Posttranslational histone modifications participate in modulating the structure and function of chromatin. Promoters of transcribed genes are enriched with K4 trimethylation and hyperacetylation on the N-terminal tail of histone H3. Recently, PHD finger proteins, like Yng1 in the NuA3 HAT complex, were shown to interact with H3K4me3, indicating a biochemical link between K4 methylation and hyperacetylation. By using a combination of mass spectrometry, biochemistry, and NMR, we detail the Yng1 PHD-H3K4me3 interaction and the importance of NuA3-dependent acetylation at K14. Furthermore, genome-wide ChIP-Chip analysis demonstrates colocalization of Yng1 and H3K4me3 in vivo. Disrupting the K4me3 binding of Yng1 altered K14ac and transcription at certain genes, thereby demonstrating direct in vivo evidence of sequential trimethyl binding, acetyltransferase activity, and gene regulation by NuA3. Our data support a general mechanism of transcriptional control through which histone acetylation upstream of gene activation is promoted partially through availability of H3K4me3, "read" by binding modules in select subunits.
The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.During and after eukaryotic transcription, RNA is packaged into RNP complexes, then modified, spliced, folded and exported into the cytoplasm through nuclear pore complexes (NPCs). There are two main forms of RNPs. Each contains a different type of RNA: mRNPs contain mRNA, and rRNPs contain ribosomal RNA (rRNA). Each also uses discrete processing factors, which associate with RNPs in a dynamic fashion to define the order of transcript maturation. The function and composition of many of these RNP complexes, however, is poorly understood.In recent years, there have been considerable efforts to study the composition of mRNPs and rRNPs in greater detail. A favored approach has been to purify these complexes from cell lysates [1][2][3][4] . But the limitations of presently available purification techniques pose a major hurdle to the study of RNA processing and export. Here we describe a method that aims to overcome these limitations and capture the dynamic interactome of maturing RNP complexes.We used this method to analyze complexes along both the ribosome and mRNA biogenesis pathways. RESULTSA strategy for isolation of RNP complexes Our strategy comprises seven steps that we optimized for efficient isolation of intact RNP complexes ( Fig. 1 and Supplementary Methods online). Collected cells are rapidly frozen in liquid nitrogen and broken open in the solid phase by milling 5 , using a planetary ball mill that produces particles of B1-2 mm in diameter (small enough for rapid and efficient RNP extraction). Because cell breakage occurs in the frozen state, we avoid damage to RNP complexes by released nucleases and proteases, as well as redistribution of proteins within the extract. The resulting frozen grindate is rapidly thawed into an RNP-compatible buffer (see Supplementary Methods), to produce an extract that is then immediately clarified by filtration. We isolate the tagged complexes using antibody-conjugated magnetic beads, rather than more commonly used antibody-conjugated resins such as Sepharose. The magnetic beads are small (2.8 mm) and so have a large surface area-to-volume ratio that increases the speed of binding. Moreover, the beads are impermeable, such that the antibodies are densely conjugated to their exposed surface; hence, there is no theoretical upper limit to the size of isolated complexes (unlike with permeable resins such as Sepharose, which are limited by their pore size). Incubation times...
Regulatory agencies have recently recommended a Quality by Design (QbD) approach for the manufacturing of therapeutic molecules. A QbD strategy requires deep understanding at the molecular level of the attributes that are crucial for safety and efficacy and for insuring that the desired quality of the purified protein drug product is met at the end of the manufacturing process. A mass spectrometry (MS)-based approach to simultaneously monitor the extensive array of product quality attributes (PQAs) present on therapeutic molecules has been developed. This multi-attribute method (MAM) uses a combination of high mass accuracy / high resolution MS data generated by Orbitrap technology and automated identification and relative quantification of PQAs with dedicated software (Pinpoint). The MAM has the potential to replace several conventional electrophoretic and chromatographic methods currently used in Quality Control to release therapeutic molecules. The MAM represents an optimized analytical solution to focus on the attributes of the therapeutic molecule essential for function and implement QbD principles across process development, manufacturing and drug disposition.
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