Understanding How Distinct Heterochromatin States Control Pioneer Factor Binding - Project Summary Precise spatiotemporal regulation of lineage-specification genes is critical not only for directing proper development in the early embryo but also for safeguarding cell fate decisions in adult tissues. Histone methyltransferases (HMTs) restrict transcription factor binding by condensing DNA into heterochromatin and thus prevents the formation of developmental disorders. This process can be reversed through the action of pioneer factors, which are capable of binding and decondensing heterochromatin. However, the mechanisms governing where and when a pioneer factor can bind to a given region remains unclear. How different states of heterochromatin may be resistant to pioneer factor binding and activity. Recent work by the Bell lab has identified a unique overlap between the histone 3 lysine 9 (H3K9) dimethyltransferase G9A/GLP and pioneer factors at lineage specification genes bound by the zinc finger protein ZFP462. However, while these overlapping regions are more accessible than those bound solely by G9A/GLP, they are not fully derepressed. In contrast, H3K9 trimethyl (H3K9me3) rich DNA established by SETDB1 remained inaccessible and devoid of pioneer factor binding. Therefore, I propose that unlike TF-resistant heterochromatin established by SETDB1, G9A/GLP creates a poised heterochromatin state which is semi-permeable for pioneer factors and thus facilitates cell fate plasticity during differentiation. To test this, I will compare the heterochromatin states established by G9A/GLP and SETDB1 at unbiased loci using two distinct methods. In aim#1, a tethering reporter strategy pioneered by the Bell lab will recruit G9A/GLP or SETDB1 to the same reporter locus through tethering minimal interaction domains (IDs). Two reporter loci will be utilized for this, one knocked into the endogenous Oct3/4 locus to investigate the established heterochromatin in the context of the OCT4 positive feedback loop and the other in a gene desert on chromosome 7 allowing for modular addition of pioneer factor binding motifs. Repression of the locus will be assessed through expression of the reporter as determined by flow cytometry and ChIP-qPCR probing of the targeted locus for the downstream histone marks and repressive machinery recruited by G9A/GLP and SETDB1. In aim#2, the ability of pioneer factors recruited by endogenous cis- regulatory elements to open heterochromatin during differentiation will be tested by a ZFP462 fusion protein with the C-terminal G9A/GLP-ID swapped with a SETDB1-ID. Through ChIP- and ATAC-sequencing, the establishment of heterochromatin and accessibility of ZFP462 target loci during directed neural stem cell differentiation will be assessed. Additionally, 10X single cell RNA-sequencing during undirected EB differentiation will determine any differences in the capacity of the ZFP462-ID fusion proteins to direct differentiation into the three germ layers. In aim#3, a 6mA footprinting analysis in conjunction with long-read genome sequencing will be performed in the chromosome 7 tethered reporter lines to identify differences in nucleosome arrangement in the different heterochromatin states, both alone and in the presence of pioneer factor binding motifs.
