ESR7: “DNA damage-driven chromatin changes and immunometabolism”

FORTH (C. Spilianakis group): DNA damage-driven chromatin changes and immunometabolism

i. Objective of research: To identify the SATB1-mediated 3D chromatin architecture changes in CD4 T cells exposed to DNA damage.

ii. Current state of the art: There is increasing evidence that chromatin structure can affect the kinetics and efficiency of DNA repair mechanisms as indicated by subtle differences in events localized in eu/hetero-chromatin. Higher-order chromatin structure is mediated by multiple architectural proteins such as SATB1 (Special AT-rich binding protein 1), which is a pleiotropic molecule with functions that evolve early in development, as it is expressed in ES cells regulating the expression of Nanog and it acts as a silencing factor for the Xist RNA. It is also expressed in CD4 cells regulating the coordinated expression of cytokine genes by forming a higher ordered chromatin structure. Moreover, SATB1 globally reprograms gene expression during metastasis by tethering hundreds of gene loci onto its regulatory network. Elucidating the SATB1 roles in “loopscape” formation by means of genome-wide approaches will unravel its role as chromatin organizer and coordinator of global gene transcription that shapes chromatin under normal and upon DNA damage conditions.

iii. Research methodology and approach: Our studies will be performed in primary CD4 cells, where SATB1 is highly expressed, isolated from either wild type or genetically modified [tissue specific CD4-Cre conditional knockout of Satb1 (the complete Satb1 knockout is postembryonic lethal)] C57BL/6 mice. CD4 cells will be ex vivo cultured either under normal or upon low-dose ionizing radiation conditions. Therefore, there are four experimental conditions that will be employed in all our experiments: wild type and conditionally deleted Satb1 CD4 cells under normal and ionizing radiation conditions. We are going to perform RNA- and aSATB1-ChIP-seq experiments using next generation sequencing with the ultimate goal to define the SATB1-mediated transcriptome in the aforementioned physiological conditions. To identify the co-regulated gene networks before and after DNA damage we will perform ChIA-PET experiments utilizing a highly specific custom-made SATB1 antibody. The cells with genetic ablation of the Satb1 gene will be utilized as the necessary negative control for our studies. To identify the protein complexes that the SATB1 protein participates under normal and upon DNA damage conditions we will employ immunoprecipitation of the protein coupled to mass spectrometry analysis.

iv. Originality and innovative aspects of the ESR project: We will employ a novel approach to obtain a high-resolution picture of the CD4 T cell chromatin organisation and identify the differences on the SATB1-mediated chromatin networks upon induction of DNA damage.

v. Integration of the ESR project to the overall research programme: our ESR will work with the Genevia group in analyzing ChIP-seq data for the SATB1 protein in CD4 cells, with the Norgenotech in the development of assays for the detection of Inflammation-driven DNA lesions and with the Garinis group for the detection immunosecretory factors in the sera of Satb1-/- and NER-defective mice.