global-level-quantification-histone-post-translational-modifications

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...

automated-sample-preparation-for-multiplexed-analysis-single-cell

Automated Sample Preparation for the Multiplexed Analysis of Single-Cell Histone Post-Translational Modifications (sc-hPTM2)

Histone post-translational modifications (hPTMs) are central regulators of chromatin organization and gene expression. Their dysregulation is implicated ...

Albert Einstein College of Medicine

To gain insights into the molecular interactions of an intracellular pathogen and its host cell, we studied the gene expression and chromatin states of human fibroblasts infected with the Apicomplexan parasite Toxoplasma gondii. We show a striking activation of host cell genes that regulate a number of cellular processes, some of which are protective of the host cell, others likely to be advantageous to the pathogen. The simultaneous capture of host and parasite genomic information allowed us to gain insights into the regulation of the T. gondii genome. We show how chromatin accessibility and transcriptional profiling together permit novel annotation of the parasite's genome, including more accurate mapping of known genes and the identification of new genes and cis-regulatory elements. Motif analysis reveals not only the known T. gondii AP2 transcription factor-binding site but also a previously-undiscovered candidate TATA box-containing motif at one-quarter of promoters. By inferring the transcription factor and upstream cell signaling responses involved in the host cell, we can use genomic information to gain insights into T. gondii's perturbation of host cell physiology. Our resulting model builds on previously-described human host cell signalling responses to T. gondii infection, linked to induction of specific transcription factors, some of which appear to be solely protective of the host cell, others of which appear to be co-opted by the pathogen to enhance its own survival.

Genomic insights into host and parasite interactions during intracellular infection by Toxoplasma gondii.

Three-dimensional (3D) cell culture has emerged as an alternative approach to 2D flat culture to model more accurately the phenotype of solid tissue in laboratories. Culturing cells in 3D more precisely recapitulates physiological conditions of tissues, as these cells reduce activities related to proliferation, focusing their energy consumption toward metabolism and homeostasis.

Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model.

Comprehensive enhancer discovery is challenging because most enhancers, especially those contributing to complex diseases, have weak effects on gene expression. Our gene regulatory network modeling identified that nonlinear enhancer gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Using human embryonic stem cell definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. We discovered a comprehensive set of enhancers for each of the core endoderm-specifying transcription factors. Many enhancers had strong effects mid-transition but weak effects post-transition, consistent with the nonlinear temporal responses to enhancer perturbation predicted by the modeling. Integrating three-dimensional genomic information, we were able to develop a CTCF-loop-constrained Interaction Activity model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and systematic enhancer discovery in both normal and pathological cell state transitions.

Dynamic network-guided CRISPRi screen identifies CTCF-loop-constrained nonlinear enhancer gene regulatory activity during cell state transitions.

Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps - 1) immobilization of a single cell on solid substrate, 2) hypotonic lysis of immobilized single cell, and 3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.

Concurrent analysis of genome and transcriptome in one single cell.

Genetic wrinkles in time.

Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps-(1) immobilization of a single cell on solid substrate, (2) hypotonic lysis of immobilized single cell, and (3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.

Histone proteins are the structural components of nucleosomes, which form chromatin. Histone proteins are typically modified with many posttranslational modifications (PTMs), which affect chromatin accessibility, and by extension, modulate gene transcription, and other DNA-related processes. Mass spectrometry has become the reference technology to quantify global levels of hundreds of histone PTMs in single experiments. The advancement of high throughput has paved the way to new possibilities, including experimental design that include large cohort of samples. In this chapter, we describe a protocol for the unbiased analysis of histone PTMs assisted by a robotic liquid handler. The implementation of a simple-to-use script for automated histone derivatization and digestion reduces the number of manual steps needed to prepare histone peptides for mass spectrometry analysis and improves consistency of resulting data.

Automated Sample Preparation for the Unbiased Analysis of Histone Posttranslational Modifications Via Mass Spectrometry.

