Chromatin is a complex network of structural and functional moieties. Different components of chromatin such as, chromatin binding proteins, post-translational modifications (PTMs), small molecules etc. define its structure and function in a spatiotemporal manner. In our recent work, we have dissected the molecular details of such a multilayered interaction. We studied how a lipid small molecule, phosphatidyl inositol 5-phosphate (PI5P), allosterically regulates the three-dimensional organization of a multidomain chromatin protein, named hUHRF1 (human Ubiquitin like with PHD and RING finger domains 1) thereby modulating its interaction with specific chromatin marks. To study this complex interplay, we have employed various biochemical and biophysical approaches together with NMR and Hydrogen-Deuterium Exchange mass spectrometry (HDXMS.

hUHRF1 has five structurally defined domains. Three of them (TTD, PHD and SRA) interact with histone H3 trimethylated on the lysine 9 residue (H3K9me3), unmodified histone H3 and hemi-methylated DNA, respectively. The RING domain has E3 ubiquitin ligase activity and the UBL domain stimulates catalytic activity of the RING domain. The different hUHRF1 domains are connected via flexible and positively charged linkers. We had previously shown that the TTD domain of hUHRF1 remains in an autoinhibited state as the Linker 4 (between SRA and RING domains) interacts on the acidic surface of TTD and blocks its aromatic cage (H3K9me3 binding pocket). PI5P interaction with the Linker 4 unblocks TTD for histone H3K9me3 binding.

We now have dissected the molecular details of hUHRF1’s conformational rearrangements upon PI5P binding. 16:0 PI5P was found to be most potent in enhancing hUHRF1’s interaction with the H3K9me3 mark. We showed that hUHRF1 has two distinct binding modes for H3K9me3. In the apo state (in absence of PI5P) hUHRF1 interacts with the unmodified N-terminus of histone H3 via its PHD domain, while a TTD-PHD dual domain mediated synergistic and cooperative binding mode was engaged in the PI5P bound state. We dissected that hUHRF1 has a composite binding interface for 16:0 PI5P involving Linker 4, Linker 2 and part of the TTD domain. The allosteric ligand thereby connects the TTD and PHD domains of hUHRF1 in a mode that establishes synergistic and cooperative interaction with the H3K9me3 mark.

hUHRF1 has been found to be upregulated in various cancers and therefore, it is a promising drug target. All approaches so far screened for small molecules targeting the different domains of hUHRF1 directly. Our work showed that targeting the allosteric regulation of the protein via its linkers could be a promising avenue for inhibiting specific functions of hUHRF1 and thereby for affecting particular disease states.