units for CHRAC, were able to substitute for NURF in the transcriptional activation of chromatin by GAL4-HSF. Control transcription experiments on free DNA templates confirmed that yeast SWI/SNF and Drosophila CHRAC did not block the transcription reaction (Figure 3B). The two remodeling activities also did not assist nucleosome perturbation by GAL4 derivatives in the MNase digestion assay (Figure 3C). Although yeast SWI/ SNF may not function in the Drosophila system, the

Preinitiation Complex Formation and Transcription after Nucleosome Remodeling Does Not Require High NURF Activity We next investigated whether a high level of nucleosome remodeling activity was continuously required to facilitate assembly of the preinitiation complex and to proceed with the remaining steps of the transcription assay. For this purpose, reconstituted chromatin was first remodeled with GAL4 derivatives, ATP, and endogenous NURF, then treated with Sarkosyl to inactivate NURF, followed by gel filtration chromatography to remove detergent and ATP, and transcription (Figure 4, top). A portion of the chromatin was analyzed by MNase digestion and Southern blotting, and the extent of GAL4mediated remodeling was confirmed to be unaltered by detergent treatment (data not shown). Sarkosyl also did not displace GAL4 from DNA as revealed by gel mobility shift (data not shown). The results revealed that inactivation of NURF after remodeling did not substantially affect the extent of transcriptional activation. Activation by GAL4-HSF was observed for the Sarkosyl-treated template and for the untreated control that also had ATP omitted during preinitiation complex formation (Figure 4, lanes 3 and 9). Hence, once remodeling has occurred, a high level of endogenous NURF (or another Sarkosyl-sensitive or ATP-dependent factor) is apparently not necessary during preinitiation complex formation and transcription to z199, as defined by primer extension. It is formally possible that NURF could be resistant to Sarkosyl in the presence of GAL4-HSF remodeled chromatin; however, this remodeling complex is unlikely to be active in the absence of ATP during formation of the preinitiation complex. It should be also noted that by design, we cannot exclude the possibility that low-level NURF or a distinct remodeling activity and ATP present in the transcription reaction may also have some contribution toward activation. Overall, the results suggest that the maintenance of a high level of NURF is unnecessary for template activation after nucleosome perturbation mediated by GAL4-HSF. Recruitment of the RNA polymerase II transcription machinery to promoters involves complex interactions between activators and the transcriptional apparatus. We were concerned whether certain components of the basal transcription machinery could be present in the S-190 extract and be recruited by GAL4-HSF during the remodeling step, thereby masking a greater requirement for ATP-dependent nucleosome disruption during preinitiation complex formation. Indeed, despite indications that the S-190 chromatin assembly extract possessed undetectable transcriptional activity (Pazin et al., 1994; unpublished data), Western analysis of the S-190 extract revealed surprising amounts of TBP, TFIIB, RAP 30, several TAFs (with the curious exception of TAF80), and the largest subunit of RNA polymerase II (IIa), all of which

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were in range of the levels found in the nuclear extract for transcription (Figure 5A; left and right panels). To minimize the potential recruitment of one or more of these components by GAL4-HSF during the remodeling reaction, we partially purified chromatin after assembly by gel filtration chromatography, before proceeding with the remodeling reaction and transcription. The chromatography eliminated or substantially reduced all examined components of the general transcription apparatus (Figure 5A, middle panel). Nonetheless, activation by GAL4-HSF when preinitiation complex formation was conducted with ATP absent (endogenous NURF not activated) was the same as that conducted with ATP present (endogenous NURF activated) (36-fold versus 35-fold; Figure 5B, lanes 3 and 6). The results indicate that recruitment of the general transcriptional apparatus by GAL4-HSF during the remodeling step is unlikely to be responsible for the reduced dependence on ATP-dependent remodeling activities during preinitiation complex formation. Discussion Efficient initiation of transcription on nucleosomal templates was first demonstrated by early studies that circumvented the requirement for nucleosome remodeling by allowing transcription factor access to DNA before the subsequent assembly of nucleosomes (Workman and Roeder, 1987; Knezetic et al., 1988; Workman et al., 1988, 1990; Becker et al., 1991). In this report, we show that a purified nucleosome remodeling complex, NURF, is able to facilitate transcriptional activation on previously assembled nucleosomes. The basic action of NURF is to activate transcription via remodeling of chromatin, an activity that could not be interchanged with that of yeast SWI/SNF. We also show that the remodeling activity of NURF is compatible with different DNA binding motifs, and that NURF may be superfluous after nucleosome remodeling has occurred.

