Role of Nucleosome Remodeling Factor NURF in Transcriptional Activation of Chromatin  Gaku Mizuguchi, Toshio Tsukiyama, Jan Wisniewski, Carl Wu  Molecular Cell  Volume 1, Issue 1, Pages 141-150 (December 1997) DOI: 10.1016/S1097-2765(00)80015-5

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 (−CL4B column, continuous presence of 3 mM ATP), 100 ng (DNA equivalents) chromatin reconstituted for 6 hr was incubated with GAL4(1–147) or GAL4-HSF 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 (+CL4B column, ATP was removed before addition of GAL4 derivatives), chromatin reconstituted for 6 hr was purified on a Sepharose CL4B spin column and incubated +/−0.5 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 μg 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 μl) 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 −900/−874 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). Molecular Cell 1997 1, 141-150DOI: (10.1016/S1097-2765(00)80015-5)

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, +/−0.5 μl NURF (100 ng/μl P11 fraction or 50 ng/μl glycerol gradient fraction) and +0.5 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. Molecular Cell 1997 1, 141-150DOI: (10.1016/S1097-2765(00)80015-5)

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 μl P-11 fraction), 0.5 μl yeast SWI/SNF, or 1.0 μl 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. Molecular Cell 1997 1, 141-150DOI: (10.1016/S1097-2765(00)80015-5)

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 (+Sarkosyl) to inactivate NURF, or no treatment (−Sarkosyl) and purification by gel filtration. Samples were next incubated for 30 min with nuclear extract (NE), +/−ATP to form preinitiation complexes, followed by addition of NTPs for 10 min of transcription. Molecular Cell 1997 1, 141-150DOI: (10.1016/S1097-2765(00)80015-5)

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 +/−ATP, followed by addition of NTPs for 10 min of transcription. Molecular Cell 1997 1, 141-150DOI: (10.1016/S1097-2765(00)80015-5)

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. Molecular Cell 1997 1, 141-150DOI: (10.1016/S1097-2765(00)80015-5)