KRAS-specific inhibition using a DARPin binding to a site in the allosteric lobe.

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function

Phosphoproteomics Screen Reveals Akt Isoform-Specific Signals Linking RNA Processing to Lung Cancer

The three Akt isoforms are functionally distinct. Here we show that their phosphoproteomes also differ, suggesting that their functional differences are due to differences in target specificity. One of the top cellular functions differentially regulated by Akt isoforms is RNA processing. IWS1, an RNA processing regulator, is phosphorylated by Akt3 and Akt1 at Ser720/Thr721. The latter is required for the recruitment of SETD2 to the RNA Pol II complex. SETD2 trimethylates histone H3 at K36 during transcription, creating a docking site for MRG15 and PTB. H3K36me3-bound MRG15 and PTB regulate FGFR-2 splicing, which controls tumor growth and invasiveness downstream of IWS1 phosphorylation. Twenty-one of the twenty-four non-small-cell-lung carcinomas we analyzed express IWS1. More importantly, the stoichiometry of IWS1 phosphorylation in these tumors correlates with the FGFR-2 splicing pattern and with Akt phosphorylation and Akt3 expression. These data identify an Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer

A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress.

Repetitive sequences derived from transposons make up a large fraction of eukaryotic genomes and must be silenced to protect genome integrity. Repetitive elements are often found in heterochromatin; however, the roles and interactions of heterochromatin proteins in repeat regulation are poorly understood. Here we show that a diverse set of C. elegans heterochromatin proteins act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. Mutants in genes encoding HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 also show functionally redundant sterility, increased germline apoptosis, DNA repair defects, and interactions with small RNA pathways. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting cep-1/p53, endogenous meiotic double strand breaks, or the expression of MIRAGE1 DNA transposons. Functional redundancy among factors and pathways underlies the importance of safeguarding the genome through multiple means

EpiChIP: gene-by-gene quantification of epigenetic modification levels

The combination of chromatin immunoprecipitation with next-generation sequencing technology (ChIP-seq) is a powerful and increasingly popular method for mapping protein–DNA interactions in a genome-wide fashion. The conventional way of analyzing this data is to identify sequencing peaks along the chromosomes that are significantly higher than the read background. For histone modifications and other epigenetic marks, it is often preferable to find a characteristic region of enrichment in sequencing reads relative to gene annotations. For instance, many histone modifications are typically enriched around transcription start sites. Calculating the optimal window that describes this enrichment allows one to quantify modification levels for each individual gene. Using data sets for the H3K9/14ac histone modification in Th cells and an accompanying IgG control, we present an analysis strategy that alternates between single gene and global data distribution levels and allows a clear distinction between experimental background and signal. Curve fitting permits false discovery rate-based classification of genes as modified versus unmodified. We have developed a software package called EpiChIP that carries out this type of analysis, including integration with and visualization of gene expression data

A native chromatin purification system for epigenomic profiling in Caenorhabditis elegans

High-resolution mapping of chromatin features has emerged as an important strategy for understanding gene regulation and epigenetic inheritance. We describe an in vivo tagging system coupled to chromatin purification for genome-wide epigenetic profiling in Caenorhabditis elegans. In this system, we coexpressed the Escherichia coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino-acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We found that the fusion BioTag::H3.3 was efficiently biotinylated in vivo. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. We found that embryonic chromatin is differentially extracted with increasing salt concentrations. Interestingly, chromatin that remains insoluble after washing in 600 mM salt is enriched at 5′ and 3′ ends, suggesting the presence of large protein complexes that render chromatin insoluble at transcriptional initiation and termination sites. We also found that H3.3 landscapes from these salt fractions display consistent features that correlate with gene activity: the most highly expressed genes contain the most H3.3. This versatile two-component approach has the potential of facilitating genome-wide chromatin dynamics and regulatory site identification in C. elegans

Modeling the relative relationship of transcription factor binding and histone modifications to gene expression levels in mouse embryonic stem cells

Transcription factor (TF) binding and histone modification (HM) are important for the precise control of gene expression. Hence, we constructed statistical models to relate these to gene expression levels in mouse embryonic stem cells. While both TF binding and HMs are highly ‘predictive’ of gene expression levels (in a statistical, but perhaps not strictly mechanistic, sense), we find they show distinct differences in the spatial patterning of their predictive strength: TF binding achieved the highest predictive power in a small DNA region centered at the transcription start sites of genes, while the HMs exhibited high predictive powers across a wide region around genes. Intriguingly, our results suggest that TF binding and HMs are redundant in strict statistical sense for predicting gene expression. We also show that our TF and HM models are cell line specific; specifically, TF binding and HM are more predictive of gene expression in the same cell line, and the differential gene expression between cell lines is predictable by differential HMs. Finally, we found that the models trained solely on protein-coding genes are predictive of expression levels of microRNAs, suggesting that their regulation by TFs and HMs may share a similar mechanism to that for protein-coding genes

An assessment of histone-modification antibody quality

We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/)

Complex exon-intron marking by histone modifications is not determined solely by nucleosome distribution

It has recently been shown that nucleosome distribution, histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (“exon-intron marking”), linking chromatin structure and function to co-transcriptional splicing in a variety of eukaryotes. Previous ChIP-sequencing studies suggested that these marking patterns reflect the nucleosomal landscape. By analyzing ChIP-chip datasets across the human genome in three cell types, we have found that this marking system is far more complex than previously observed. We show here that a range of histone modifications and Pol II are preferentially associated with exons. However, there is noticeable cell-type specificity in the degree of exon marking by histone modifications and, surprisingly, this is also reflected in some histone modifications patterns showing biases towards introns. Exon-intron marking is laid down in the absence of transcription on silent genes, with some marking biases changing or becoming reversed for genes expressed at different levels. Furthermore, the relationship of this marking system with splicing is not simple, with only some histone modifications reflecting exon usage/inclusion, while others mirror patterns of exon exclusion. By examining nucleosomal distributions in all three cell types, we demonstrate that these histone modification patterns cannot solely be accounted for by differences in nucleosome levels between exons and introns. In addition, because of inherent differences between ChIP-chip array and ChIP-sequencing approaches, these platforms report different nucleosome distribution patterns across the human genome. Our findings confound existing views and point to active cellular mechanisms which dynamically regulate histone modification levels and account for exon-intron marking. We believe that these histone modification patterns provide links between chromatin accessibility, Pol II movement and co-transcriptional splicing

ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity

Transcriptional control is dependent on a vast network of epigenetic modifications. One epigenetic mark of particular interest is tri-methylation of lysine 27 on histone H3 (H3K27me3), which is catalysed and maintained by Polycomb Repressive Complex 2 (PRC2). Although this histone mark is studied widely, the precise relationship between its local pattern of enrichment and regulation of gene expression is currently unclear. We have used ChIP-seq to generate genome-wide maps of H3K27me3 enrichment, and have identified three enrichment profiles with distinct regulatory consequences. First, a broad domain of H3K27me3 enrichment across the body of genes corresponds to the canonical view of H3K27me3 as inhibitory to transcription. Second, a peak of enrichment around the transcription start site (TSS) is commonly associated with ‘bivalent’ genes, where H3K4me3 also marks the TSS. Finally and most surprisingly, we identified an enrichment profile with a peak in the promoter of genes that is associated with active transcription. Genes with each of these three profiles were found in different proportions in each of the cell types studied. The data analysis techniques developed here will be useful for the identification of common enrichment profiles for other histone modifications that have important consequences for transcriptional regulation