Trayectorias whitenses.

3.2.1 Identify Proteins that may be Involved in the Recruitment of DNMT3B and TET1 to Bivalent Promoters

It is unclear why certain genomic loci are more prone to dynamic changes in DNA methylation and what are the molecular mechanisms that underlie competition between the TET and DNMT proteins to determine methylation levels at these loci. Focusing specifically on bivalent promoters it would be interesting to determine whether recruitment of the TET and DNMT proteins by a third protein may explain the competition between TET and DNMT (Appendix 2A). The first candidate we chose to study is the

polycomb repressive complex, PRC2. PRC2 itself is present at bivalent loci as it is responsible for deposition of the H3K27me3 mark. Furthermore previous studies have found interaction between PRC2 and TET1 and PRC2 and DNMT3A/DNMT3B in mESCs (Neri et al., 2013a; Neri et al., 2013b). We generated a 3x flag tagged TET1 hESC line to first perform co- immunoprecipitation experiments (Appendix 2B). We detected interaction between PRC2 and DNMT3B but limited interaction between PRC2 and TET1 (Appendix 2C). It is possible that TET1 is recruited by another protein instead of PRC2. It would thus be useful to perform mass spectrometry analysis of the 3xFlag-TET1 line to identify potential interacting partners that may function to recruit TET1 to specific genomic regions.

We decided to investigate further the interaction between PRC2 and DNMT3B. We had already observed that inactivating DNMT3B in the TKO background reduced methylation at bivalent loci, among other regions. We wanted to determine whether inactivation of PRC2 in the TKO background might provide more precise loss of DNA hypermethylation at bivalent promoters as it may abrogate DNMT3B recruitment specifically to these regions. We decided to target two different components of PRC2, the catalytic component EZH2 and the DNA binding component SUZ12. We generated CRISPRs that could produce indel mutations at the start codons of both EZH2 and SUZ12; however, upon targeting we obtained few clones that had frameshift mutations in both alleles (Appendix 3A). It is possible that inactivating EZH2 has a lethal phenotype as suggested by previous studies (Collinson et al., 2016). We continued to culture the WT and TKO lines that showed frameshift mutations in both alleles of SUZ12.

the lines lie in a SUZ12 pseudogene that shares the first 12 exons of the functioning SUZ12 gene (Appendix 3B). We are currently generating

additional targeting CRISPR gRNAs, to reattempt the inactivation of SUZ12 in the TKO background. We aim to identify interacting partners of TET1 and DNMT3B and determine whether they function to recruit these proteins to bivalent promoters. This may explain the specificity of hypermethylation that we observe in TKO hESCs.

3.2.2 Investigate Changes in Chromatin Environment Upon TET Inactivation

We focused on changes in DNA methylation upon inactivation of the TET genes. It is likely however that there are additional changes in the chromatin as there is significant crosstalk between DNA methylation and other epigenetic modifications. We aim to investigate this further using genome-wide ChIP-Seq analysis in WT and TKO hESCs to determine whether the distribution of certain histone marks (H3K4me1, H3K4me3, H3K27me3, H3K27ac) and chromatin modifiers (EZH1, SUZ12, DNMT1, DNMT3A) are altered upon TET inactivation. We will also perform ATAC- Seq to determine if there are changes in the chromatin accessibility for biologically relevant loci.

3.2.3 Investigate the Mechanisms Underlying Heterogeneity in the Gain of Methylation after TET Inactivation

Among bivalent promoters we observed large variability in the gain of methylation. Approximately half of the bivalent promoters in two hESC backgrounds showed greater than 5% increase in methylation between TKO

and WT lines, and among these approximately 10% showed a greater than 60% increase in methylation. It would be interesting to explore why these particular promoters were vulnerable to such large increases in methylation. It is possible that transcription factor binding, local chromatin environment or chromatin 3D structure or configuration may influence whether a particular locus is protected from or vulnerable to aberrant hypermethylation. The TKO hESCs offer a useful platform as we could manipulate these variables in TKO hESCs to explore whether we can cause hypermethylation at previously resistant loci.

3.2.4 Investigate Hypermethylation Patterns and Consequences in Other Progenitor Populations Upon TET Inactivation

It is unclear whether the observations we made at the hESC stage regarding the hypermethylation of bivalent promoters is also observed in other progenitor cell types. Furthermore it is unknown whether

hypermethylation at bivalent promoters in other progenitors would also produce differentiation defects due to impaired activation of the

differentiation genes associated with these promoters. hESCs are an ideal platform to study this question because they have the potential to

differentiate into a variety of tissues. We are currently working on an inducible system that would enable us to immediately inactivate the TET genes at different stages of differentiation and thus observe the effect of the loss of the TET proteins in different cellular contexts. In our first attempt to generate such a platform we infected our iCRISPR hESCs (in which Cas9 is doxycycline inducible) with a lentiviral construct containing the three TET gRNAs in tandem, each under the control of its own individual U6 promoter

(Appendix 4A). After establishing one clone we found that doxycycline treatment led to progressive depletion of 5hmC, consistent with the loss of the TET proteins (Appendix 4B). Upon T7 endonuclease analysis we found that indel mutations were generated in the TET1 and TET3 loci, but not at the TET2 locus (Appendix 4C). We are currently investigating why there is no functional TET2 gRNA and exploring alternative strategies to develop a true inducible TKO line.

We believe the question of whether our observations are reproducible in other progenitor cells is particularly important due to the prevalence of mutations in the DNA methylation and demethylation pathways in a variety of cancers. Ideally we would want to investigate cell populations that are particularly prone to transformation upon disruption of TET or TET- interacting pathways, such as hematopoietic stem cells and neural stem cells. A better understanding of the molecular mechanisms that drive cancer progression could potentially enable the design of more efficacious

therapeutics.