Histone tails modulate nucleosome mobility and regulate ATP-dependent nucleosome sliding by NURF

Abstract

Nucleosome Remodeling Factor (NURF) is an ATP-dependent nucleosome remodeling complex that alters chromatin structure by catalyzing nucleosome sliding, thereby exposing DNA sequences previously associated with nucleosomes. We systematically studied how the unstructured N-terminal residues of core histones (the N-terminal histone tails) influence nucleosome sliding. We used bacterially expressed Drosophila histones to reconstitute hybrid nucleosomes lacking one or more histone N-terminal tails. Unexpectedly, we found that removal of the N-terminal tail of histone H2B promoted uncatalyzed nucleosome sliding during native gel electrophoresis. Uncatalyzed nucleosome mobility was enhanced by additional removal of other histone tails but was not affected by hyperacetylation of core histones by p300. In addition, we found that the N-terminal tail of the histone H4 is specifically required for ATP-dependent catalysis of nucleosome sliding by NURF. Alanine scanning mutagenesis demonstrated that H4 residues 16-KRHR-19 are critical for the induction of nucleosome mobility, revealing a histone tail motif that regulates NURF activity. An exchange of histone tails between H4 and H3 impaired NURF-induced sliding of the mutant nucleosome, indicating that the location of the KRHR motif in relation to global nucleosome structure is functionally important. Our results provide functions for the N-terminal histone tails in regulating the mobility of nucleosomes.

Figure 1

(A) Influence of histone tails on nucleosome assembly and positioning. Mononucleosomes lacking histone N-terminal tails were reconstituted on a radiolabeled 359-bp hsp70 promoter fragment and electrophoresed on a 4% native polyacrylamide gel in TE buffer. The four major differentially migrating nucleosome species are indicated: N1, N2, N3, and N4. Di, dinucleosome species, confirmed by sedimentation on glycerol gradients. N* is a reconstitution artifact (see text). (B) Electrophoresis of tailless nucleosomes in TGE buffer, as in A.

Figure 2

Two-dimensional native gels showing dynamics of nucleosome sliding. Electrophoresis of 359-bp hsp70 mononucleosomes containing WT and mutant histones was first conducted in TE buffer (4% polyacrylamide gel). Entire gel lanes containing the separated nucleoprotein species identical to Fig. 1B were excised and applied horizontally for electrophoresis on 5% polyacrylamide gels in TGE buffer. The electrophoretic positions of N1–N4, N*, and free DNA are indicated.

Figure 3

(A) Characterization of mobilized H2B tailless nucleosome. Mononucleosomes containing WT recombinant histones and lacking histone H2B N-terminal tails (GH2B) were reconstituted on a radiolabeled 359-bp hsp70 promoter fragment and electrophoresed on a 4% native polyacrylamide in TGE buffer. N* is a reconstitution artifact (see text). (B) Histone composition of GH2B nucleosomes migrating to the N3 region. The corresponding native gel band (A, lane 2) was excised and analyzed by SDS/PAGE and silver staining. (C) N* is greatly reduced in 100 mM NaCl. hsp70 mononucleosomes (359 bp) lacking histone H2B tails were incubated for 1 h at room temperature in TE buffer containing 100 mM NaCl. The sample was electrophoresed on a 4% native polyacrylamide gel in TE buffer. (D and E) Mononucleosomes containing recombinant histones (WT) or histones lacking H2B N-terminal tails (GH2B) were reconstituted on radiolabeled 357-bp 5S and 256-bp 5S DNA fragments and electrophoresed as above in TGE buffer.

Figure 4

Requirement of histone H4 N-terminal tail for NURF activity. (A) Stimulation of ATPase activity of NURF by nucleosomes. ATPase assays contained WT nucleosomes or nucleosomes lacking histone tails, as indicated. ATPase activity was measured by hydrolysis of [α-P³²]ATP to [α-P³²]ADP and visualized by TLC. ATPase assays were conducted in the presence of GST or GST-H4 tail proteins, as indicated. (B) Nucleosome sliding assay. hsp70 mononucleosomes (359 bp) reconstituted from WT or mutant histones were incubated with NURF in the presence or absence of ATP and electrophoresed (4% polyacrylamide gel) in TE buffer. WT mononucleosomes were reacted with NURF and ATP in the presence of GST or GST-H4 tail proteins, as indicated, before electrophoresis.

Figure 5

(A) Alanine scanning mutagenesis of the histone H4 tail and exchange of the H3 and H4 tails. SDS/PAGE (15% polyacrylamide) and Coomassie blue staining of histone octamers assembled from purified WT and mutant histones, as indicated. A, alanine; t, tail; G, globular domain. (B) Importance of histone H4 residues 16-KRHR-19 for NURF-mediated nucleosome sliding. hsp70 mononucleosomes (359 bp) reconstituted from WT or mutant histones, as indicated, were incubated with NURF in the presence or absence of ATP, electrophoresed on a 4% native polyacrylamide gel in TE buffer, and visualized by autoradiography.