Dynamics as a cause for the nanoscale organization of the genome

Chromatin ‘blobs’ were recently identified by live super-resolution imaging of labeled nucleosomes as pervasive but fleeting structural entities. However, the mechanisms leading to the formation of these blobs and their functional implications are unknown. We explore here whether causal relationships exist between parameters that characterize the chromatin blob dynamics and structure, by adapting a framework for spatio-temporal Granger-causality inference. Our analysis reveals that chromatin dynamics is a key determinant for both blob area and local density. Such causality, however, could be demonstrated only in 10–20% of the nucleus, suggesting that chromatin dynamics and structure at the nanometer scale are dominated by stochasticity. We show that the theory of active semiflexible polymers can be invoked to provide potential mechanisms leading to the organization of chromatin into blobs. Our results represent a first step toward elucidating the mechanisms that govern the dynamic and stochastic organization of chromatin in the cell nucleus.</p

Identification of high affinity Tbf1p-binding sites within the budding yeast genome

The yeast TBF1 gene is essential for mitotic growth and encodes a protein that binds the human telomere repeats in vitro, although its cellular function is unknown. The sequence of the DNA-binding domain of Tbf1p is more closely related to that of the human telomeric proteins TRF1 and TRF2 than to any yeast protein sequence, yet the functional homologue of TRF1 and TRF2 is thought to be Rap1p. In this study we show that the Tbf1p DNA-binding domain can target the Gal4 transactivation domain to a (TTAGGG)(n) sequence inserted in the yeast genome, supporting the model that Tbf1p binds this sub-telomeric repeat motif in vivo. Immunofluorescence of Tbf1p shows a spotty pattern throughout the interphase nucleus and along synapsed chromosomes in meiosis, suggesting that Tbf1p binds internal chromosomal sites in addition to sub-telomeric regions. PCR-assisted binding site selection was used to define a consensus for high affinity Tbf1p-binding sites. Compilation of 50 selected oligonucleotides identified the consensus TAGGGTTGG. Five potential Tbf1p-binding sites resulting from a search of the total yeast genome were tested directly in gel shift assays and shown to bind Tbf1p efficiently in vitro, thus confirming this as a valid consensus for Tbf1p recognition

Differential Requirement of DNA Replication Factors for Subtelomeric ARS Consensus Sequence Protosilencers in Saccharomyces cerevisiae

The establishment of silent chromatin requires passage through S-phase, but not DNA replication per se. Nevertheless, many proteins that affect silencing are bona fide DNA replication factors. It is not clear if mutations in these replication factors affect silencing directly or indirectly via deregulation of S-phase or DNA replication. Consequently, the relationship between DNA replication and silencing remains an issue of debate. Here we analyze the effect of mutations in DNA replication factors (mcm5-461, mcm5-1, orc2-1, orc5-1, cdc45-1, cdc6-1, and cdc7-1) on the silencing of a group of reporter constructs, which contain different combinations of “natural” subtelomeric elements. We show that the mcm5-461, mcm5-1, and orc2-1 mutations affect silencing through subtelomeric ARS consensus sequences (ACS), while cdc6-1 affects silencing independently of ACS. orc5-1, cdc45-1, and cdc7-1 affect silencing through ACS, but also show ACS-independent effects. We also demonstrate that isolated nontelomeric ACS do not recapitulate the same effects when inserted in the telomere. We propose a model that defines the modes of action of MCM5 and CDC6 in silencing

Subtelomeric ACS-containing Proto-silencers Act as Antisilencers in Replication Factors Mutants in Saccharomyces cerevisiae

Subtelomeric genes are either fully active or completely repressed and can switch their state about once per 20 generations. This meta-stable telomeric position effect is mediated by strong repression signals emitted by the telomere and relayed/enhanced by weaker repressor elements called proto-silencers. In addition, subtelomeric regions contain sequences with chromatin partitioning and antisilencing activities referred to as subtelomeric antisilencing regions. Using extensive mutational analysis of subtelomeric elements, we show that ARS consensus sequence (ACS)-containing proto-silencers convert to antisilencers in several replication factor mutants. We point out the significance of the B1 auxiliary sequence next to ACS in mediating these effects. In contrast, an origin-derived ACS does not convert to antisilencer in mutants and its B1 element has little bearing on silencing. These results are specific for the analyzed ACS and in addition to the effects of each mutation (relative to wild type) on global silencing. Another line of experiments shows that Mcm5p possesses antisilencing activity and is recruited to telomeres in an ACS-dependent manner. Mcm5p persists at this location at the late stages of S phase. We propose that telomeric ACS are not static proto-silencers but conduct finely tuned silencing and antisilencing activities mediated by ACS-bound factors