The N-terminal tail of histone H2A binds to two distinct sites within the nucleosome core - PubMed (original) (raw)
The N-terminal tail of histone H2A binds to two distinct sites within the nucleosome core
K M Lee et al. Proc Natl Acad Sci U S A. 1997.
Abstract
Each of the core histone proteins within the nucleosome has a central "structured" domain that comprises the spool onto which the DNA superhelix is wrapped and an N-terminal "tail" domain in which the structure and molecular interactions have not been rigorously defined. Recent studies have shown that the N-terminal domains of core histones probably contact both DNA and proteins within the nucleus and that these interactions play key roles in the regulation of nuclear processes (such as transcription and replication) and are critical in the formation of the chromatin fiber. An understanding of these complex mechanisms awaits identification of the DNA or protein sites within chromatin contacted by the tail domains. To this end, we have developed a site-specific histone protein-DNA photocross-linking method to identify the DNA binding sites of the N-terminal domains within chromatin complexes. With this approach, we demonstrate that the N-terminal tail of H2A binds DNA at two defined locations within isolated nucleosome cores centered around a position approximately 40 bp from the nucleosomal dyad and that this tail probably adopts a defined structure when bound to DNA.
Figures
Figure 1
Schematic of APB modification. (A) Position of cysteine substitutions within the N-terminal domain of H2A. (B) Chemical structure of APB.
Figure 2
(A) Extent of APB modification of cysteine-substituted H2A. H2AA12C/H2Bwt dimers were incubated in the presence or absence of APB as indicated and subsequently reacted with 14C-labeled N-ethylmaleimide . Proteins used for the reconstitutions were separated on SDS/PAGE, and the gel was stained with Coomassie blue and then autoradiographed, as indicated. (B) Nucleoprotein gel analysis of irradiated nucleosomes. Nucleosomes containing APB-modified H2A were either loaded onto the gel (lane 2) or first irradiated and then loaded (lane 3). The positions of naked DNA (lane 1) and the nucleosome complex within the gel are indicated. (C) Protein–DNA cross-linking is dependent on APB modification and UV irradiation. Nucleosomes containing labeled DNA and either APB-modified or unmodified H2A were irradiated with UV light, products were separated by SDS/PAGE, and the gel was autoradiographed. Lanes: 1, UV-irradiated wild-type nucleosomes; 2, unirradiated APB-modified nucleosomes; 3, APB-modified and UV-irradiated nucleosomes. The positions of uncross-linked naked DNA and protein–DNA cross-linked products are indicated.
Figure 3
Location of cross-links between APB-modified H2AA12C and nucleosome DNA. Nucleosomes were reconstituted with the 215-bp _Eco_RI-_Dde_I fragment 5S DNA fragment and core histones including either APB-modified H2AA12C or wild-type H2A. Cross-linking was carried out and the DNA analyzed on sequencing gels as described in Materials and Methods. (Top) Samples from nucleosomes reconstituted with the _Eco_RI-_Dde_I DNA fragment 5′-end labeled at the _Eco_RI (labels the top strand). Lanes: 1, G-specific sequencing reaction markers; 2 and 3, cross-linking within nucleosomes containing wild-type H2A and APB-modified H2AA12C, respectively. (Bottom) As in the top gel except nucleosomes were reconstituted with the _Eco_RI-_Dde_I DNA fragment 5′-end labeled at the _Dde_I site (labels the bottom strand). Lanes: 1, G-specific markers; 2, cross-linking within nucleosomes containing APB-modified H2AA12C.
Figure 4
Location of cross-links between APB-modified H2AG2C and nucleosome DNA. Samples were reconstituted with the 154 bp _Eco_RI-_Rsa_I 5S DNA fragment and prepared as in Fig. 3. (Top) samples from nucleosomes reconstituted with the _Eco_RI-_Rsa_I DNA fragment labeled at the 5′ end of the top strand at the _Eco_RI site. Lanes: 1, G-specific sequencing reaction markers; 2, hydroxyl radical footprint of the nucleosomes; 3 and 4, cross-linking within nucleosomes containing wild-type H2A and APB-modified H2AG2C, respectively. (Bottom) As in the top gel except the DNA was labeled at 3′ end of the bottom strand of the _Eco_RI-_Rsa_I DNA fragment at the _Eco_RI site. Lanes 1 and 2, products from nucleosomes containing APB-modified H2AG2C and wild-type H2A, respectively.
Figure 5
Location of cross-linked sites with random sequence core particles containing APB-modified H2AG2C or H2AA12C. Cross-linked products from core particles were prepared and analyzed by sequencing gel electrophoresis and autoradiography as described in Materials and Methods. (A) Autoradiograph of products from core particles containing wild-type H2A (lane 1), H2AG2C (lane 2), and H2AA12C (lane 3). The position of bands is indicated relative to the distance from the dyad axis of symmetry in nucleotides. Only one symmetrical half of the nucleosome core is shown as indicated by the schematic (Left). The hydroxyl radical footprint of nucleosome core particles is shown in lane 4 for reference. (B) Densitometric scans of gel in A. Distance from dyad axis of symmetry is indicated as in A.
Figure 6
Summary of cross-links. (A) Linear representation of cross-linking by one H2A N-terminal tail domain within 5S (Upper) or random sequence (Lower) nucleosome cores. Sites of cross-linking from cross-linking probe positioned at the second (H2AG2C) or 12th amino acid residue (H2AA12C) are indicated by the filled or open arrows, respectively. Distance in base pairs from the center (dyad) of the nucleosome is indicated. (B) Positions of cross-links by one H2A N-terminal tail domain within one superhelical turn of nucleosomal DNA. Top view of the nucleosome showing the locations of cross-links as in A. Only protein and DNA in the top half of the nucleosome are shown. Core histone α-helices (2) are represented by columns and other secondary structures as thin tubes as in Felsenfeld (27). The mobile histone tail domains are indicated by the dashed lines, and residues within the N-terminal tail of H2A are indicated by 3.5-Å spheres. The positions of the 2nd and 12 residues are indicated by the closed and open spheres, respectively. Histones H3, H4, H2B, and H2A are shaded dark to light gray, respectively. Note that a small portion of H3 from the bottom half of the octamer is shown. Numbers indicate distance from nucleosomal dyad as in A.
References
- Richmond T J, Finch J T, Rushton B, Rhodes D, Klug A. Nature (London) 1984;311:532–536. - PubMed
- van Holde K E. Chromatin. New York: Springer; 1989.
- Clark D J, Kimura T. J Mol Biol. 1990;211:883–896. - PubMed
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