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Readout of DNA methylation

Periodic Reporting for period 4 - ReaDMe (Readout of DNA methylation)

Reporting period: 2020-07-01 to 2020-12-31

The cells in our body vary widely in function even though they share the same DNA template. Their diversity arises from the different use of their genes, which is controlled by the process of transcription. Proteins that recognize DNA motifs, called transcription factors (TF)bind to regulatory regions that ultimately lead to gene activity. Our DNA is packaged in chromatin, which blocks access for TFs to DNA. In addition DNA is modified methylation of the bas cytosine, which can change binding of TFs. DNA methylation and the local chromatin structure represent an epigenetic layer that is thought to define cell type specific gene regulation programs. Moreover many of the regulators of DNA methylation and chromatin are functionally linked to diseases.
Yet how these epigenetic modifications of DNA are interpreted by TFs remains poorly understood.
The ERC project ReaDMe has the ambitious goal to systematically define the sensitivity of TFs to local levels of DNA methylation in vivo.
To reach this goal ReadMe applies novel genomics, genome editing and proteomics tools to comprehensively identify chromatin bound factors that respond to DNA methylation.
As a first approach, we interrogate changes in the global regulatory landscape upon removal of DNA methylation using genetic deletion of the responsible enzymes. Here we expect that transcription factors that are sensitive to DNA methylation will change their genomic binding, which we test by testing TF binding throughout the genome. These experiments are done in undifferentiated, so called stem cells but also in neurons that we derive from these cells. This enables us to directly compare pluripotent and committed cells Secondly, we combine highly parallel chromosomal insertions with targeted footprinting to determine the link between DNA sequence context, methylation density and TF binding. In a third approach we define the global chromatin proteome as a function of DNA methylation. Through the use of a novel and orthogonal proteomics assay, we will characterize DNA methylation sensitive changes in the chromatin-bound proteome. Candidate factors predicted from all approaches will be validated and functionally described through genome-wide binding as well as loss of function analysis.
Together these experiments will increase our understanding how epigenetic changes in DNA crosstalk to the the binding of TFs and in turn should aid in understanding how epigenetic misregulation is linked to disease.
Following our success in describing the changes in the global binding of transcription factors in stem cells (Domcke, Bardet et al., Nature 2015) we collaborated with the Taipale lab to define in vitro the sensitivity of several hundred human transcription factors to DNA methylation and in vivo for selected cases in presence or absence of DNA methylation and hydroxymethylation. This identified a surprisingly large number of factors that are slightly attracted to methylated cytosines within their target motif (Yin et al., Science 2017).
We successfully established novel protocols to monitor TF binding using a combination of footprinting enzymes. As a proof of concept we have used these approaches to quantitatively describe the turnover of RNA polymerase at paused genes (Krebs et al., Molecular Cell 2017).
These approaches have also contributed to a study where we defined the contribution of CGs to the output of promoters (Hartl et al., Genome Research, 2019).
One key technology development within ReadMe was the successful establishment of a protocol that enables comprehensive monitoring of DNA bound proteins. This has been achieved recently and a proof of concept paper showing the ulitity of the approach will be published in the coming weeks (Ginno et al., Nature Communications, 2018). More recently we have published the first comprehensive genomic map of DNA methylation in our genome using a sophisticated approach of genetic deletion and time-course monitoring of DNA methylation changes (Ginno et al., Nature Communication, 2020). In addition we have generated neurons that lack DNA methylation enabling us to identify several methylation sensitive transcription factors that operate in neurons and have identified a factor that activates endogenous retrovirus in the absence of DNA methylation (Kaluscha et al., in prepation. We furthermore have identified a totally novel transcription factor important for CpG island regulation yet highly sensitive to DNA methylation (Grand et al., under review).
Thus ReadMe has reached its experimental milestones and achieved several novel insights. In addition to above and upcoming publications we have communicated these results through several presentations at international scientific meetings (including virtual meetings during the pandemic) and seminar visits in institutes. Selected achievements are further promoted on the internet and on social media using the twitter account of the FIM as well as that of the Schübeler lab.
Our achievements thus far led to several high impact publications as detailed above. Within ReadMe we have been able to identify several novel factors that are sensitive to DNA methylation in stem and differentiated cells including factors that show enhanced binding to DNA methylation. We identified endogenous transcription factors that activate retroelements in absence of DNA methylation findings help to explain how epigenetic modifications are involved in the silencing and activation of selfish DNA.
We have identified chromatin factors that depend on absence of DNA methylation and hydroxymethylation for binding. We further have shown that sites of active DNA Methlylation turnover are those occupied by transcription factors and have identified several novel factors sensitive to this epigenetic mark. This was made possible by combining novel genomics and proteomics tools combined with targeted genomic modification.
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