Cell cycle dynamics of lamina associated DNA

Abstract

words)In mammalian interphase nuclei more than one thousand large genomic regions are positioned at the nuclear lamina (NL). These lamina associated domains (LADs) are involved in gene regulation and may provide a backbone for the overall folding of interphase chromosomes. While LADs have been characterized in great detail, little is known about their dynamics during interphase, in particular at the onset of G1 phase and during DNA replication. To study these dynamics, we developed an antibody-based variant of the DamID technology (named pA-DamID) that allows us to map and visualize genome -NL interactions with high temporal resolution. Application of pA-DamID combined with synchronization and cell sorting experiments reveals that LAD -NL contacts are generally rapidly established early in G1 phase. However, LADs on the distal ~25 Mb of most chromosomes tend to contact the NL first and then gradually detach, while centromere-proximal LADs accumulate gradually at the NL. Furthermore, our data indicate that S-phase chromatin shows transiently increased lamin interactions. These findings highlight a dynamic choreography of LAD -NL contacts during interphase progression, and illustrate the usefulness of pA-DamID to study the dynamics of genome compartmentalization.genome-wide mapping of NL interactions, DamID has been the major method [1,22]. DamID is based on expression of a fusion protein of Dam and a NL protein (e.g. Lamin B1), which results in gradual accumulation of adenine methylation ( m6 A) on DNA that contacts the NL [23]. This m6 A labeled DNA is then amplified and sequenced. An added advantage of DamID is that the m6 A tags can be detected by a GFP-labeled m6 A-Tracer protein [8]. This enables visualization of LAD -NL contacts in situ, which greatly assists in the interpretation of genome-wide DamID mapping data.However, a major limitation of DamID is its poor temporal resolution. The activation of Dam and deposition of m6 A requires at least several hours [8,24], precluding detailed analysis of the NL interaction dynamics. To overcome these limitations, we developed pA-DamID -a hybrid of DamID and the CUT&RUN method [25]. This allows for both mapping and visualization of NL contacts with high temporal resolution. Using pA-DamID, we show that after mitosis NL contacts do not initiate at defined loci, but rather are widespread with an enrichment at LADs on distal regions of chromosomes. Furthermore, small LADs appear to be gradually displaced from the NL by larger LADs. Additionally, we found that replicating DNA shows transiently increasing lamin contacts. Results Principle of pA-DamID to map and visualize NL associated DNAThe pA-DamID method is a hybrid of the CUT&RUN and DamID technologies (Fig. 1A). In CUT&RUN, cells are permeabilized and incubated with an antibody against a nuclear protein of interest, followed by protein A (pA) fused to micrococcal nuclease (MNase). Subsequent activation of the tethered MNase by Ca ++ ions results in excision of DNA sequences that are in molecular proximity to the pro...

A model-based analysis reveals three-dimensional genome organization heterogeneity and functional sub-compartments in cell populations

“…We found that during early G1 the chromatin associated with the reforming NL are the sub-telomeric regions. This observation was also observed using the novel protein A-DamID approach described by van Steensel and colleagues (26). Interestingly, one sub-compartment, HCT116 C10_7, predicted by CscoreTool-M is very similar to the early G1 LADs mapped by our cTSA-seq.…”

Dynamic chromosomal interactions and control of heterochromatin positioning by Ki-67

“…We hypothesized that these transitions would be accompanied by a shift in genomic interactions. To test this, we synchronized RPE cells in metaphase and harvested cells at several time points during the cell cycle (van Schaik et al, 2020) to profile Ki-67 interactions. Remarkably, chromosome X does not follow these trends.…”

Phase Separation and Correlated Motions in Motorized Genome

“…We note that, for stem cells and cancer cells, which both have larger volumes, LADs, in fact, are less localized towards the periphery. 102,103 Though a disruption in lamin expression may cause the displacement of LADs from the nuclear envelope, 65,[104][105][106][107] the non-equilibrium mechanisms could contribute to the changes in LADs localization as well.…”