The Telomere Binding Protein TRF2 Induces Chromatin Compaction

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures

Structural plasticity of single chromatin fibers revealed by torsional manipulation

Magnetic tweezers are used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin 3-D architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, which are determined by the crossing status of the entry/exit DNAs (positive, null or negative). Torsional strain, in displacing that equilibrium, extensively reorganizes the fiber architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin, and may provide the ground to better understand the dynamic binding of most chromatin-associated proteins.Comment: 18 pages, 7 figures, Supplementary information available at http://www.nature.com/nsmb/journal/v13/n5/suppinfo/nsmb1087_S1.htm

Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction

The previously identified LRS (Loss of rDNA Silencing) domain of the nucleosome is critically important for silencing at both ribosomal DNA and telomeres. To understand the function of the LRS surface in silencing, we performed an EMS mutagenesis screen to identify suppressors of the H3 A75V LRS allele. We identified dominant and recessive mutations in histones H3, H4, and dominant mutations in the BAH (Bromo Adjacent Homology) domain of SIR3. We further characterized a surface of Sir3p critical for silencing via the LRS surface. We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles. Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4. Mutations in H3 and H4 genes that lose silencing tend to make the LRS surface more electronegative, whereas mutations that increase silencing make it less electronegative. Conversely, suppressors of LRS alleles in either SIR3, histone H3, or H4 also tend to make their respective surfaces less electronegative. Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain. Based on these findings, we propose an electrostatic model for how an extensive surface on the Sir3p BAH domain may regulate docking onto the LRS surface

Assessment of major and minor groove DNA interactions by the zinc fingers of Xenopus transcription factor IIIA.

Zinc finger proteins of the Cys2His2 class are DNA sequence-specific transcription factors. Previous structural studies of zinc finger protein-DNA complexes have shown that amino acids in the finger tip and alpha-helix regions within individual finger domains make base-specific contacts with the major groove of DNA. The nine finger protein transcription factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair region of the 5S RNA gene through major groove interactions with two sets of three fingers (fingers 1-3 and 7-9) and with finger 5. Previous studies have suggested that zinc fingers 4 and 6 each bind in or across the minor groove to bridge these major groove-binding zinc fingers. Here it is shown that a polypeptide containing zinc fingers 1-5 (zf1-5) binds oligonucleotides with modifications in the major groove of the finger 4 binding site with wild-type affinity. Mutagenesis and binding site selection studies were performed to determine whether high affinity DNA binding by zf1-5 requires a particular sequence in the binding site for finger 4. Several mutations in this region of the 5S gene reduced the DNA-binding affinity of zf1-5; however, selection and amplification binding assays did not recover the wild-type finger 4 binding site sequence from a pool of mixed sequence oligonucleotides. Rather, a purine-rich sequence on the top strand was highly selected within the finger 4 binding site. We suggest that high affinity DNA binding by zinc finger 4 may be dictated by a sequence-specific DNA structure rather than by a unique DNA sequence. Deletion of finger 4 from zf1-5 results in a protein with poor binding affinity, demonstrating the importance of finger 4 in proper alignment of neighboring fingers with the DNA, and/or the importance of correct protein-protein interactions between fingers