Estudios estructurales y caracterización de la unión al DNA del dominio C-terminal de la histona H1. Efecto de la fosforilación

Abstract

H1 linker histones are thought to be primarily responsible for the condensation of the 30 nm chromatin fibre. Histone H1 preferentially binds to scaffold-associated regions (SARs). Here we show that the mammalian somatic subtypes H1a,b,c,d,e and H1_ and themale germline-specific subtype H1t, all preferentially bind to SARs. 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. The carboxyl-terminal domain of linker histone H1 subtypes H1º (C-H1º) and H1t (C-H1t) has little structure in aqueous solution but becomes extensively folded upon interaction with DNA. The secondary structure elements present in the bound carboxylterminal domain include α-helix, β-structure, turns, and open loops. The addition of TFE also induces secondary structure in the C-terminal domain that is very similar to the structure of the DNA bound. Examination of the changes in the amide I components in the 20-80 °C temperature interval showed that the secondary structure of the DNA-bound C-H1t is for the most part extremely stable. The H1 carboxyl-terminal domain appears to belong to the so-called disordered proteins, undergoing coupled binding and folding. In the cellular environment macromolecules and small molecule solutes are present at high concentrations so that a significant fraction of the intracellular space is not available to other macromolecules.The C-terminal domains C-H1º and C-H1t are significantly structured in the presence of Ficoll 70 (30%) and PEG (30%) The proportions of secondary structure motifs were comparable to those of the DNA-bound domain. The small-angle X-ray scattering showed that in crowding agents the C-terminus had the compaction of a globular state. Progressive dissipation of the secondary structure and a lineal increase in partial heat capacity (Cp) with temperature together with increased binding of ANS indicated that the C-terminus is not cooperatively folded in crowded conditions. These results indicate that the C-terminus in crowding agents is in a molten globule state. Folding of the C-terminus in crowded conditions may increase the rate of the transition toward the DNA bound state and facilitate H1 diffusion inside cell nuclei. Histone H1 is phosphorylated in a cell cycle-dependent manner by cyclin-dependent kinases (CDKs). The highest number of phosphorylated sites is found in mitosis. The majority of the phosphorylation sites for CDKs are located on the C-terminal domain. Complete phosphorylation of C-H1º is associated to a major structural change. This structural rearrengement implies the loss of almost all the α-helix and a large increase in β-structure. The extent of the conformational change appears to be dependent on triphosphorylation and the protein/DNA ratio. The final state of the structural change consists in an all-β protein at ratios near saturation. Phosphorylation of one or two site have distintic structural effects. Phosphorylation of T118 affects the secondary structure the most, with a decrease of the α-helix and the appearence of random coil. Charge neutralization provided by DNA phosphate groups is an important factor in the folding of the C-terminal domain of histone H1 when bound to DNA. Alkaline pH induces secondary structure in C-H1º unphosphorylated and triphosphorylated that reflects the structural changes associated to phosphorylation.

Date of Award	25 Jan 2008
Original language	Spanish
Supervisor	Inmaculada Ponte Marull (Director) & Pedro Suau León (Director)