Restoration of locus coeruleus noradrenergic transmission during sleep.

Sleep is indispensable for health and wellbeing, but its basic function remains elusive. The locus coeruleus (LC) powerfully promotes arousal by releasing noradrenaline. We found that noradrenaline transmission is reduced by prolonged wakefulness and restored during sleep.

Activation of locus coeruleus noradrenergic neurons rapidly drives homeostatic sleep pressure.

Homeostatic sleep regulation is essential for optimizing the amount and timing of sleep, but the underlying mechanism remains unclear. Optogenetic activation of locus coeruleus noradrenergic neurons immediately increased sleep propensity following transient wakefulness. Fiber photometry showed that repeated optogenetic or sensory stimulation caused rapid declines of locus coeruleus calcium activity and noradrenaline release.

Local rewiring of genome-nuclear lamina interactions by transcription.

Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning toward the nuclear interior. How this process works and how it impacts flanking chromosomal regions are poorly understood.

Control of DNA replication timing in the 3D genome.

The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later.

Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication.

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT.

Cellular senescence induces replication stress with almost no affect on DNA replication timing.

Organismal aging entails a gradual decline of normal physiological functions and a major contributor to this decline is withdrawal of the cell cycle, known as senescence. Senescence can result from telomere diminution leading to a finite number of population doublings, known as replicative senescence (RS), or from oncogene overexpression, as a protective mechanism against cancer. Senescence is associated with large-scale chromatin re-organization and changes in gene expression.

Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.

This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.

Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors.

The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system (E-BACs) consisting of ∼200 kb human bacterial artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) stable segregation elements. E-BACs were stably maintained as autonomous mini-chromosomes in EBNA1-expressing HeLa or human induced pluripotent stem cells (hiPSCs) and established distinct RT patterns.

DNA replication timing alterations identify common markers between distinct progeroid diseases.

Progeroid syndromes are rare genetic disorders that phenotypically resemble natural aging. Different causal mutations have been identified, but no molecular alterations have been identified that are in common to these diseases. DNA replication timing (RT) is a robust cell type-specific epigenetic feature highly conserved in the same cell types from different individuals but altered in disease.

Large-Scale Chromatin Structure-Function Relationships during the Cell Cycle and Development: Insights from Replication Timing.

Chromosome architecture has received a lot of attention since the recent development of genome-scale methods to measure chromatin interactions (Hi-C), enabling the first sequence-based models of chromosome tertiary structure. A view has emerged of chromosomes as a string of structural units (topologically associating domains; TADs) whose boundaries persist through the cell cycle and development. TADs with similar chromatin states tend to aggregate, forming spatially segregated chromatin compartments.

Many paths lead chromatin to the nuclear periphery.

It is now well accepted that defined architectural compartments within the cell nucleus can regulate the transcriptional activity of chromosomal domains within their vicinity. However, it is generally unclear how these compartments are formed. The nuclear periphery has received a great deal of attention as a repressive compartment that is implicated in many cellular functions during development and disease.

Topologically associating domains and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program.

Mammalian genomes are partitioned into domains that replicate in a defined temporal order. These domains can replicate at similar times in all cell types (constitutive) or at cell type-specific times (developmental). Genome-wide chromatin conformation capture (Hi-C) has revealed sub-megabase topologically associating domains (TADs), which are the structural counterparts of replication domains.

Complex correlations: replication timing and mutational landscapes during cancer and genome evolution.

A recent flurry of reports correlates replication timing (RT) with mutation rates during both evolution and cancer. Specifically, point mutations and copy number losses correlate with late replication, while copy number gains and other rearrangements correlate with early replication. In some cases, plausible mechanisms have been proposed.