Local and Large-Scale Conformational Dynamics in Unfolded Proteins and IDPs. I. Effect of Solvent Viscosity and Macromolecular Crowding.

Protein/solvent interactions largely influence protein dynamics, particularly motions in unfolded and intrinsically disordered proteins (IDPs). Here, we apply triplet-triplet energy transfer (TTET) to investigate the coupling of internal protein motions to solvent motions by determining the effect of solvent viscosity (η) and macromolecular crowding on the rate constants of loop formation (kc) in several unfolded polypeptide chains including IDPs. The results show that the viscosity dependence of loop formation depends on amino acid sequence, loop length, and co-solute size. Below a critical size (rc), co-solutes exert a maximum effect, indicating that under these conditions microviscosity experienced by chain motions matches macroviscosity of the solvent. rc depends on chain stiffness and reflects the length scale of the chain motions, i.e., it is related to the persistence length. Above rc, the effect of solvent viscosity decreases with increasing co-solute size. For co-solutes typically used to mimic cellular environments, a scaling of kc ∝ η-0.1 is observed, suggesting that dynamics in unfolded proteins are only marginally modulated in cells. The effect of solvent viscosity on kc in the small co-solute limit (below rc) increases with increasing chain length and chain flexibility. Formation of long and very flexible loops exhibits a kc ∝ η-1 viscosity dependence, indicating full solvent coupling. Shorter and less flexible loops show weaker solvent coupling with values as low as kc ∝ η-0.75 ± 0.02. Coupling of formation of short loops to solvent motions is very little affected by amino acid sequence, but solvent coupling of long-range loop formation is decreased by side chain sterics.

Local and Large-Scale Conformational Dynamics in Unfolded Proteins and IDPs. II. Effect of Temperature and Internal Friction.

Internal dynamics of proteins are essential for protein folding and function. Dynamics in unfolded proteins are of particular interest since they are the basis for many cellular processes like folding, misfolding, aggregation, and amyloid formation and also determine the properties of intrinsically disordered proteins (IDPs). It is still an open question of what governs motions in unfolded proteins and whether they encounter major energy barriers. Here we use triplet-triplet energy transfer (TTET) in unfolded homopolypeptide chains and IDPs to characterize the barriers for local and long-range loop formation. The results show that the formation of short loops encounters major energy barriers with activation energies (Ea) up to 18 kJ/mol (corrected for effects of temperature on water viscosity) with very little dependence on amino acid sequence. For poly(Gly-Ser) and polySer chains the barrier decreases with increasing loop size and reaches a limiting value of 4.6 ± 0.4 kJ/mol for long and flexible chains. This observation is in accordance with the concept of internal friction encountered by chain motions due to steric effects, which is high for local motions and decreases with increasing loop size. Comparison with the results from the viscosity dependence of loop formation shows a negative correlation between Ea and the sensitivity of the reaction to solvent viscosity (α) in accordance with the Grote-Hynes theory of memory friction. The Arrhenius pre-exponential factor (A) also decreases with increasing loop size, indicating increased entropic costs for loop formation. Long-range loop formation in the investigated sequences derived from IDPs shows increased Ea and A compared with poly(Gly-Ser) and polySer chains. This increase is exclusively due to steric effects that cause additional internal friction, whereas intramolecular hydrogen bonds, dispersion forces, and charge interactions do not affect the activation parameters.

ZFP462 safeguards neural lineage specification by targeting G9A/GLP-mediated heterochromatin to silence enhancers.

ZNF462 haploinsufficiency is linked to Weiss-Kruszka syndrome, a genetic disorder characterized by neurodevelopmental defects, including autism. Though conserved in vertebrates and essential for embryonic development, the molecular functions of ZNF462 remain unclear. We identified its murine homologue ZFP462 in a screen for mediators of epigenetic gene silencing. Here we show that ZFP462 safeguards neural lineage specification of mouse embryonic stem cells (ESCs) by targeting the H3K9-specific histone methyltransferase complex G9A/GLP to silence meso-endodermal genes. ZFP462 binds to transposable elements that are potential enhancers harbouring pluripotency and meso-endoderm transcription factor binding sites. Recruiting G9A/GLP, ZFP462 seeds heterochromatin, restricting transcription factor binding. Loss of ZFP462 in ESCs results in increased chromatin accessibility at target sites and ectopic expression of meso-endodermal genes. Taken together, ZFP462 confers lineage and locus specificity to the broadly expressed epigenetic regulator G9A/GLP. Our results suggest that aberrant activation of lineage non-specific genes in the neuronal lineage underlies ZNF462-associated neurodevelopmental pathology.

Pathway-Based High-Throughput Chemical Screen Identifies Compounds That Decouple Heterochromatin Transformations.

Heterochromatin protein 1 (HP1) facilitates the formation of repressive heterochromatin domains by recruiting histone lysine methyltransferase enzymes to chromatin, resulting in increased levels of histone H3K9me3. To identify chemical inhibitors of the HP1-heterochromatin gene repression pathway, we combined a cell-based assay that utilized chemical-mediated recruitment of HP1 to an endogenous active gene with high-throughput flow cytometry. Here we characterized small molecule inhibitors that block HP1-mediated heterochromatin formation. Our lead compounds demonstrated dose-dependent inhibition of HP1-stimulated gene repression and were validated in an orthogonal cell-based system. One lead inhibitor was improved by a change in stereochemistry, resulting in compound 2, which was further used to decouple the inverse relationship between H3K9 and H3K4 methylation states. We identified molecular components that bound compound 2, either directly or indirectly, by chemical affinity purification with a biotin-tagged derivative, followed by quantitative proteomic techniques. In summary, our pathway-based chemical screening approach resulted in the discovery of new inhibitors of HP1-mediated heterochromatin formation while identifying exciting new molecular interactions in the pathway to explore in the future. This modular platform can be expanded to test a wide range of chromatin modification pathways yielding inhibitors that are cell permeable and function in a physiologically relevant setting.

Canonical PRC1 controls sequence-independent propagation of Polycomb-mediated gene silencing.

Polycomb group (PcG) proteins play critical roles in the epigenetic inheritance of cell fate. The Polycomb Repressive Complexes PRC1 and PRC2 catalyse distinct chromatin modifications to enforce gene silencing, but how transcriptional repression is propagated through mitotic cell divisions remains a key unresolved question. Using reversible tethering of PcG proteins to ectopic sites in mouse embryonic stem cells, here we show that PRC1 can trigger transcriptional repression and Polycomb-dependent chromatin modifications. We find that canonical PRC1 (cPRC1), but not variant PRC1, maintains gene silencing through cell division upon reversal of tethering. Propagation of gene repression is sustained by cis-acting histone modifications, PRC2-mediated H3K27me3 and cPRC1-mediated H2AK119ub1, promoting a sequence-independent feedback mechanism for PcG protein recruitment. Thus, the distinct PRC1 complexes present in vertebrates can differentially regulate epigenetic maintenance of gene silencing, potentially enabling dynamic heritable responses to complex stimuli. Our findings reveal how PcG repression is potentially inherited in vertebrates.