Myeloid-T cell interplay and cell state transitions associated with checkpoint inhibitor response in melanoma.

BACKGROUND: The treatment of melanoma, the deadliest form of skin cancer, has greatly benefited from immunotherapy. However, many patients do not show a durable response, which is only partially explained by known resistance mechanisms. METHODS: We performed single-cell RNA sequencing of tumor immune infiltrates and matched peripheral blood mononuclear cells of 22 checkpoint inhibitor (CPI)-naive stage III-IV metastatic melanoma patients. After sample collection, the same patients received CPI treatment, and their response was assessed. FINDINGS: CPI responders showed high levels of classical monocytes in peripheral blood, which preferentially transitioned toward CXCL9-expressing macrophages in tumors. Trajectories of tumor-infiltrating CD8+ T cells diverged at the level of effector memory/stem-like T cells, with non-responder cells progressing into a state characterized by cellular stress and apoptosis-related gene expression. Consistently, predicted non-responder-enriched myeloid-T/natural killer cell interactions were primarily immunosuppressive, while responder-enriched interactions were supportive of T cell priming and effector function. CONCLUSIONS: Our study illustrates that the tumor immune microenvironment prior to CPI treatment can be indicative of response. In perspective, modulating the myeloid and/or effector cell compartment by altering the described cell interactions and transitions could improve immunotherapy response. FUNDING: This research was funded by Roche Pharma Research and Early Development.

Tumor-agnostic transcriptome-based classifier identifies spatial infiltration patterns of CD8+T cells in the tumor microenvironment and predicts clinical outcome in early-phase and late-phase clinical trials.

BACKGROUND: The immune status of a patient's tumor microenvironment (TME) may guide therapeutic interventions with cancer immunotherapy and help identify potential resistance mechanisms. Currently, patients' immune status is mostly classified based on CD8+tumor-infiltrating lymphocytes. An unmet need exists for comparable and reliable precision immunophenotyping tools that would facilitate clinical treatment-relevant decision-making and the understanding of how to overcome resistance mechanisms. METHODS: We systematically analyzed the CD8 immunophenotype of 2023 patients from 14 phase I-III clinical trials using immunohistochemistry (IHC) and additionally profiled gene expression by RNA-sequencing (RNA-seq). CD8 immunophenotypes were classified by pathologists into CD8-desert, CD8-excluded or CD8-inflamed tumors using CD8 IHC staining in epithelial and stromal areas of the tumor. Using regularized logistic regression, we developed an RNA-seq-based classifier as a surrogate to the IHC-based spatial classification of CD8+tumor-infiltrating lymphocytes in the TME. RESULTS: The CD8 immunophenotype and associated gene expression patterns varied across indications as well as across primary and metastatic lesions. Melanoma and kidney cancers were among the strongest inflamed indications, while CD8-desert phenotypes were most abundant in liver metastases across all tumor types. A good correspondence between the transcriptome and the IHC-based evaluation enabled us to develop a 92-gene classifier that accurately predicted the IHC-based CD8 immunophenotype in primary and metastatic samples (area under the curve inflamed=0.846; excluded=0.712; desert=0.855). The newly developed classifier was prognostic in The Cancer Genome Atlas (TCGA) data and predictive in lung cancer: patients with predicted CD8-inflamed tumors showed prolonged overall survival (OS) versus patients with CD8-desert tumors (HR 0.88; 95% CI 0.80 to 0.97) across TCGA, and longer OS on immune checkpoint inhibitor administration (phase III OAK study) in non-small-cell lung cancer (HR 0.75; 95% CI 0.58 to 0.97). CONCLUSIONS: We provide a new precision immunophenotyping tool based on gene expression that reflects the spatial infiltration patterns of CD8+ lymphocytes in tumors. The classifier enables multiplex analyses and is easy to apply for retrospective, reverse translation approaches as well as for prospective patient enrichment to optimize the response to cancer immunotherapy.

Profiling the heterogeneity of colorectal cancer consensus molecular subtypes using spatial transcriptomics.

The consensus molecular subtypes (CMS) of colorectal cancer (CRC) is the most widely-used gene expression-based classification and has contributed to a better understanding of disease heterogeneity and prognosis. Nevertheless, CMS intratumoral heterogeneity restricts its clinical application, stressing the necessity of further characterizing the composition and architecture of CRC. Here, we used Spatial Transcriptomics (ST) in combination with single-cell RNA sequencing (scRNA-seq) to decipher the spatially resolved cellular and molecular composition of CRC. In addition to mapping the intratumoral heterogeneity of CMS and their microenvironment, we identified cell communication events in the tumor-stroma interface of CMS2 carcinomas. This includes tumor growth-inhibiting as well as -activating signals, such as the potential regulation of the ETV4 transcriptional activity by DCN or the PLAU-PLAUR ligand-receptor interaction. Our study illustrates the potential of ST to resolve CRC molecular heterogeneity and thereby help advance personalized therapy.

Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies.