A key driver of Glioblastoma (GBM) heterogeneity and therapy resistance is the capacity of glioma stem-like cells (GSCs) to hijack developmental signaling programs. However, it remains unclear how GSCs regulate these adapted developmental signaling pathways and how these pathways might be therapeutically exploited. The arginine methyltransferase, CARM1, has been shown to play critical roles in maintaining stem cell pluripotency, preventing differentiation, and recently was discovered to be upregulated in Glioblastoma. To date, there is little to no understanding of the role that CARM1 plays in regulating developmental processes in Glioblastoma. To address this gap in knowledge, we applied a multi-omics approach to characterize developmental processes that are specifically regulated by CARM1 in GSCs. We found that loss of CARM1 results in dysregulation of several developmental markers: ARX, GFAP, NGFR, PDGFRA and results in both a proteomic and transcriptomic shift towards the radial glia cell lineage. Moreover, CARM1 depleted cells reprogram their signaling to develop an increased survival dependency on NGFR/NTRK signaling and are hypersensitive to the FDA approved brain penetrant NTRK inhibitor-Entrectinib. Mechanistically, we find that NFIA is a CARM1 substrate and can repress NGFR signaling just as CARM1 does, and thus the CARM1/NFIA relationship is likely a key regulator of NGFR/NTRK signaling in GSCs. Altogether, we demonstrate that CARM1 regulates the cell lineage of GSCs at the transcriptomic and proteomic level, and naturally represses NGFR/NTRK signaling-likely through CARM1 dependent methylation of NFIA. Further, CARM1 depletion leads GSCs to develop a survival dependency on NGFR/NTRK signaling and creates a therapeutic vulnerability to NTRK inhibition.

Loss of CARM1 alters the developmental programming of Glioma stem-like cells and creates a druggable NGFR/NTRK dependency.

Chaperone-mediated autophagy (CMA) declines in ageing and neurodegenerative diseases. Loss of CMA in neurons leads to neurodegeneration and behavioural changes in mice but the role of CMA in neuronal physiology is largely unknown. Here we show that CMA deficiency causes neuronal hyperactivity, increased seizure susceptibility and disrupted calcium homeostasis. Pre-synaptic neurotransmitter release and NMDA receptor-mediated transmission were enhanced in CMA-deficient females, whereas males exhibited elevated post-synaptic AMPA-receptor activity. Comparative quantitative proteomics revealed sexual dimorphism in the synaptic proteins degraded by CMA, with preferential remodelling of the pre-synaptic proteome in females and the post-synaptic proteome in males. We demonstrate that genetic or pharmacological CMA activation in old mice and an Alzheimer's disease mouse model restores synaptic protein levels, reduces neuronal hyperexcitability and seizure susceptibility, and normalizes neurotransmission. Our findings unveil a role for CMA in regulating neuronal excitability and highlight this pathway as a potential target for mitigating age-related neuronal decline.

Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome.

Somatic mutations accumulate with age in human tissues. Clonal amplification of some mutations causes cancers and other diseases. However, it is unclear if random mutation accumulation affects cellular function without clonal amplification. We tested this in cell culture, avoiding the limitation that mutation accumulation  leads to cancer. We performed single-cell whole-genome sequencing of fibroblasts from DNA-mismatch-repair-deficient   mice and controls after long-term passaging. While maintaining the same growth rates, in the   fibroblasts, single-nucleotide variants increased up until >50,000 per cell, with small insertions and deletions plateauing at ∼16,000 per cell. We provide evidence for genome-wide negative selection and large-scale mutation-driven population changes, including significant clonal expansion of preexisting mutations and widespread cell-strain-specific hotspots, likely caused by positive selection of mutations in specific genes. Since negative selection to prevent mutations with adverse effects  during aging is difficult to envision, these results suggest a causal role of somatic mutations in age-related cell functional decline.

Divergent accumulation patterns of SNVs and INDELs reveal negative selection in noncancerous cells.

