The preferential binding of histone H1 to DNA scaffold-associated regions is determined by its C-terminal domain

Abstract

Histone H1 preferentially binds and aggregates scaffold-associated regions (SARs) via the numerous homopolymeric oligo(dA).oligo(dT) tracts present within these sequences. Here we show that the mammalian somatic subtypes H1a,b,c,d,e and H1 degrees and the male germline-specific subtype H1t, all preferentially bind to the Drosophila histone SAR. Experiments with the isolated domains show that whilst the C-terminal domain maintains strong and preferential binding, the N-terminal and globular domains show weak binding and poor specificity for the SAR. The preferential binding of SAR by the H1 molecule thus appears to be determined by its highly basic C-terminal domain. Salmine, a typical fish protamine, which could have its evolutionary origin in histone H1, also shows preferential binding to the SAR. The interaction of distamycin, a minor groove binder with high affinity for homopolymeric oligo(dA).oligo(dT) tracts, abolishes preferential binding of the C-terminal domain of histone H1 and protamine to the SAR, suggesting the involvement of the DNA minor groove in the interaction.

Figure 1

Preferential binding of histone H1 subtypes to the Drosophila SAR. The subtypes H1a–e, H1° and H1t (lanes 2–15) show preferential binding to the SAR fragment compared with the pUC19 fragment. Once the SAR fragment is saturated, H1 binds to pUC19 (lanes 16 and 17). The subtype is indicated above. The protein/DNA ratio (w/w) is indicated. I, input mixture of the SAR and pUC19 fragments; P, pellet; S, supernatant.

Figure 2

Preferential binding of the histone H1 C-terminal domain to the SAR fragment. (A) C-terminal domain of H1° (CH1°); (B) C-terminal domain of H1e (CH1e); (C, D and E) C-terminal domain of H1t (CH1t). DNA fragments are indicated: SAR (SAR fragment of 657 bp), pUC19 (pUC19 fragment of 587 bp), epUC19 (extended pUC19 fragment of 763 bp). The concentration of NaCl is indicated in parenthesis. The protein/DNA ratio (w/w) is indicated. I, input mixture of the fragments; P, pellet; S, supernatant.

Figure 3

Sequence identity between the C-terminal domains of mouse histone H1 subtypes. (A) Alignment of the C-terminal domain sequences of H1a–e, H1° and H1t. Multiple alignment was performed using the ClustalW resource under DNASTAR. The conserved positions are indicated by an asterisk (^*). (B) Percentage similarity table.

Figure 4

Preferential binding of the histone H1 C-terminal domain and protamine to the SAR in the presence of AT-rich DNA. The AT-rich DNA was obtained by polymerization of 50 bp sequence containing 75% AT and lacking A-tracts as described in Materials and Methods. (A) Analysis of the ligated DNA by 2% agarose gel electrophoresis; 1, DNA molecular size markers; 2, ligation reaction. The limits of the size distribution of oligomers used in the binding experiments were from 500 to 5000 bp. (B) Preferential binding of the C-terminal domain of H1t to the SAR. (C) Preferential binding of protamine to the SAR. The concentration of NaCl is indicated in parenthesis. The protein/DNA ratio (w/w) is indicated. I, input mixture of the DNA fragments, the discrete band corresponds to the SAR and the smear to the AT-rich fragments; P, pellet; S, supernatant.

Figure 5

Binding of the N-terminal peptides and the globular domain of histone H1 to the SAR and pUC19 fragments. (A) and (B) N-terminal peptide of H1e (NE-1); (C) and (D) N-terminal peptide of H1° (NH-1); (E) globular domain of H1° (GH1°); and (F) globular domain of H5 (GH5). The concentration of NaCl is indicated in parenthesis. The protein/DNA ratio (w/w) is indicated. I, input mixture of the SAR and pUC19 fragments; P, pellet; S, supernatant.

Figure 6

Preferential binding of protamine to the SAR fragment. DNA fragments are indicated: SAR (SAR fragment of 657 bp), pUC19 (pUC19 fragment of 587 bp), epUC19 (extended pUC19 fragment of 763 bp). The concentration of NaCl is indicated in parenthesis. The protein/DNA ratio (w/w) is indicated. I, input mixture of the fragments; P, pellet; S, supernatant.

Figure 7

Distamycin inhibits the preferential binding of the histone H1 C-terminal domain and protamine to the SAR fragment. (A) C-terminal domain of H1t (CH1t); (B) salmon protamine. The concentration of NaCl is indicated in parenthesis. The concentration of distamycin and the protein/DNA ratio (w/w) are indicated. I, input mixture of the SAR and pUC19 fragments; P, pellet; S, supernatant.