Uncoupling histone modification crosstalk by engineering lysine demethylase LSD1.

Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation, but its functional significance in cells has been difficult to discern. Previous enzymatic studies revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine-specific demethylase 1 (LSD1). In the present study, we engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation. K562 cells with the Y391K LSD1 CRISPR knockin show decreased expression of a set of genes associated with cellular adhesion and myeloid leukocyte activation. Chromatin profiling revealed that the cis-regulatory regions of these silenced genes display a higher level of H3 Lys14 acetylation, and edited K562 cells show diminished H3 mono-methyl Lys4 near these silenced genes, consistent with a role for enhanced LSD1 demethylase activity. These findings illuminate the functional consequences of disconnecting histone modification crosstalk for a key epigenetic enzyme.

Coenzyme A biosynthesis: mechanisms of regulation, function and disease.

The tricarboxylic acid cycle, nutrient oxidation, histone acetylation and synthesis of lipids, glycans and haem all require the cofactor coenzyme A (CoA). Although the sources and regulation of the acyl groups carried by CoA for these processes are heavily studied, a key underlying question is less often considered: how is production of CoA itself controlled? Here, we discuss the many cellular roles of CoA and the regulatory mechanisms that govern its biosynthesis from cysteine, ATP and the essential nutrient pantothenate (vitamin B5), or from salvaged precursors in mammals. Metabolite feedback and signalling mechanisms involving acetyl-CoA, other acyl-CoAs, acyl-carnitines, MYC, p53, PPARα, PINK1 and insulin- and growth factor-stimulated PI3K-AKT signalling regulate the vitamin B5 transporter SLC5A6/SMVT and CoA biosynthesis enzymes PANK1, PANK2, PANK3, PANK4 and COASY. We also discuss methods for measuring CoA-related metabolites, compounds that target CoA biosynthesis and diseases caused by mutations in pathway enzymes including types of cataracts, cardiomyopathy and neurodegeneration (PKAN and COPAN).

PI3K drives the de novo synthesis of coenzyme A from vitamin B5.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K-PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

Large-scale, in-house production of viral transport media to support SARS-CoV-2 PCR testing in a multi-hospital healthcare network during the COVID-19 pandemic.

The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media (VTM) due to widespread demand for SARS-CoV-2 RT-PCR testing. In response to this ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our hospital network. As our diagnostic laboratory was not equipped for reagent production, we took advantage of space and personnel that became available due to closure of the research division of our medical center. We utilized a formulation of VTM described by the CDC that was simple to produce, did not require filtration for sterilization, and used reagents that were available from commercial suppliers. Performance of VTM was evaluated by several quality assurance measures. Based on Ct values of spiking experiments, we found that our VTM supported highly consistent amplification of the SARS-CoV-2 target (coefficient of variation = 2.95%) using the Abbott RealTime SARS-CoV-2 EUA assay on the Abbott m2000 platform. VTM was also found to be compatible with multiple swab types and, based on accelerated stability studies, able to maintain functionality for at least four months at room temperature. We further discuss how we met logistical challenges associated with large-scale VTM production in a crisis setting including use of staged, assembly line for VTM transport tube production.

Large-Scale, In-House Production of Viral Transport Media To Support SARS-CoV-2 PCR Testing in a Multihospital Health Care Network during the COVID-19 Pandemic.

The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media (VTM) due to widespread demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription-PCR (RT-PCR) testing. In response to this ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our hospital network. As our diagnostic laboratory was not equipped for reagent production, we took advantage of space and personnel that became available due to closure of the research division of our medical center. We utilized a formulation of VTM described by the CDC that was simple to produce, did not require filtration for sterilization, and used reagents that were available from commercial suppliers. Performance of VTM was evaluated by several quality assurance measures. Based on cycle threshold (CT ) values of spiking experiments, we found that our VTM supported highly consistent amplification of the SARS-CoV-2 target (coefficient of variation = 2.95%) using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization (EUA) assay on the Abbott m2000 platform. VTM was also found to be compatible with multiple swab types and, based on accelerated stability studies, able to maintain functionality for at least 4 months at room temperature. We further discuss how we met logistical challenges associated with large-scale VTM production in a crisis setting, including use of a staged assembly line for VTM transport tube production.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments.

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.