Summary Chromatin remodelers are ATP-driven machines that assemble, slide, and remove nucleosomes from DNA, but how the ATPase motors of remodelers are regulated is poorly understood. Here we show that the double chromodomain unit of the Chd1 remodeler blocks DNA binding and activation of the ATPase motor in the absence of nucleosome substrates. The Chd1 crystal structure reveals that an acidic helix joining the chromodomains can pack against a DNA-binding surface of the ATPase motor. Disruption of the chromodomain-ATPase interface prevents discrimination between nucleosomes and naked DNA and reduces the reliance on the histone H4 tail for nucleosome sliding. We propose that the chromodomains allow Chd1 to distinguish between nucleosomes and naked DNA by physically gating access to the ATPase motor, and we hypothesize that related ATPase motors may employ a similar strategy to discriminate among DNA-containing substrates.

Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses1,2. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signaling has been implicated in human birth defects and cancer3-7. Hh signaling is mediated by its N-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle8-10. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog/CDO and the vertebrate-specific proteins Hip and Gas111. Drosophila Ihog and its vertebrate homologs CDO and BOC contain multiple immunoglobulin (Ig) and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner12,13. Surprisingly, pull-down experiments suggest that mammalian Sonic hedgehog (ShhN) binds a nonorthologous FNIII repeat of CDO12,14. We report here biochemical, biophysical, and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hot spot for mediating interactions between ShhN and CDO, Ptc, Hip, and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.

Human SET7/9 is a protein lysine methyltransferase (PKMT) that methylates histone H3, the tumor suppressor p53 and the TBP-associated factor TAF10. To elucidate the determinants of its substrate specificity, we have solved the enzyme's structure bound to a TAF10 peptide and examined its ability to methylate histone H3, TAF10 and p53 substrates bearing either mutations or covalent modifications within their respective methylation sites. Collectively, our data reveal that SET7/9 recognizes a conserved K/R-S/T/A motif preceding the lysine substrate and has a propensity to bind aspartates and asparagines on the C-terminal side of the lysine target. We then used a sequence-based approach with this motif to identify novel substrates for this PKMT. Among the putative targets is TAF7, which is methylated at Lys5 by the enzyme in vitro. These results demonstrate the predictive value of the consensus motif in identifying novel substrates for SET7/9.