Dual protease type XIII/pepsin digestion offers superior resolution and overlap for the analysis of histone tails by HX-MS.

The N-terminal regions of histone proteins (tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications (PTMs) present on these regions contribute to transcriptional regulation. While hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed to analyze histones due to the poor N-terminal coverage obtained using pepsin. Here, we test the applicability of a dual protease type XIII/pepsin digestion column to this class of proteins. We optimize online digestion conditions using the H4 monomer, and extend the method to the analysis of histones in monomeric states and nucleosome core particles (NCPs). We show that the dual protease column generates many short and overlapping N-terminal peptides. We evaluate our method by performing hydrogen exchange experiments of NCPs for different time points and present full coverage of the tails at excellent resolution. We further employ electron transfer dissociation and showcase an unprecedented degree of overlap across multiple peptides that is several fold higher than previously reported methods. The method we report here may be readily applied to the HX-MS investigation of histone dynamics and to the footprints of histone binding proteins on nucleosomes.

Chasing Tails: Cathepsin-L Improves Structural Analysis of Histones by HX-MS.

The N-terminal regions (tails) of histone proteins are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications present on these regions contribute to transcriptional regulation. Considering their biological significance, relatively few structural details have been established for histone tails, mainly because of their inherently disordered nature. Although hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed in this context, presumably because of the poor N-terminal coverage provided by pepsin. Inspired from histone-clipping events, we profiled the activity of cathepsin-L under HX-MS quench conditions and characterized its specificity employing the four core histones (H2A, H2B, H3 and H4). Cathepsin-L demonstrated cleavage patterns that were substrate- and pH-dependent. Cathepsin-L generated overlapping N-terminal peptides about 20 amino acids long for H2A, H3, and H4 proving its suitability for the analysis of histone tails dynamics. We developed a comprehensive HX-MS method in combination with pepsin and obtained full sequence coverage for all histones. We employed our method to analyze histones H3 and H4. We observe rapid deuterium exchange of the N-terminal tails and cooperative unfolding (EX1 kinetics) in the histone-fold domains of histone monomers in-solution. Overall, this novel strategy opens new avenues for investigating the dynamic properties of histones that are not apparent from the crystal structures, providing insights into the structural basis of the histone code.

Down-syndrome-induced senescence disrupts the nuclear architecture of neural progenitors.

Down syndrome (DS) is a genetic disorder driven by the triplication of chromosome 21 (T21) and characterized by a wide range of neurodevelopmental and physical disabilities. Transcriptomic analysis of tissue samples from individuals with DS has revealed that T21 induces a genome-wide transcriptional disruption. However, the consequences of T21 on the nuclear architecture and its interplay with the transcriptome remain unknown. In this study, we find that unlike human induced pluripotent stem cells (iPSCs), iPSC-derived neural progenitor cells (NPCs) exhibit genome-wide "chromosomal introversion," disruption of lamina-associated domains, and global chromatin accessibility changes in response to T21, consistent with the transcriptional and nuclear architecture changes characteristic of senescent cells. Treatment of T21-harboring NPCs with senolytic drugs alleviates the transcriptional, molecular, and cellular dysfunctions associated with DS. Our findings provide a mechanistic link between T21 and global transcriptional disruption and indicate that senescence-associated phenotypes may play a key role in the neurodevelopmental pathogenesis of DS.

A multi-omic analysis of MCF10A cells provides a resource for integrative assessment of ligand-mediated molecular and phenotypic responses.

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis.

Proteomic profiling dataset of chemical perturbations in multiple biological backgrounds.

While gene expression profiling has traditionally been the method of choice for large-scale perturbational profiling studies, proteomics has emerged as an effective tool in this context for directly monitoring cellular responses to perturbations. We previously reported a pilot library containing 3400 profiles of multiple perturbations across diverse cellular backgrounds in the reduced-representation phosphoproteome (P100) and chromatin space (Global Chromatin Profiling, GCP). Here, we expand our original dataset to include profiles from a new set of cardiotoxic compounds and from astrocytes, an additional neural cell model, totaling 5300 proteomic signatures. We describe filtering criteria and quality control metrics used to assess and validate the technical quality and reproducibility of our data. To demonstrate the power of the library, we present two case studies where data is queried using the concept of "connectivity" to obtain biological insight. All data presented in this study have been deposited to the ProteomeXchange Consortium with identifiers PXD017458 (P100) and PXD017459 (GCP) and can be queried at https://clue.io/proteomics .

Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis.

Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.

Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

Triggered Release of Encapsulated Cargo from Photoresponsive Polyelectrolyte Nanocomplexes.

Combining the numerous advantages of using light as a stimulus, simple free radical random copolymerization, and the easy, all-aqueous preparation of polyelectrolyte complexes (PECs), we prepared photolabile PEC nanoparticles and demonstrated their rapid degradation under UV light. As a proof of concept demonstration, the dye Nile Red was encapsulated in the PECs and successfully released into the surrounding solution as the polyelectrolyte nanocomplex carriers dissolved upon light irradiation.