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 folding of interphase chromosomes. Little is known about the dynamics of LADs during interphase, in particular at the onset of G1 phase and during DNA replication. 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.

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...

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We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5–10%), re-differentiated monolayers with 11–27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen-progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium.