Abstract
BackgroundMetazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined.ResultsWe apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired.ConclusionOur work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.
Highlights
DNA replication is an exquisitely regulated process
Replication origins initiate separately at the periphery of a topologically associating domains (TADs) In order to investigate the role of chromatin structure in origin selection, we chose to directly visualize how replication initiation is spatially organized and regulated within individual replication domains (RDs) using stochastic optical reconstruction microscopy (STORM) imaging
Morphological characterization showed that the radii of gyration of TAD1 and TAD2 are about 200 nm (Additional file 1: Figure S2), which is consistent with previous work [27, 36]
Summary
DNA replication is an exquisitely regulated process. Its deregulation may lead to genome instability and tumorigenesis [1]. While a mammalian cell has a total of ~ 250,000 potential replication origins, it only uses a small subset (~ 10%) to duplicate the whole genome [2,3,4,5] It has been under debate whether the selection of origins is random or regulated. Single cellbased measurements, including the classic DNA combing assays [6,7,8] and the recent single-cell sequencing studies [9, 10], showed that cells rarely use the same cohort of origins to duplicate the genome Both single cell and population-averaged origin mapping experiments have confirmed that not all origins are equal, and they rather have differential probabilities of firing, namely origin efficiency [4], against a random origin selection mechanism. Various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined
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