Figure 3. Drosophila CHRAC and Yeast SWI/SNF Do Not Substitute for NURF (A) Experimental scheme is diagrammed at the top. Primer extension analysis showing transcriptional activation of chromatin templates by GAL4 derivatives. Chromatin assembled for 6 hr was treated with 0.05% Sarkosyl, purified by gel filtration, and incubated with GAL4 derivatives and ATP, without chromatin remodeling factors, or with purified NURF (0.5 ml P-11 fraction), 0.5 ml yeast SWI/SNF, or 1.0 ml of 1:10 dilution of Drosophila CHRAC (Mono Q fraction) for 30 min to allow nucleosome remodeling. Chromatin templates were then incubated for 30 min with nuclear extract (NE) to form preinitiation complexes, followed by addition of NTPs for 10 min of transcription. Equivalent amounts of NURF and CHRAC, or NURF and yeast SWI/SNF were employed, as determined by Western blotting of ISWI and by analysis of ATPase units upon stimulation by DNA (SWI/ SNF) and nucleosomes (NURF). (B) Primer extension analysis of transcription reactions using free pGIE-0 DNA templates performed as in Figure 1, with the inclusion of yeast SWI/SNF and Drosophila CHRAC, as above. (C) MNase digestion assay. Chromatin samples processed as for transcription (6.5 hr) were analyzed by MNase digestion and sequential Southern blot hybridization as in Figure 1.

NURF Is Able to Activate Preassembled Chromatin Demonstration of the action of NURF requires treatment of the reconstituted chromatin with 0.05% Sarkosyl, which inactivates not only NURF but other remodeling activities as well. However, the ability of purified NURF to restore template activation to z70% of the untreated sample when introduced at levels that are likely to be near the physiological range suggests that NURF comprises a substantial portion of the total chromatin remodeling activity in the crude Drosophila embryo extract. Two other Drosophila chromatin remodeling complexes carrying the ISWI subunit, CHRAC (Varga-Weisz et al., 1997) and ACF (Ito et al., 1997), each with an ATP-dependent nucleosome spacing activity distinct from NURF, were reported since the submission of this manuscript. We have found that CHRAC, despite its similarity to NURF as a Sarkosyl-sensitive factor, does not substitute for NURF for nucleosome remodeling or transcriptional activation. ACF was shown to facilitate transcription of a chromatin template, which was revealed in the presence of substantial amounts the histone chaperone NAP1 (Ito et al., 1997). It will be of interest to ascertain

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Figure 4. Preinitiation Complex Formation and Transcription after Nucleosome Remodeling Does Not Require High NURF Activity Experimental scheme is diagrammed at the top. Primer extension analysis showing transcriptional activation of chromatin templates by GAL4 derivatives. Chromatin assembled for 6 hr was incubated with GAL4 derivatives, and nucleosome remodeling was allowed to proceed for 0.5 hr with endogenous NURF, followed by treatment with 0.05% Sarkosyl (1Sarkosyl) to inactivate NURF, or no treatment (2Sarkosyl) and purification by gel filtration. Samples were next incubated for 30 min with nuclear extract (NE), 1/2ATP to form preinitiation complexes, followed by addition of NTPs for 10 min of transcription.

whether ACF can substitute for NURF in our assay. The yeast SWI/SNF complex was also found not to substitute for NURF when introduced at comparable units of ATPase activity. Although it is possible that yeast SWI/ SNF may be incompatible with the Drosophila system, it is also possible that each distinct chromatin remodeling complex has a specialized function in transcription or in another chromosomal activity. Once nucleosome remodeling has occurred, a high level of NURF activity is apparently not necessary for recruitment of the RNA polymerase II transcription machinery to form the preinitiation complex or for transcription of the first hundred nucleotides. However, because our transcription assay employed a crude nuclear extract with weak NURF activity, the possibility that the low activity of NURF is helpful for these late events is not excluded, nor is the potential contribution from other remodeling activities in the extract. It will be of interest to address this issue by the use of a purified RNA polymerase II transcription apparatus devoid of NURF. Finally, as the level of activation so far achieved from the chromatin template represents at best only 10%–20% of the maximal transcription observed on naked DNA, it will be of interest to identify other chromatin disordering activities that need to be brought to play to in order to reach the full transcriptional potential of the UASG-E4 promoter. These may include histone acetyltransferases, which are inoperative in our reactions lacking the acetyl-CoA substrate.

