Investigation of the variability in the assessment of digital chest X-ray image quality.

A large database of digital chest radiographs was developed over a 14-month period. Ten radiographic technologists and five radiologists independently evaluated a stratified subset of images from the database for quality deficiencies and decided whether each image should be rejected. The evaluation results showed that the radiographic technologists and radiologists agreed only moderately in their assessments. When compared against each other, radiologist and technologist reader groups were found to have even less agreement than the inter-reader agreement within each group. Radiologists were found to be more accepting of limited-quality studies than technologists. Evidence from the study suggests that the technologists weighted their reject decisions more heavily on objective technical attributes, while the radiologists weighted their decisions more heavily on diagnostic interpretability relative to the image indication. A suite of reject-detection algorithms was independently run on the images in the database. The algorithms detected 4 % of postero-anterior chest exams that were accepted by the technologist who originally captured the image but which would have been rejected by the technologist peer group. When algorithm results were made available to the technologists during the study, there was no improvement in inter-reader agreement in deciding whether to reject an image. The algorithm results do, however, provide new quality information that could be captured within a site-wide, reject-tracking database and leveraged as part of a site-wide QA program.

6 Structure of SET domain protein lysine methyltransferases.

Methylation of lysine residues is now understood to constitute a key component of the complex signaling paradigm referred to as the histone code hypothesis. Rapid progress has been made in the structural and functional biology of the enzymes responsible for this modification. TheseSET proteins are based on a common fold that appears unique to this family. The structure of the SET domain is such that peptide substrates bind on one surface, whereas the AdoMet cofactor binds on the opposite side of the domain. Remarkably, the target lysine residue gains access to the cofactor by passing through a channel that runs through the SET domain connecting these two binding surfaces. Different SET enzymes carry out mono-, di-, or tri-methylation of their targets, and these modifications give rise to distinctive biological readouts. Ternary complexes of several SET enzymes reveal how the size and bonding patterns of residues flanking the active site determine the multiplicity of methylation that occurs. Indeed, the methylation multiplicity of some of these enzymes has been engineered by specifically mutating residues close to the active site. The catalytic activity of SET enzymes depends on adjacent domains at theirN- and C-termini. The N-flanking domains seem important for structural stability but the C-flanking domains are necessary for the completion of the active sites of these enzymes. Recent NMR studies have shown that these C-flanking domains are flexible and that their ordering, driven by substrate binding, is an important part of the catalytic cycle of these enzymes.

Specificity and mechanism of the histone methyltransferase Pr-Set7.

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.

Regulation of p53 activity through lysine methylation.

p53 is a tumour suppressor that regulates the cellular response to genotoxic stresses. p53 is a short-lived protein and its activity is regulated mostly by stabilization via different post-translational modifications. Here we report a novel mechanism of p53 regulation through lysine methylation by Set9 methyltransferase. Set9 specifically methylates p53 at one residue within the carboxyl-terminus regulatory region. Methylated p53 is restricted to the nucleus and the modification positively affects its stability. Set9 regulates the expression of p53 target genes in a manner dependent on the p53-methylation site. The crystal structure of a ternary complex of Set9 with a p53 peptide and the cofactor product S-adenosyl-l-homocysteine (AdoHcy) provides the molecular basis for recognition of p53 by this lysine methyltransferase.

SET domains and histone methylation.

The realisation that SET domains, which are found in numerous proteins involved in chromatin regulation, catalyse the methylation of lysine residues has led to intense interest in their cellular, biochemical and structural properties. The structures of five SET domain proteins have been reported over the past year. SET domains possess a novel fold, and use adjacent domains for both structural stabilisation and the completion of their active sites. The cofactor S-adenosyl-L-methionine and peptide substrates bind on opposite faces of the SET domain. Remarkably, the sidechain of the target lysine approaches the transferred methyl group through a narrow channel that passes through the middle of the domain.

Structure and catalytic mechanism of the human histone methyltransferase SET7/9.

Acetylation, phosphorylation and methylation of the amino-terminal tails of histones are thought to be involved in the regulation of chromatin structure and function. With just one exception, the enzymes identified in the methylation of specific lysine residues on histones (histone methyltransferases) belong to the SET family. The high-resolution crystal structure of a ternary complex of human SET7/9 with a histone peptide and cofactor reveals that the peptide substrate and cofactor bind on opposite surfaces of the enzyme. The target lysine accesses the active site of the enzyme and the S-adenosyl-l-methionine (AdoMet) cofactor by inserting its side chain into a narrow channel that runs through the enzyme, connecting the two surfaces. Here we show from the structure and from solution studies that SET7/9, unlike most other SET proteins, is exclusively a mono-methylase. The structure indicates the molecular basis of the specificity of the enzyme for the histone target, and allows us to propose a model for the methylation reaction that accounts for the role of many of the residues that are invariant across the SET family.

Crystal structure and functional analysis of the histone methyltransferase SET7/9.

Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.