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The naive T cell compartment undergoes multiple changes across age that associate with altered susceptibility to infection and autoimmunity. In addition to the acquisition of naive-like memory T cell subsets, mouse studies describe substantial molecular reprogramming of the naive compartment in adults compared with adolescents. However, these alterations are not well delineated in human aging. Using a new trimodal single cell technology (TEA-seq), we discovered that the composition and transcriptional and epigenetic programming of the naive T cell compartment in children (11-13 yrs) is distinct from that of older adults (55-65 yrs). Naive CD4 T cells, previously considered relatively resistant to aging, exhibited far more pronounced molecular reprogramming than naive CD8 T cells, in which alterations are preferentially driven by shifts in naive-like memory subsets. These data reveal the complex nature of the naive T cell compartment that may contribute to differential immune responses across the spectrum of human age.One Sentence Summary:The naive CD8 and CD4 T cell compartments in humans are heterogeneous and impacted differently with age, in which naive CD8 T cell subsets dramatically shift in composition and true naive CD4 T cells display significant molecular re-programming.

Summary Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021)., Graphical abstract, Highlights • Details a robust pipeline for the profiling of human PBMC • Outline a cell thaw protocol compatible with flow cytometry and single cell RNA-seq • Focus on batch effect reduction with a bridging cell control and commercial buffers • Increased throughput with automated liquid handling and cell counting, Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput.

SARS-CoV-2 has infected over 160 million and caused more than 3 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive activation, including an early cellular and proteomic signature that correlated with the amplitude of virus-specific humoral responses after day 30. We characterized signals associated with recovery and convalescence to define a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

Summary Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMC up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states., Graphical abstract, Highlights • Studies of human blood cells and plasma are highly sensitive to process variability • Time variability distorts biology in cutting-edge single-cell and multiplex assays • Longitudinal, multi-modal, and aligned data enable data qualification and exploration • Dataset holds potential novel, multi-modal biological correlations and hypotheses, Molecular Physiology ; Immunology ; Proteomics ; Transcriptomics

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to extracellular signals, and human disease states. scATAC-seq has been particularly challenging due to the large size of the human genome and processing artefacts resulting from DNA damage that are an inherent source of background signal. Downstream analysis and integration of scATAC-seq with other single-cell assays is complicated by the lack of clear phenotypic information linking chromatin state and cell type. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases the signal-to-noise ratio and allows simultaneous measurement of cell surface markers: Integrated Cellular Indexing of Chromatin Landscape and Epitopes (ICICLE-seq). We extended this approach using a droplet-based multiomics platform to develop a trimodal assay to simultaneously measure Transcriptomic state (scRNA-seq), cell surface Epitopes, and chromatin Accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

SUMMARY The DUX4 transcription factor is briefly expressed in the early cleavage-stage embryo, where it induces an early wave of zygotic gene transcription, whereas its mis-expression in skeletal muscle causes the muscular dystrophy facioscapulohumeral dystrophy (FSHD). Here, we show that DUX4 induces the expression of the histone variants H3.X and H3.Y. We have used a myoblast cell line with doxycycline-inducible DUX4 to show that these histone variants are incorporated throughout the body of DUX4-induced genes. Following a brief pulse of DUX4, these histones contribute to greater perdurance and to enhanced re-activation of DUX4 target gene expression. These findings provide a model for H3.X/Y as a chromatin mechanism that facilitates the expression of DUX4 target genes subsequent to a brief pulse of DUX4 expression., Graphical Abstract, In Brief Resnick et al. show that the DUX4-induced histone variants, H3.X and H3.Y, incorporate into the chromatin of DUX4-induced genes, making them more sensitive to subsequent expression. This suggests a mechanism for how the brief expression of DUX4 can establish a memory of its transcriptional network.

Cleavage under targets and release using nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used chromatin immunoprecipitation (ChIP) protocols in resolution, signal-to-noise ratio and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and has been used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here, we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data when starting with only 100 cells for a histone modification and 1,000 cells for a transcription factor. From cells to purified DNA, CUT&RUN requires less than a day at the laboratory bench and requires no specialized skills.

Transcriptional changes occur presymptomatically and throughout Huntington9s disease (HD), motivating the study of transcriptional regulatory networks (TRNs) in HD. We reconstructed a genome‐scale model for the target genes of 718 transcription factors (TFs) in the mouse striatum by integrating a model of genomic binding sites with transcriptome profiling of striatal tissue from HD mouse models. We identified 48 differentially expressed TF‐target gene modules associated with age‐ and CAG repeat length‐dependent gene expression changes in Htt CAG knock‐in mouse striatum and replicated many of these associations in independent transcriptomic and proteomic datasets. Thirteen of 48 of these predicted TF‐target gene modules were also differentially expressed in striatal tissue from human disease. We experimentally validated a specific model prediction that SMAD3 regulates HD‐related gene expression changes using chromatin immunoprecipitation and deep sequencing (ChIP‐seq) of mouse striatum. We found CAG repeat length‐dependent changes in the genomic occupancy of SMAD3 and confirmed our model9s prediction that many SMAD3 target genes are downregulated early in HD.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and can be used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench. In summary, CUT&RUN has several advantages over ChIP-seq: (1) The method is performed in situ in non-crosslinked cells and does not require chromatin fragmentation or solubilization; (2) The intrinsically low background allows low sequence depth and identification of low signal genomic features invisible to ChIP; (3) The simple procedure can be completed within a day and is suitable for robotic automation; (4) The method can be used with low cell numbers compared to existing methodologies; (5) A simple spike-in strategy can be used for accurate quantitation of protein-DNA interactions. As such, CUT&RUN represents an attractive replacement for ChIPseq, which is one of the most popular methods in biological research.

SUMMARYCleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely-used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and can be used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench.

CpG islands (CGIs) are prominent in the mammalian genome owing to their GC-rich base composition and high density of CpG dinucleotides1,2. Most human gene promoters are embedded within CGIs that lack DNA methylation and coincide with sites of histone H3 lysine 4 trimethylation (H3K4me3), irrespective of transcriptional activity3,4. In spite of these intriguing correlations, the functional significance of non-methylated CGI sequences with respect to chromatin structure and transcription is unknown. By performing a search for proteins that are common to all CGIs, here we show high enrichment for Cfp1, which selectively binds to non-methylated CpGs in vitro5,6. Chromatin immunoprecipitation of a mono-allelically methylated CGI confirmed that Cfp1 specifically associates with non-methylated CpG sites in vivo. High throughput sequencing of Cfp1-bound chromatin identified a notable concordance with non-methylated CGIs and sites of H3K4me3 in the mouse brain. Levels of H3K4me3 at CGIs were markedly reduced in Cfp1-depleted cells, consistent with the finding that Cfp1 associates with the H3K4 methyltransferase Setd1 (refs 7, 8). To test whether non-methylated CpG-dense sequences are sufficient to establish domains of H3K4me3, we analysed artificial CpG clusters that were integrated into the mouse genome. Despite the absence of promoters, the insertions recruited Cfp1 and created new peaks of H3K4me3. The data indicate that a primary function of non-methylated CGIs is to genetically influence the local chromatin modification state by interaction with Cfp1 and perhaps other CpG-binding proteins.

Chromatin immunoprecipitation (ChIP) and its derivatives are the main techniques used to determine transcription factor binding sites. However, conventional ChIP with sequencing (ChIP-seq) has problems with poor resolution, and newer techniques require significant experimental alterations and complex bioinformatics. Previously, we have used a new crosslinking ChIP-seq protocol (X-ChIP-seq) to perform high-resolution mapping of RNA Polymerase II (Skene et al., 2014). Here, we build upon this work and compare X-ChIP-seq to existing methodologies. By using micrococcal nuclease, which has both endo- and exo-nuclease activity, to fragment the chromatin and thereby generate precise protein–DNA footprints, high-resolution X-ChIP-seq achieves single base-pair resolution of transcription factor binding. A significant advantage of this protocol is the minimal alteration to the conventional ChIP-seq workflow and simple bioinformatic processing.

RNA polymerase II (PolII) transcribes RNA within a chromatin context, with nucleosomes acting as barriers to transcription. Despite these barriers, transcription through chromatin in vivo is highly efficient, suggesting the existence of factors that overcome this obstacle. To increase the resolution obtained by standard chromatin immunoprecipitation, we developed a novel strategy using micrococcal nuclease digestion of cross-linked chromatin. We find that the chromatin remodeler Chd1 is recruited to promoter proximal nucleosomes of genes undergoing active transcription, where Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover. The expression of a dominant negative form of Chd1 results in increased stalling of PolII past the entry site of the promoter proximal nucleosomes. We find that Chd1 evicts nucleosomes downstream of the promoter in order to overcome the nucleosomal barrier and enable PolII promoter escape, thus providing mechanistic insight into the role of Chd1 in transcription and pluripotency. DOI: http://dx.doi.org/10.7554/eLife.02042.001, eLife digest DNA is tightly packaged in a material called chromatin inside the cell nucleus. To produce proteins this DNA must first be transcribed to produce a molecule of messenger RNA, which is then translated to make a protein. To assist with this process cells ‘unpack’ certain regions of the DNA so that enzymes that catalyze the different steps in this process can have access to the DNA. A protein called Chd1 is involved in the unpacking process in yeast, but its role in more complex animals is not clear. Now, Skene et al. have shown that this protein is needed to allow the enzyme that catalyzes the transcription of DNA—an enzyme called RNA polymerase II—to do its job. Chd1 acts to unpack the tightly packaged DNA from chromatin, thus allowing the transcription of the DNA to proceed. In the absence of Chd1 activity, RNA polymerase II stalls at the gene promoter—the region of DNA that starts the transcription of a particular gene. This work highlights how the packaging of DNA in the cell is highly dynamic and controls fundamental biological processes. Skene et al. modified a well-known genetic technique called ChIP-seq. Previous ChIP-seq protocols typically provided a blurry, low-resolution map of where proteins bound to chromatin. Skene et al. used an enzyme to ‘chew back’ the DNA to reveal the exact ‘footprints’ of the Chd1 protein and the RNA polymerase II enzyme on the chromatin in mice. It will be possible to adapt this new protocol to map the positions of other proteins, which will help to improve our understanding of the ways in which chromatin regulates access to DNA. DOI: http://dx.doi.org/10.7554/eLife.02042.002