NURF Assists Disparate DNA Binding Motifs in Remodeling Chromatin Our previous studies identified NURF as an ATP-dependent protein complex that assisted GAGA transcription factor-mediated reconfiguration of nucleosomes (Tsukiyama et al., 1994; Tsukiyama and Wu, 1995). The in vitro activity of the purified complex was effective not only for GAGA factor but also for HSF (Tsukiyama and Wu, 1995). In this paper, we show that purified NURF is also able to assist the GAL4 DNA binding domain in disordering nucleosomes. Hence, at least three different DNA binding motifs: the Zn finger/basic region of GAGA factor (Omichinski et al., 1997), the winged helix-turn-helix of HSF (Damberger et al., 1994; Vuister et al., 1994), and the zinc cluster of GAL4 (Marmorstein et al., 1992) can remodel nucleosomes when assisted by NURF. These findings, together with the relative abundance of NURF (Tsukiyama and Wu, 1995) and the absence of physical interactions between NURF and the GAGA factor (M. Martinez-Balbas and T. T., unpublished data), suggest a model whereby the transient, nonspecific action of NURF on chromatin creates a window of opportunity for nucleosome disorder that is exploited by sequencespecific DNA binding motifs. Such a model, where DNA binding motifs interacting with clustered cognate elements are assisted by the independent action of NURF to overcome nucleosome organization, is different from one involving the physical recruitment of the SWI/SNF remodeling complex (for a review, see Struhl, 1996). The

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directly remodeling nucleosome structure, or for stimulating productive transcription through downstream nucleosomes. Activation of heterologous promoters by GAL4 represents a model transcriptional switch (Gann et al., 1992), which is widely exploited as a powerful tool for directing ectopic gene expression in Drosophila (Brand and Dormand, 1995). As such, the requirement for NURF in chromatin remodeling and transcriptional activation revealed in our present studies of the UASG-E4 promoter are likely to reflect bona fide requirements, at least for those natural promoters where the multiple cis-regulatory elements are in very close proximity to the TATA box and transcription start site (Figure 6). It will be of interest to define the chromatin remodeling requirements when the upstream activating elements are crippled or moved to a remote location and to analyze genes in the context of a natural arrangement of promoters and enhancers. Experimental Procedures

Figure 5. General Transcription Factors in Chromatin Assembly Extract Do Not Appear to Contribute to PIC Formation during Remodeling (A) Western blot analyses of general transcription factors. Left, chromatin assembly reaction. Middle, assembled chromatin after CL4B gel filtration. Right, purified chromatin incubated with nuclear extract NE(G25) depleted of ATP by Sephadex G25 chromatography. Samples (100 ng DNA equivalents) were analyzed using antibodies to the indicated proteins. There is a moderate level of ISWI protein fractionating with the reconstituted chromatin and a substantial level of ISWI protein in NE, despite low NURF activity. It is unclear whether ISWI in the NE is part of a complete NURF complex whose activity is inhibited, or whether another essential NURF subunit is absent. (B) Primer extension analysis showing transcriptional activation of chromatin templates. Reconstituted chromatin (6 hr) was first purified by CL4B gel filtation to remove general transcription factors; a quantity of NURF (ISWI subunit) copurifies with chromatin in the excluded volume. Purified chromatin was next incubated with GAL4 derivatives and ATP for nucleosome remodeling, and the chromatin was repurified to remove ATP. Chromatin was then incubated with NE (G25) for 30 min to form preinitiation complexes 1/2ATP, followed by addition of NTPs for 10 min of transcription.

additional recruitment of NURF by as yet unidentified DNA binding factors to increase local activity may also be accomodated by our model. An Activating Region Is Essential for In Vitro Transcription of Chromatin While both GAL4(1–147) and GAL4-HSF were found to be capable of disordering nucleosomes when assisted by NURF, only GAL4-HSF showed strong activation of the hybrid UAS G-E4 promoter. These results indicate that a strong activating region linked to the GAL4 DNA binding domain is absolutely essential, perhaps for recruitment of the RNA polymerase II apparatus (Gaudreau et al., 1997) along with the potential nucleosome disordering activities of TFIID (Mizzen et al., 1996; Xie et al., 1996). The activating region may also be needed for recruitment of other chromatin remodeling activities, for

