Abstract
Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation.
Highlights
Covalent DNA and histone modifications play a key role in epigenetic gene regulation and have been intensively investigated over the past decades
We show that designer transcription activatorlike effectors (dTALEs) can be used to visualize the dynamics of major satellite repeats in mouse embryonic stem cells (ESCs) throughout the cell cycle and to characterize their in vivo binding kinetics
The distinct subnuclear localization and high copy number of major satellite repeats constitute an ideal model system to test the applicability of dTALEs for in vivo tracing of DNA sequences
Summary
Covalent DNA and histone modifications play a key role in epigenetic gene regulation and have been intensively investigated over the past decades. To study the dynamics of chromosomal loci, Lac operator repeats were inserted and traced with Lac repressor green fluorescent protein (GFP) fusion proteins [8] With this genetic tag, rapid movements of a DNA chromosome region were observed in response to gene activation [9,10]. The base preference of the individual repeats is specified by amino acids 12 and 13, referred to as repeat variable diresidues (RVDs) [28,29] This straightforward correlation between RVD and bound nucleotide allows the fast and efficient generation of DNA binding modules for any user-defined target sequence leading to a broad application of designer TALEs (dTALEs) in genome engineering and as artificial transcription factors [23,24,25,26,27,30,31,32]. We show that dTALEs can be used to visualize the dynamics of major satellite repeats in mouse embryonic stem cells (ESCs) throughout the cell cycle and to characterize their in vivo binding kinetics
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