Skip to main content
Fig. 4 | Epigenetics & Chromatin

Fig. 4

From: Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription

Fig. 4

hNAP1 and SET evict H1 from chromatin. a, b hNAP1 and SET remove endogenous H1 and rH10 from chromatin templates. Immobilized chromatin templates (0.33 ng/µl) were incubated with CEM NE (upper panel) (~0.55 µg/µl), or NE supplemented with rH10 (lower panel) (0.067 µM; ~fourfold excess rH10 relative to octamer) in the absence or presence of hNAP1 (panel a) or SET (panel b) (0.67 µM each). The binding assays were designed to replicate the conditions of the in vitro transcription reactions shown in Fig. 2 (hNAP1) and 3 (SET) (i.e., transcription in the presence of NE, or NE supplemented with exogenous rH10). For each panel, input NE (15 %) and rH10 (100 %) are shown (lane 3). Note that input H1 appears similar due to saturating amounts of protein. H1 binding was assessed by Western blot using an antibody against rH10. c hNAP1 and SET remove H1 incorporated into chromatin. The 900 bp immobilized promoter fragment was assembled into H10-chromatin, followed by incubation with activators, Ac-CoA, and histone chaperones, as indicated. For reference, input amounts (100 %) of histones (octamer, rH10), activators (Tax, CREB, p300), and chaperones were analyzed in parallel (lanes 79). Note that in the reactions containing activators, p300, Ac-CoA, and histone chaperones, p300 acetylation caused diffuse migration of the H3 band (lanes 4, 6). Under the conditions of the experiment, only histone H1 was evicted from the promoter template. Reactions were analyzed by SDS-PAGE followed by Coomassie staining. Molecular weight size markers (kDa) are shown (lane 1). d hNAP1 and SET directly bind rH10 as demonstrated by GST pull-down assay. Input rH10 (50 %) (lane 5) and molecular weight size markers (kDa) (lane 1) are shown. Binding reactions were analyzed by SDS-PAGE followed by Coomassie staining. e SET binding to full-length p300 is abolished in the presence of enhancer-bound Tax/pCREB complex. GST pull-down assays were performed to measure the binding of p300 to SET in the absence or presence of HTLV-1 promoter-bound activators (Tax/pCREB). Full-length p300 bound to GST-SET alone (lanes 2, 3), but was disrupted in the presence of the Tax/pCREB-bound promoter DNA (which serves as a high affinity binding site for p300) (lanes 4, 5). The p300/SET interaction was unaffected under the same conditions, but in the absence of pCREB (lanes 6, 7). Input p300 (100 %) is shown (lane 1). Binding was analyzed by Western blot using an antibody against p300

Back to article page