Most mammalian gene promoters are embedded within genomic regions called CpG islands, characterized by elevated levels of nonmethylated CpG dinucleotides. Here, we describe recent work demonstrating that CpG islands act as specific nucleation sites for the zinc finger CxxC domain-containing proteins CFP1 and KDM2A. Importantly, both CFP1 and KDM2A are associated with enzymatic activities that modulate specific histone lysine methylation marks. The action of these zinc finger CxxC domain proteins therefore imposes a defined chromatin architecture on CpG islands that distinguishes these important regulatory elements from the surrounding genome. The functional consequence of this CpG island-directed chromatin environment is discussed.

Most histones are assembled into nucleosomes during replication to package genomic DNA. However, several variant histones are deposited independently of replication at particular regions of chromosomes. Such histone variants include cenH3, which forms the nucleosomal foundation for the centromere, and H3.3, which replaces histones that are lost during dynamic processes that disrupt nucleosomes. Furthermore, various H2A variants participate in DNA repair, gene regulation and other processes that are, as yet, not fully understood. Here, we review recent studies that have implicated histone variants in maintaining pluripotency and as causal factors in cancer and other diseases.

A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon. See research article: http://www.epigeneticsandchromatin.com/content/5/1/17

Trimethylation of histone H3 Lys 4 (H3K4me3) is a mark of active and poised promoters. The Set1 complex is responsible for most somatic H3K4me3 and contains the conserved subunit CxxC finger protein 1 (Cfp1), which binds to unmethylated CpGs and links H3K4me3 with CpG islands (CGIs). Here we report that Cfp1 plays unanticipated roles in organizing genome-wide H3K4me3 in embryonic stem cells. Cfp1 deficiency caused two contrasting phenotypes: drastic loss of H3K4me3 at expressed CGI-associated genes, with minimal consequences for transcription, and creation of “ectopic” H3K4me3 peaks at numerous regulatory regions. DNA binding by Cfp1 was dispensable for targeting H3K4me3 to active genes but was required to prevent ectopic H3K4me3 peaks. The presence of ectopic peaks at enhancers often coincided with increased expression of nearby genes. This suggests that CpG targeting prevents “leakage” of H3K4me3 to inappropriate chromatin compartments. Our results demonstrate that Cfp1 is a specificity factor that integrates multiple signals, including promoter CpG content and gene activity, to regulate genome-wide patterns of H3K4me3.

The development of new strategies to deplete maternal histone proteins in vivo and in vitro has led to the discovery of unexpected roles of histones in forming a functional nuclear envelope.

Translation initiation site usage on the human rhinovirus 2 internal ribosome entry site (IRES) has been examined in a mixed reticulocyte lysate/HeLa cell extract system. There are two relevant AUG triplets, both in a base-paired hairpin structure (domain VI), with one on the 5′ side at nucleotide (nt) 576, base paired with the other at nt 611, which is the initiation site for polyprotein synthesis. A single residue was inserted in the apical loop to put AUG-576 in frame with AUG-611, and in addition another in-frame AUG was introduced at nt 593. When most of the IRES was deleted to generate a monocistronic mRNA, the use of these AUGs conformed to the scanning ribosome model: improving the AUG-576 context increased initiation at this site and decreased initiation at downstream sites, whereas the converse was seen when AUG-576 was mutated to GUA; and AUG-593, when present, took complete precedence over AUG-611. Under IRES-dependent conditions, by contrast, much less initiation occurred at AUG-576 than in a monocistronic mRNA with the same AUG-576 context, mutation of AUG-576 decreased initiation at downstream sites by ∼70%, and introduction of AUG-593 did not completely abrogate initiation at AUG-611, unless the apical base pairing in domain VI was destroyed by point mutations. These results indicate that ribosomes first bind at the AUG-576 site, but instead of initiating there, most of them are transferred to AUG-611, the majority by strictly linear scanning and a substantial minority by direct transfer, which is possibly facilitated by the occasional persistence of base pairing in the apical part of the domain VI stem.

MeCP2 is a nuclear protein with an affinity for methylated DNA that can recruit histone deacetylases. Deficiency or excess of MeCP2 causes severe neurological problems, suggesting that the number of molecules per cell must be precisely regulated. We quantified MeCP2 in neuronal nuclei and found that it is nearly as abundant as the histone octamer. Despite this high abundance, MeCP2 associates preferentially with methylated regions, and high-throughput sequencing showed that its genome-wide binding tracks methyl-CpG density. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. Neither change is detectable in glia, where MeCP2 occurs at lower levels. The mutant brain also shows elevated transcription of repetitive elements. Our data argue that MeCP2 may not act as a gene-specific transcriptional repressor in neurons, but might instead dampen transcriptional noise genome-wide in a DNA methylation-dependent manner.

Background: Formaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined.Methodology: Using a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells. The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process. We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking, also fails to capture these transient interactions.Conclusions/Significance: Our findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research.