Differences in nanoscale organization of regulatory active and inactive human chromatin.

Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. Analyzing genome-wide data from 733 human cell and tissue samples, we identified 2 prototypical regions that exhibit high or absent hypersensitivity to deoxyribonuclease I, respectively. These regulatory active or inactive regions were examined in the lymphoblast cell line K562 by using high-throughput super-resolution microscopy. In both regions, we systematically measured the physical distance of 2 fluorescence in situ hybridization spots spaced by only 5 kb of DNA. Unexpectedly, the resulting distance distributions range from very compact to almost elongated configurations of more than 200-nm length for both the active and inactive regions. Monte Carlo simulations of a coarse-grained model of these chromatin regions based on published data of nucleosome occupancy in K562 cells were performed to understand the underlying mechanisms. There was no parameter set for the simulation model that can explain the microscopically measured distance distributions. Obviously, the chromatin state given by the strength of internucleosomal interaction, nucleosome occupancy, or amount of histone H1 differs from cell to cell, which results in the observed broad distance distributions. This large variability was not expected, especially in inactive regions. The results for the mechanisms for different distance distributions on this scale are important for understanding the contacts that mediate gene regulation. Microscopic measurements show that the inactive region investigated here is expected to be embedded in a more compact chromatin environment. The simulation results of this region require an increase in the strength of internucleosomal interactions. It may be speculated that the higher density of chromatin is caused by the increased internucleosomal interaction strength.

The Active Mechanism of Nucleosome Depletion by Poly(dA:dT) Tracts In Vivo.

Poly(dA:dT) tracts cause nucleosome depletion in many species, e.g., at promoters and replication origins. Their intrinsic biophysical sequence properties make them stiff and unfavorable for nucleosome assembly, as probed by in vitro nucleosome reconstitution. The mere correlation between nucleosome depletion over poly(dA:dT) tracts in in vitro reconstituted and in in vivo chromatin inspired an intrinsic nucleosome exclusion mechanism in vivo that is based only on DNA and histone properties. However, we compile here published and new evidence that this correlation does not reflect mechanistic causation. (1) Nucleosome depletion over poly(dA:dT) in vivo is not universal, e.g., very weak in S. pombe. (2) The energy penalty for incorporating poly(dA:dT) tracts into nucleosomes is modest (<10%) relative to ATP hydrolysis energy abundantly invested by chromatin remodelers. (3) Nucleosome depletion over poly(dA:dT) is much stronger in vivo than in vitro if monitored without MNase and (4) actively maintained in vivo. (5) S. cerevisiae promoters evolved a strand-biased poly(dA) versus poly(dT) distribution. (6) Nucleosome depletion over poly(dA) is directional in vivo. (7) The ATP dependent chromatin remodeler RSC preferentially and directionally displaces nucleosomes towards 5' of poly(dA). Especially distribution strand bias and displacement directionality would not be expected for an intrinsic mechanism. Together, this argues for an in vivo mechanism where active and species-specific read out of intrinsic sequence properties, e.g., by remodelers, shapes nucleosome organization.