Capitalizing on disaster: Establishing chromatin specificity behind the replication fork

Abstract

Eukaryotic genomes are packaged into nucleosomal chromatin, and genomic activity requires the precise localization of transcription factors, histone modifications and nucleosomes. Classic work described the progressive reassembly and maturation of bulk chromatin behind replication forks. More recent proteomics has detailed the molecular machines that accompany the replicative polymerase to promote rapid histone deposition onto the newly replicated DNA. However, localized chromatin features are transiently obliterated by DNA replication every S phase of the cell cycle. Genomic strategies now observe the rebuilding of locus-specific chromatin features, and reveal surprising delays in transcription factor binding behind replication forks. This implies that transient chromatin disorganization during replication is a central juncture for targeted transcription factor binding within genomes. We propose that transient occlusion of regulatory elements by disorganized nucleosomes during chromatin maturation enforces specificity of factor binding.

Keywords: chromatin; epigenetics.

Figure 1

Chromatin dynamics around the replication fork. In front of the replication fork, charge neutralization by histone acetylation loosens nucleosomes while positive supercoiling promotes unwinding of DNA from histone octamers. Histone chaperones are localized at the fork for efficient histone transfer. Newly replicated chromatin gradually matures so that chromatin close behind the fork is transiently disordered, and chromatin structure is only completed further behind the fork.

Figure 2

Restoration of NDRs behind the replication fork in Drosophila and Saccharomyces. In Drosophila, transcription factor binding sites (blue) are occluded by nucleosomes (yellow) immediately behind the replication fork, and are only cleared on more mature chromatin. In budding yeast, transcription factor binding sites are cleared of nucleosomes immediately behind the replication fork, allowing rapid factor binding.

Figure 3

The MINCE-seq technique for mapping newly replicated chromatin. MINCE-seq maps nucleosome (yellow) and transcription factor (blue) binding positions using metabolic labeling of newly replicated DNA (red). DNA is labeled in vivo with the thymidine analog 5′-ethynyl-2′-deoxyuridine (EdU) and chromatin is fragmented using Micrococcal Nuclease (MNase). Labeled DNA is biotinylated by “Click” chemistry, and captured on streptavidin beads. Paired-end deep sequencing maps protein protection, and the length of the protected fragments distinguishes nucleosomes (~150 bp) from bound transcription factors (<50 bp).

Figure 4

Transient nucleosome disorganization behind the replication fork restricts transcription factor binding. The transcription factor binding free energy landscape on naked DNA (top) is indicated, where the dashed line represents an effective free energy cutoff for stable binding. Spurious sites (light blue) have binding energies lower than the cutoff, but these sites outnumber functional high-affinity sites (dark blue), and occasional spurious sites may have intermediate affinities (blue). In mature chromatin, spurious sites are occluded by nucleosomes (yellow) while true functional sites are accessible. Immediately after replication all binding sites are occluded by nucleosomes, and only true sites have sufficient affinity for transcription factors to compete with nucleosomes.