Bacterial Expression and Purification of GAL4(1–147) and GAL4-HSF To construct plasmid pGM1, which directs expression of amino acids 1–147 of GAL4, a NdeI site was introduced into the initiating ATG codon of GAL4 in plasmid pG (Wisniewski et al., 1996) by standard PCR mutagenesis. The NdeI-BamHI fragment corresponding to DNA sequences encoding residues 1–147 of GAL4 was inserted into the plasmid pET3 (Novagen) digested with NdeI and BamHI. Plasmid pGM7 expressing the GAL4-HSF fusion protein was constructed by inserting the XhoI-BamHI fragment of construct I (321–691) (Wisniewski et al., 1996), which encodes amino acids 95– 147 of GAL4, and the carboxy-terminal 291 amino acids of Drosophila HSF of plasmid construct I (321–691) into plasmid pGM1 digested with XhoI and BamHI. Expression was induced in E. coli BL21 (DE3) pLysE (Novagen). Cells were grown at 378C in LB medium, 100 mg/ml ampicillin to an OD600 of 0.5, induced with 0.4 mM IPTG, and grown for an additional 2 hr at 308C. After centrifugation, the cell pellet was resuspended in 1/20 of the original culture volume in 0.3 M KCl-HEMGNZ buffer (25 mM HEPES-KOH [pH7.6], 0.1 mM EDTA, 12.5 mM MgCl2, 10% (v/v) glycerol, 0.1% (v/v) NP40, 20 mM ZnSO 4, 1 mM dithiothreitol, 0.25 mM phenylmethylsulfonyl fluoride, 1 mM sodium bisulfite). The cell suspension was sonicated and the lysate was clarified by centrifugation. The supernatant was diluted with HEMGNZ so that the final KCl concentration was 200 mM and then applied to HeparinSepharose CL6B (Pharmacia) column equilibrated with 0.2 M KClHEMGNZ. The column was washed with 0.2 M KCl-HEMGNZ and then successively eluted with 0.6 M and 1.0 M KCl-HEMGNZ. GAL4 derivatives were eluted in the 0.6 M KCl fraction. The eluate was dialyzed against 0.1 M KCl-HEMGNZ and applied to Streptavidin-Dynabeads M280 (Dynal) bound to a 59 terminal biotinylated GAL4 recognition site (59 -GATCCAGATCGGAGTACTGTCCTCCGG TACA-39). The beads were washed with 0.1 M KCl-HEMGNZ and 0.2 M KCl-HEMGNZ and protein was eluted with 1.0 M KClHEMGNZ. The purity of eluted GAL4 derivatives was more than 95%, as judged by SDS-PAGE and Coomassie blue staining. The binding capacity of each GAL4 derivative was determined by DNaseI footprint analysis and by the gel mobility shift assay. DNA binding units were defined according to the minimal amount of protein required to saturate the DNA. One DNA binding unit of GAL4 derivative is equivalent to 0.5 pmol protein; this amount was added to 100 ng of plasmid chromatin for the nucleosome disruption and in vitro transcription assays. Under these conditions, the molar ratio between each GAL4 binding site and GAL4 protein is 1:2. Nucleosome Assembly Reaction and Sarkosyl Treatment Chromatin was assembled using a Drosophila embryo S-190 extract (Kamakaka et al., 1993) and plasmid pGIE-0 as a template; this plasmid contains five GAL4-binding sites and a TATA box from the Adenovirus type 5 E4 promoter (Pazin et al., 1994). In a standard

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Figure 6. Summary and Model of Requirements for Chromatin Remodeling and Formation of the Preinitiation Complex Minus represents a low or no requirement. N. A., not addressed. The overlapping ovals represent a statistical distribution of nucleosomes. Black object represents GAL4-HSF. PIC, preinitiation complex, striped object. The region of DNase I hypersensitivity is indicated by brackets (data not shown). Nucleosome rearrangement is indicated by the absence of nucleosome objects over the DNase I hypersensitive sites, but this is not necessarily meant to imply total eviction of the histones.

