Summary HP1 proteins are central to the assembly and spread of heterochromatin containing histone H3K9 methylation. The chromodomain (CD) of HP1 proteins specifically recognizes the methyl mark on H3 peptides, but the same extent of specificity is not observed within chromatin. The chromoshadow domain of HP1 proteins promotes homodimerization, but this alone cannot explain heterochromatin spread. Using the S. pombe HP1 protein, Swi6, we show that recognition of H3K9 methylated chromatin in vitro relies on a newly identified interface between two CDs. This interaction causes Swi6 to tetramerize on a nucleosome, generating two vacant CD sticky ends. On nucleosomal arrays, methyl-mark recognition is highly sensitive to inter-nucleosomal distance, suggesting that the CD sticky ends bridge nearby methylated nucleosomes. Strengthening the CD-CD interaction enhances silencing and heterochromatin spread in vivo. Our findings suggest that recognition of methylated nucleosomes and HP1 spread on chromatin are structurally coupled, and imply that methylation and nucleosome arrangement synergistically regulate HP1 function.

Glucocorticoid receptor binds to genomic response elements and regulates gene transcription with cell- and gene-specificity. Within a response element, the precise sequence to which the receptor binds has been implicated in directing its structure and activity. We use NMR chemical shift difference mapping to show that non-specific interactions with particular base positions within the binding sequence, such as those of the “spacer”, affect the conformation of distinct regions of the rat glucocorticoid receptor DNA binding domain. These regions include the DNA-binding surface, the “lever arm” and the dimerization interface, suggesting an allosteric pathway that signals between the DNA binding sequence and the associated dimer partner. Disrupting this path by mutating the dimer interface alters sequence-specific conformations, DNA-binding kinetics and transcriptional activity. Our study demonstrates that glucocorticoid receptor dimer partners collaborate to read DNA shape and to direct sequence specific gene activity.

The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism—enabled by the functional coupling between a reader and a catalytic domain in KDM5A—suggests a model for the spread of demethylation on chromatin.

The signaling phosphatidylinositol lipids PI(4,5)P 2 (PIP 2 ) and PI (3,4,5)P 3 (PIP 3 ) bind nuclear receptor 5A family (NR5As), but their regulatory mechanisms remain unknown. Here, the crystal structures of human NR5A1 (steroidogenic factor-1, SF-1) ligand binding domain (LBD) bound to PIP 2 and PIP 3 show the lipid hydrophobic tails sequestered in the hormone pocket, as predicted. However, unlike classic nuclear receptor hormones, the phosphoinositide head groups are fully solvent-exposed and complete the LBD fold by organizing the receptor architecture at the hormone pocket entrance. The highest affinity phosphoinositide ligand PIP 3 stabilizes the coactivator binding groove and increases coactivator peptide recruitment. This receptor-ligand topology defines a previously unidentified regulatory protein-lipid surface on SF-1 with the phosphoinositide head group at its nexus and poised to interact with other proteins. This surface on SF-1 coincides with the predicted binding site of the corepressor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X), and importantly harbors missense mutations associated with human endocrine disorders. Our data provide the structural basis for this poorly understood cluster of human SF-1 mutations and demonstrates how signaling phosphoinositides function as regulatory ligands for NR5As.T he existence of nuclear, nonmembrane pools of signaling phosphorylated derivatives of phosphatidylinositols or phosphoinositides (PIP n ) was reported over two decades ago (1-3). Consistent with these early reports, lipid-modifying enzymes responsible for phosphoinositide metabolism were also found in the nucleus (4-7); however, the function of PIP n in this cellular compartment remains poorly defined. The nuclear receptors (NRs) steroidogenic factor 1 (SF-1, NR5A1) and liver receptor homolog 1 (LRH-1, NR5A2) bind phosphoinositides as well as other phospholipids in their large hydrophobic pockets (8-13). The ability of NR5As to interact with PIP n is well-conserved with the Caenorhabditis elegans ortholog nhr-25 able to bind both PIP 2 and PIP 3 (14). That phosphoinositides might serve as endogenous NR5A ligands is suggested by the fact that elevating cellular pools of PIP 3 increases SF-1 activity (15) and that impairing PIP 3 uptake decreases SF-1 activity (12). Further, when purified from mammalian cells, the phosphoinositide PIP 2 is found associated with SF-1 and can be modified by the lipid kinase, IPMK, as well as the lipid phosphatase, PTEN (13). Taken together, these data suggest that signaling phosphoinositides are biologically relevant ligands for SF-1.Phosphoinositide ligands diverge chemically from classic NR hormones in that they contain a long, extended hydrophobic moiety and a prominent hydrophilic head group, which is inherently incompatible with the hydrophobic core of the NR5A ligand-binding pocket. Our previous structural analyses of SF-1 bound to phosphatidylcholine suggest that the acyl tails of phosphoinositides should be sequestered...

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