TALEs fused to chromatin modifiers for epigenetic engineering at satellite DNA repeats in mouse and human centromeric regions

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Ms Judith Lopes (INSERM)


Centromeric regions of eukaryotic chromosomes contain large numbers of tandemly repeated DNA sequences, also called satellite DNA sequences. These sequences represent the main component of constitutive heterochromatin, a compact type of chromatin. They also support the attachment of the kinetochore, thereby contributing to the stability of the genome. Although centromeric repeats are left out from most chromatin studies, several investigations have pointed to the presence of specific epigenetics marks, such as DNA methylation, the presence of specific histone variants, hypoacetylation and histone modifications. If there is no doubt about the involvement of various chromatin structures in the functional role of centromeres, the real contribution of each specific epigenetic mark and the interplay between chromatin structure and genome stability, remain poorly known. We developed an epigenetic engineering technique that will aim to induce chromatin modifications specifically at satellite repeats at centromeric loci in human and mouse cells. The production of transcriptional activator-like effectors (TALEs) fused to fluorescent proteins, targeting a 18 bp sequence highly represented in human alpha-satellites repeats or targeting major satellite repeats in mouse cells, permitted us to validate, by immunofluorescence experiments, the correct recruitment of TALEs fusion proteins in these compact heterochromatin regions. The fusion of chromatin modifiers (JMJD2B and JMJD2D histone demethylases) at the C-terminal region of the TALEs, induces a decrease of the immuno-staining of the H3K9me3 histone marks at the targeted regions in TALE-positive cells. In mouse fibroblast cells, we can observe that the decrease of the trimethyl mark of H3K9 is associated with the appearance of the di- and mono-methyl marks of H3K9. Furthermore, this lost of H3K9me3 marks is also associated with modifications of chromocenter structures, in a TALE concentration dependent manner. These results comfort us for the use of TALEs fusion proteins for epigenetic engineering at tandem repeats in human and mouse centromeric regions. This will permit us to decipher the role of this histone mark in centromere integrity and chromosome stability.

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Ms Judith Lopes (INSERM)

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