Anjanabha Saha

ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression

Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules. Here we identify a novel class of methylated H3K4 effector domains—the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a–HDAC1 histone deacetylase complex, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a–HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.

Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation

Conjugation of ubiquitin-like protein Nedd8 to cullins (neddylation) is essential for the function of cullin-RING ubiquitin ligases (CRLs). Here, we show that neddylation stimulates CRL activity by multiple mechanisms. For the initiator ubiquitin, the major effect is to bridge the approximately 50 A gap between naked substrate and E2 approximately Ub bound to SCF. The gap between the acceptor lysine of ubiquitinated substrate and E2 approximately Ub is much smaller, and, consequentially, the impact of neddylation on transfer of subsequent ubiquitins by Cdc34 arises primarily from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires >or= 4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation and suggest that modification of other ubiquitin ligases with ubiquitin or ubiquitin-like proteins may likewise have major functional consequences.

Chromatin remodeling through directional DNA translocation from an internal nucleosomal site

The RSC chromatin remodeler contains Sth1, an ATP-dependent DNA translocase. On DNA substrates, RSC/Sth1 tracks along one strand of the duplex with a 3′ → 5′ polarity and a tracking requirement of one base, properties that may enable directional DNA translocation on nucleosomes. The binding of RSC or Sth1 elicits a DNase I–hypersensitive site approximately two DNA turns from the nucleosomal dyad, and the binding of Sth1 requires intact DNA at this location. Results with various nucleosome substrates suggest that RSC/Sth1 remains at a fixed position on the histone octamer and that Sth1 conducts directional DNA translocation from a location about two turns from the nucleosomal dyad, drawing in DNA from one side of the nucleosome and pumping it toward the other. These studies suggest that nucleosome mobilization involves directional DNA translocation initiating from a fixed internal site on the nucleosome.

The nuclear actin‐related proteins Arp7 and Arp9: a dimeric module that cooperates with architectural proteins for chromatin remodeling

Nuclear actin‐related proteins (ARPs) are essential components of chromatin remodeling and modifying complexes, but their functions and relationship to actin remain elusive. The yeast SWI/SNF and RSC complexes contain Arp7 and Arp9, and are shown to form a stable heterodimer with the properties of a functional module. Arp7 and Arp9 rely on their actin‐related regions for heterodimerization, and their unique C‐termini cooperate for assembly into RSC. We suggest that regulated ARP–ARP (and possibly ARP–β–actin) heterodimerization might be a conserved feature of chromatin complexes. A RSC complex lacking Arp7/9 was isolated that displays robust nucleosome remodeling activity, suggesting a separate essential role for ARPs in the regulation of chromatin structure. A screen for suppressors of arp mutations yielded the DNA bending architectural transcription factor Nhp6, which interacts with RSC complex physically and functionally and shows facilitated binding to nucleosomes by RSC. We propose that Arp7/9 dimers function with DNA bending proteins to facilitate proper chromatin architecture and complex–complex interactions.

Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction method

Chromatin remodeling complexes (remodelers) are large, multisubunit macromolecular assemblies that use ATP hydrolysis to alter the structure and positioning of nucleosomes. The mechanisms proposed for remodeler action on nucleosomes are diverse, and require structural evaluation and insights. Previous reconstructions of remodelers using electron microscopy revealed interesting features, but also significant discrepancies, prompting new approaches. Here, we use the orthogonal tilt reconstruction method, which is well suited for heterogeneous samples, to provide a reconstruction of the yeast RSC (remodel the structure of chromatin) complex. Two interesting features are revealed: first, we observe a deep central cavity within RSC, displaying a remarkable surface complementarity for the nucleosome. Second, we are able to visualize two distinct RSC conformers, revealing a major conformational change in a large protein “arm,” which may shift to further envelop the nucleosome. We present a model of the RSC-nucleosome complex that rationalizes the single molecule results obtained by using optical tweezers and also discuss the mechanistic implications of our structures.

Mechanisms for nucleosome movement by ATP-dependent chromatin remodeling complexes.

Chromatin remodeling complexes (remodelers) are a set of diverse multi-protein machines that reposition and restructure nucleosomes. Remodelers are specialized, containing unique proteins that assist in targeting, interaction with modified nucleosomes, and performing specific chromatin tasks. However, all remodelers contain an ATPase domain that is highly similar to known DNA translocases/helicases, suggesting that DNA translocation is a property common to all remodelers. Here we examine the different reactions they perform in vitro, focusing on the SWI/SNF and the ISWI complexes, and explore how DNA translocation might be utilized to execute various remodeling processes.