Automethylation-induced conformational switch in Clr4 (Suv39h) maintains epigenetic stability.

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression1-4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H35-11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help to limit the spread of inappropriate H3K9me12-15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me-which would lead to aberrant gene silencing and epigenetic instability-is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (also known as Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains and inhibition of growth, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue16 suggest that the mechanism described here is broadly conserved.

A microRNA negative feedback loop downregulates vesicle transport and inhibits fear memory.

The SNARE-mediated vesicular transport pathway plays major roles in synaptic remodeling associated with formation of long-term memories, but the mechanisms that regulate this pathway during memory acquisition are not fully understood. Here we identify miRNAs that are up-regulated in the rodent hippocampus upon contextual fear-conditioning and identify the vesicular transport and synaptogenesis pathways as the major targets of the fear-induced miRNAs. We demonstrate that miR-153, a member of this group, inhibits the expression of key components of the vesicular transport machinery, and down-regulates Glutamate receptor A1 trafficking and neurotransmitter release. MiR-153 expression is specifically induced during LTP induction in hippocampal slices and its knockdown in the hippocampus of adult mice results in enhanced fear memory. Our results suggest that miR-153, and possibly other fear-induced miRNAs, act as components of a negative feedback loop that blocks neuronal hyperactivity at least partly through the inhibition of the vesicular transport pathway.

Mapping intact protein isoforms in discovery mode using top-down proteomics.

A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.

Efficient readout of posttranslational codes on the 50-residue tail of histone H3 by high-resolution MS/MS.

Histone modifications are highly linked to DNA methylation and together they exert epigenetic control over many activities in the cell including gene transcription. Using a streamlined mass spectrometric approach to determine changes in modification states in the first 50 residues of histone H3, we found a decrease in the global methylation states of H3.1 at Lys 9, Lys 14, and Lys 27 after inhibition of DNA methyltransferases by 5-aza-2'-deoxycytidine. Collisional ion dissociation methods proved adequate to determine site-specific H3 posttranslational modifications (PTMs) because ample backbone bonds are cleaved between each modification site and PTMs were stable to MS/MS using threshold fragmentation in a linear ion trap (LTQ). Our assay allows for a quick profiling and site-specific interrogation of modification states on the first 50 residues of H3 and is directly applicable to H3.1, H3.2, or H3.3 using most OrbiTrap, FT ICR, or TOF mass spectrometers.

Decoding protein modifications using top-down mass spectrometry.

Top-down mass spectrometry is an emerging technology which strives to preserve the post-translationally modified forms of proteins present in vivo by measuring them intact, rather than measuring peptides produced from them by proteolysis. The top-down technology is beginning to capture the interest of biologists and mass spectrometrists alike, with a main goal of deciphering interaction networks operative in cellular pathways. Here we outline recent approaches and applications of top-down mass spectrometry as well as an outlook for its future.

Gene-specific characterization of human histone H2B by electron capture dissociation.

The basis set of protein forms expressed by human cells from the H2B gene family was determined by Top Down Mass Spectrometry. Using Electron Capture Dissociation for MS/MS of H2B isoforms, direct evidence for the expression of unmodified H2B.Q, H2B.A, H2B.K/T, H2B.J, H2B.E, H2B.B, H2B.F, and monoacetylated H2B.A was obtained from asynchronous HeLa cells. H2B.A was the most abundant form, with the overall expression profile not changing significantly in cells arrested in mitosis by colchicine or during mid-S, mid-G2, G2/M, and mid-G1 phases of the cell cycle. Modest hyperacetylation of H2B family members was observed after sodium butyrate treatment.