Studies of herpes simplex virus (HSV) entry uncovered a previously unappreciated "outside-in" signaling pathway whereby activation of the calcium (Ca) responsive enzyme phospholipid scramblase 1 (PLSCR1), which is known to trigger the bidirectional movement of phosphatidylserines (PS) between the inner and outer leaflet of the plasma membrane, also induces the translocation and subsequent extracellular activation of intracellular proteins, including Akt. We hypothesized that HIV-1, which has been shown to elicit scramblase TMEM16F-mediated PS externalization, may trigger a similar "outside-in" signaling cascade involving exofacial kinase activity to promote its entry into CD4+ T cells. To study this process, we utilized a cell impermeable staurosporine analogue, alkyl-CIMSS, which is a broadly active kinase inhibitor that blocks HSV-induced exofacial Akt phosphorylation and HSV infection. Using multiple cell types including TZM-bl, Jurkat T cells, and human peripheral blood mononuclear cells (PBMCs), we show that, in contrast to the effects on HSV, treatment of cells with alkyl-CIMSS enhances HIV-1 infection post-entry that is not dependent on TMEM16F. To identify potential biological processes that are responsive to alkyl-CIMSS, we performed bulk RNA-sequencing and whole cell proteomics and found that alkyl-CIMSS treatment of cells robustly upregulates the cell surface density of the proteoglycan glypican-1 (GPC1). Lentiviral delivery of GPC1 overexpression and shRNA knockdown constructs reveal that the presence and absence of GPC1 independently of alkyl-CIMSS treatment significantly impact HIV-1 infection, with the effect on infection corresponding to GPC1 expression. Further, we demonstrate that the influence of GPC1 on HIV-1 infection is in part mediated by TGF-β signaling. Collectively, these findings implicate a cell surface protein susceptible to alkyl-CIMSS in restricting HIV-1 infection and identify GPC1 as a novel modulator of HIV-1 infection.

Glypican-1 upregulation elicited in response to a cell-impermeable kinase inhibitor and its overexpression enhance HIV-1 infection.

Single-cell proteomics confidently quantifies cellular heterogeneity, however quantification of post-translational modifications, such as those deposited on histone proteins, remains elusive. Here, we develop a robust mass spectrometry-based method for the unbiased analysis of single-cell histone post-translational modifications (sc-hPTM). sc-hPTM identifies both single- and combinatorial histone post-translational modifications (67 peptidoforms in total), which includes nearly all frequently studied histone post-translational modifications with comparable reproducibility to traditional bulk experiments. As a proof of concept, we treat cells with sodium butyrate, a histone deacetylase inhibitor, and demonstrate that our method can i) distinguish between treated and untreated cells, ii) identify sub-populations of cells with heterogeneous response to the treatment, and iii) reveal differential co-regulation of histone post-translational modifications in the context of drug treatment. The sc-hPTM method enables comprehensive investigation of chromatin heterogeneity at single-cell resolution and provides a further understanding of the histone code.

ArticleTotal : 2	Year
Publication title Cited by 7	2022
Publication title Cited by 1	2025

Article	Year
Genomic insights into host and parasite interactions during intracellular infection by Toxoplasma gondii. PloS one\| PubMed ID: 36178892	2022
Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model. Epigenetics & chromatin\| PubMed ID: 36411440	2022
Dynamic network-guided CRISPRi screen identifies CTCF-loop-constrained nonlinear enhancer gene regulatory activity during cell state transitions. Nature genetics\| PubMed ID: 37488417	2023
Concurrent analysis of genome and transcriptome in one single cell. Research square\| PubMed ID: 37577506	2023
Genetic wrinkles in time. Nature reviews. Molecular cell biology\| PubMed ID: 37620588	2023
Concurrent analysis of genome and transcriptome in one single cell. BMC research notes\| PubMed ID: 39285281	2024
Automated Sample Preparation for the Unbiased Analysis of Histone Posttranslational Modifications Via Mass Spectrometry. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 40257557	2025
Loss of CARM1 alters the developmental programming of Glioma stem-like cells and creates a druggable NGFR/NTRK dependency. bioRxiv : the preprint server for biology\| PubMed ID: 40321217	2025
Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome. Nature cell biology\| PubMed ID: 41087566	2025
Divergent accumulation patterns of SNVs and INDELs reveal negative selection in noncancerous cells. Innovation (Cambridge (Mass.))\| PubMed ID: 41113919	2025
Glypican-1 upregulation elicited in response to a cell-impermeable kinase inhibitor and its overexpression enhance HIV-1 infection. bioRxiv : the preprint server for biology\| PubMed ID: 41278678	2025
Mass spectrometry-based profiling of single-cell histone post-translational modifications to dissect chromatin heterogeneity. Nature communications\| PubMed ID: 41387402	2025

Ronald Cutler

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