We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis , which is known to possess histone‐like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle‐positioning code and are excluded from gene‐regulatory DNA suggesting a functional organization. Beads‐on‐a‐string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone‐fold proteins in a variety of multimeric forms.

Being distinct from bacteria and eukaryotes, Archaea constitute a third domain of living things. The DNA replication, transcription, and translation machineries of Archaea are more similar to those of eukaryotes, whereas the genes involved in metabolic processes show more similarity to their bacterial counterparts. We report here that TK0471/TrmB-like 2 (TrmBL2), in addition to histone, is a novel type of abundant chromosomal protein in the model euryarchaeon Thermococcus kodakarensis. The chromosome of T. kodakarensis can be separated into regions enriched either with histone, in which the genetic material takes on a “beads-on-a-string” appearance, or with TK0471/TrmBL2, in which it assumes a thick fibrous structure. TK0471/TrmBL2 binds to both coding and intergenic regions and represses transcription when bound to the promoter region. These results show that the archaeal chromosome is organized into heterogeneous structures and that TK0471/TrmBL2 acts as a general chromosomal protein as well as a global transcriptional repressor.

The nematode Caenorhabditis elegans, after completing its developmental stages and a brief reproductive period, spends the remainder of its adult life as an organism consisting exclusively of post-mitotic cells. Here we show that telomere length varies considerably in clonal populations of wild-type worms, and that these length differences are conserved over at least ten generations, suggesting a length regulation mechanism in cis. This observation is strengthened by the finding that the bulk telomere length in different worm strains varies considerably. Despite the close correlation of telomere length and clonal cellular senescence in mammalian cells, nematodes with long telomeres were neither long lived, nor did worm populations with comparably short telomeres exhibit a shorter life span. Conversely, long-lived daf-2 and short-lived daf-16 mutant animals can have either long or short telomeres. Telomere length of post-mitotic cells did not change during the aging process, and the response of animals to stress was found independent of telomere length. Collectively, our data indicate that telomere length and life span can be uncoupled in a post-mitotic setting, suggesting separate pathways for replication-dependent and -independent aging. The worm Caenorhabditis elegans has historically been used as a powerful model to study organismal aging. After a brief reproductive period, an adult worm consists of 959 post-mitotic cells. Telomeres, the natural ends of linear chromosomes, have long been implicated in the aging process of mitotic cells, and telomere shortening has been proposed to be a limiting factor in cell division. The authors show that telomere length of C. elegans chromosomes is independent of organismal aging. Worm telomeres were characterized in detail, and found to vary considerably in individual clones. These differences were conserved over several populations, suggesting that telomere length is controlled by similar mechanisms in worms and mammals, emphasizing the value of C. elegans as a model for telomere biology. However, telomere length has not been found to determine the potential life span of the animals, since worms with short telomeres lived as long as worms with long telomeres. Vice versa, worms that have an altered life span due to mutations in the insulin receptor pathway do not show any changes in telomere length. Telomere length was also found to be constant in isolated, aging worm populations, suggesting that organismal aging can be uncoupled from mitotic aging.

Architectural DNA proteins play important roles in the chromosomal DNA organization and global gene regulation in living cells. However, physiological functions of some DNA-binding proteins from archaea remain unclear. Recently, several abundant DNA-architectural proteins including histones, Alba, and TrmBL2 have been identified in model euryarchaeon Thermococcus kodakarensis. Although histones and Alba proteins have been previously characterized, the DNA binding properties of TrmBL2 and its interplay with the other major architectural proteins in the chromosomal DNA organization and gene transcription regulation remain largely unexplored. Here, we report single-DNA studies showing that at low ionic strength (<300 mm KCl), TrmBL2 binds to DNA largely in non-sequence-specific manner with positive cooperativity, resulting in formation of stiff nucleoprotein filamentous patches, whereas at high ionic strength (>300 mm KCl) TrmBL2 switches to more sequence-specific interaction, suggesting the presence of high affinity TrmBL2-filament nucleation sites. Furthermore, in vitro assays indicate the existence of DNA binding competition between TrmBL2 and archaeal histones B from T. kodakarensis, which can be strongly modulated by DNA supercoiling and ionic strength of surrounding solution. Overall, these results advance our understanding of TrmBL2 DNA binding properties and provide important insights into potential functions of architectural proteins in nucleoid organization and gene regulation in T. kodakarensis.

In the telomere region of human chromosomes, the (TTAGGG)n sequence stretches over several kilobases and forms a distinct higher‐order structure with various proteins. Telomere repeat binding factors (TRFs) bind specifically to this sequence and play critical roles in the maintenance of telomere structure and function. Here, we prepared a series of linear DNA carrying a stretch of telomeric sequence ((TTAGGG)n, ∼1.8 (kb) with different end‐structures and observed their higher‐order complexes with TRFs by atomic force microscopy. TRF2 molecules exclusively bound to the telomeric DNA region at several different places simultaneously mainly as a dimer, and often mediated DNA loop formation by forming a tetramer at the root. These multiple‐binding, multimerization and DNA loop formation by TRF2 were observed regardless of the DNA‐end structure (blunt, 3′‐overhanging, telomeric, non‐telomeric). However, when the DNA end carried the telomeric‐3′‐overhanging region, the loop was frequently formed at the end of the DNA. Namely, the TRF2‐mediated DNA loop formation is independent of the end‐structure and the 3′‐overhanging TTAGGG sequence is responsible for the stabilization of the loop. TRF1 also bound to the telomeric DNA as a dimer, but did not mediate DNA loop formation by itself. These results provide a new insight into the molecular mechanism of DNA end‐loop formation by TRFs.

The regulation of DNA replication and transcription is achieved by dynamic structural changes of chromatin in which a series of proteins will acquire accessibility to specific regions of the DNA strand. A combination of biochemistry and nano-technology is essential to address questions regarding the structural basis for such macromolecular mechanisms. In the present study, we established an efficient salt-dialysis method of chromatin reconstitution and employed atomic force microscopy (AFM) as a single-molecule-imaging technique, to monitor the efficiency of the reconstitution. At first, the reconstitution efficiency with short DNA molecules of several kilo-base pairs was low, although the salt dialysis yielded a "beads-on-a-string" structure of oligonucleosomes with each nucleosome trapping 158+/-27 bp DNA. However, the efficiency for nucleosome formation became higher when longer DNA molecules with a super-helical constraint were used. A statistical analysis of the obtained AFM images identified a first-order relationship between the efficiency of the reconstitution and the length of the super-coiled DNA used. A high efficiency of approximately 290 bp/nucleosome that is close to the in vivo situation was obtained with a approximately 100 kbp template DNA. This enabled the structure-function studies of long chromatin molecules under well-defined conditions.