chromatin assembly reaction, S-190 extract (60 ml; 1.5 mg protein) was incubated with purified Drosophila core histones (0.8–1.2 mg) (Simon and Felsenfeld, 1979) and Buffer R (10 mM HEPES-KOH [pH7.6], 0.5 mM EGTA, 1.5 mM MgCl2, 10% [v/v] glycerol, 10 mM KCl, 10 mM b-glycerophosphate, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) in a total volume of 90 ml at 48C for 30 min. Plasmid DNA (10 mg/ml), ATP (3 mM), creatine phosphate (30 mM), creatin phosphokinase (1 mg/ml), and MgCl2 (7 mM) were added in a final volume of 100 ml, and the chromatin assembly reaction was carried out at 268C for 6 hr. For the purification of assembled chromatin, Sepharose CL4B (Pharmacia) was used in a SizeSep-400 spin column (Pharmacia) preequilibrated with Elution Buffer (10 mM HEPES-KOH (pH7.6), 0.5 mM EGTA, 5 mM MgCl2, 10% (v/v) glycerol, 50 mM KCl, 10 mM b-glycerophosphate, 1 mM dithiothreitol, and 0.5 mg/ml BSA for double spin column separations) to remove small molecules including ATP. BSA (Boehringer-Mannheim) was added to the eluted chromatin fractions to a final concentration of 0.5 mg/ml. The amount of DNA in chromatin fraction was estimated by agarose gel electrophoresis and ethidium bromide staining with DNA concentration standard. The yield after spin-column purification is 60%–70%. For Sarkosyl treatment, 5% (v/v) stock solution of Sarkosyl was added to the assembly reaction mixture to a final concentration of 0.05% and incubated at room temperature for 5 min. The Sarkosyltreated chromatin was then applied to SizeSep-400 spin column, and DNA in the eluted chromatin fraction (z120 ml) was quantitated as described above. Aliquots (20 ml) of the chromatin fractions were each used for a set of micrococcal nuclease digestion or in vitro transcription assays. In Vitro Transcription of Reconstituted Chromatin The soluble nuclear fraction (nuclear extract, NE) was prepared from 0–12 hr Drosophila embryos as described, except that 0.1 M KCl was used in the nuclear extraction buffer instead of 0.4 M potassium glutamate (Kamakaka et al., 1991; Kamakaka and Kadonaga., 1994). Transcription from the chromatin template was analyzed by primer extension analysis as described previously (Becker et al., 1991). Typically, 100 ng of chromatin (10 ml of crude assembled chromatin, or 20 ml of spin column-purified chromatin) was preincubated in a 100 ml (final volume) containing 20 mM HEPES-KOH (pH 7.6), 5 mM MgCl2 , 40 mM KCl, 2.5% (v/v) polyvinyl alcohol (final concentration), 60–80 mg NE protein for 30 min at 268C to form preinitiation complexes, and the templates were then transcribed for 10 min at 268C with the addition of ribonucleotide triphosphates (0.55 mM final). Transcripts were detected using a 32P-labeled AdE4 primer (172/ 199) in a primer extension assay, and cDNA products were analyzed on a 6% denaturing polyacrylamide gel (Kerrigan et al., 1991). Quantitation of cDNA products was performed by Fuji Bio-Image Analyzer (Fuji). For depletion of endogenous ATP from NE, the extract was chromatographed on a Sephadex G25 (Pharmacia) gel filtration column.

Peak fractions of protein were pooled and used for in vitro transcription reaction. Purification of NURF NURF was purified from nuclear extracts of 0–12 hr Drosophila embryos up to the phosphocellulose P11 step or the glycerol gradient step as described previously (Tsukiyama and Wu, 1995). Microccocal Nuclease Digestion and Assay for NURF Activity Microccocal nuclease digestion analysis and sequential Southern blot hybridization were done as described previously (Becker and Wu, 1992; Pazin et al., 1994; Tsukiyama et al., 1994). For the NURF activity assay, 20 ml (100 ng of DNA equivalent) of Sarkosyl-treated chromatin, 0.5 ml of NURF fraction, ATP (final 0.5 mM), and 0.5 pmol of GAL4 derivative were mixed and adjusted to 30 ml with Elution Buffer. The reaction was incubated at 268C for 30 min and subjected to microccocal nuclease digestion or in vitro transcription. SDS-PAGE and Western Blotting Protein samples were electrophoresed on 8% or 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes according to standard procedures. Western blotting was performed as described (Tsukiyama et al., 1995). Acknowledgments We thank M. Pazin and J. Kadonaga for plasmid pGIE-0; R. Tjian, A. Greenleaf, Y. Nakatani, and J. Kadonaga for antibodies; M. Zhong for assistance in DNA affinity chromatography; Philippe Georgel for comparative analysis of ATPase activity of NURF and SWI/SNF; Patrick Varga-Weisz and Peter Becker for purified Drosophila CHRAC; Craig Peterson for purified yeast SWI/SNF; R. Sandaltzopoulos for critical experimental suggestions; and members of our laboratory for helpful comments and suggestions on the manuscript. This work was supported by the Intramural Research Program of the National Cancer Institute. Received May 25, 1997; revised September 3, 1997. References Armstrong, J.A., and Emerson, B.M. (1996). NF-E2 disrupts chromatin structure at human b-globin locus control region hypersensitive site 2 in vitro. Mol. Cell. Biol. 16, 5634–5644. Becker, P.B. (1994). The establishment of active promoters in chromatin. Bioessays 16, 541–547. Becker, P.B., and Wu, C. (1992). Cell-free system for assembly of transcriptionally repressed chromatin from Drosophila embryos. Mol. Cell. Biol. 12, 2241–2249.

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