PURPOSE: Target-dependent TCB activity can result in the strong and systemic release of cytokines that may develop into cytokine release syndrome (CRS), highlighting the need to understand and prevent this complex clinical syndrome. EXPERIMENTAL DESIGN: We explored the cellular and molecular players involved in TCB-mediated cytokine release by single-cell RNA-sequencing of whole blood treated with CD20-TCB together with bulk RNA-sequencing of endothelial cells exposed to TCB-induced cytokine release. We used the in vitro whole blood assay and an in vivo DLBCL model in immunocompetent humanized mice to assess the effects of dexamethasone, anti-TNFα, anti-IL6R, anti-IL1R, and inflammasome inhibition, on TCB-mediated cytokine release and antitumor activity. RESULTS: Activated T cells release TNFα, IFNγ, IL2, IL8, and MIP-1ß, which rapidly activate monocytes, neutrophils, DCs, and NKs along with surrounding T cells to amplify the cascade further, leading to TNFα, IL8, IL6, IL1ß, MCP-1, MIP-1α, MIP-1ß, and IP-10 release. Endothelial cells contribute to IL6 and IL1ß release and at the same time release several chemokines (MCP-1, IP-10, MIP-1α, and MIP-1ß). Dexamethasone and TNFα blockade efficiently reduced CD20-TCB-mediated cytokine release whereas IL6R blockade, inflammasome inhibition, and IL1R blockade induced a less pronounced effect. Dexamethasone, IL6R blockade, IL1R blockade, and the inflammasome inhibitor did not interfere with CD20-TCB activity, in contrast to TNFα blockade, which partially inhibited antitumor activity. CONCLUSIONS: Our work sheds new light on the cellular and molecular players involved in cytokine release driven by TCBs and provides a rationale for the prevention of CRS in patients treated with TCBs. See related commentary by Luri-Rey et al., p. 4320.

PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Combinations of Toll-like receptor 8 agonist TL8-506 activate human tumor-derived dendritic cells.

BACKGROUND: Dendritic cells (DCs) are professional antigen presenting cells that initiate immune defense to pathogens and tumor cells. Human tumors contain only few DCs that mostly display a non-activated phenotype. Hence, activation of tumor-associated DCs may improve efficacy of cancer immunotherapies. Toll-like receptor (TLR) agonists and interferons are known to promote DC maturation. However, it is unclear if DCs in human tumors respond to activation signals and which stimuli induce the optimal activation of human tumor DCs. METHODS: We first screened combinations of TLR agonists, a STING agonist and interferons (IFNs) for their ability to activate human conventional DCs (cDCs). Two combinations: TL8-506 (a TLR8 agonist)+IFN-γ and TL8-506+Poly(I:C) (a TLR3 agonist) were studied in more detail. cDC1s and cDC2s derived from cord blood stem cells, blood or patient tumor samples were stimulated with either TL8-506+IFN-γ or TL8-506+Poly(I:C). Different activation markers were analyzed by ELISA, flow cytometry, NanoString nCounter Technology or single-cell RNA-sequencing. T cell activation and migration assays were performed to assess functional consequences of cDC activation. RESULTS: We show that TL8-506 synergized with IFN-γ or Poly(I:C) to induce high expression of different chemokines and cytokines including interleukin (IL)-12p70 in human cord blood and blood cDC subsets in a combination-specific manner. Importantly, both combinations induced the activation of cDC subsets in patient tumor samples ex vivo. The expression of immunostimulatory genes important for anticancer responses including CD40, IFNB1, IFNL1, IL12A and IL12B were upregulated on stimulation. Furthermore, chemokines associated with CD8+ T cell recruitment were induced in tumor-derived cDCs in response to TL8-506 combinations. In vitro activation and migration assays confirmed that stimulated cDCs induce T cell activation and migration. CONCLUSIONS: Our data suggest that cord blood-derived and blood-derived cDCs are a good surrogate to study treatment responses in human tumor cDCs. While most cDCs in human tumors display a non-activated phenotype, TL8-506 combinations drive human tumor cDCs towards an immunostimulatory phenotype associated with Th1 responses on stimulation. Hence, TL8-506-based combinations may be promising candidates to initiate or boost antitumor responses in patients with cancer.

Besca, a single-cell transcriptomics analysis toolkit to accelerate translational research.

Single-cell RNA sequencing (scRNA-seq) revolutionized our understanding of disease biology. The promise it presents to also transform translational research requires highly standardized and robust software workflows. Here, we present the toolkit Besca, which streamlines scRNA-seq analyses and their use to deconvolute bulk RNA-seq data according to current best practices. Beyond a standard workflow covering quality control, filtering, and clustering, two complementary Besca modules, utilizing hierarchical cell signatures and supervised machine learning, automate cell annotation and provide harmonized nomenclatures. Subsequently, the gene expression profiles can be employed to estimate cell type proportions in bulk transcriptomics data. Using multiple, diverse scRNA-seq datasets, some stemming from highly heterogeneous tumor tissue, we show how Besca aids acceleration, interoperability, reusability and interpretability of scRNA-seq data analyses, meeting crucial demands in translational research and beyond.

LncRNA Ctcflos orchestrates transcription and alternative splicing in thermogenic adipogenesis.

The recruitment of thermogenic brite adipocytes within white adipose tissue attenuates obesity and metabolic comorbidities, arousing interest in understanding the underlying regulatory mechanisms. The molecular network of brite adipogenesis, however, remains largely unresolved. In this light, long noncoding RNAs (lncRNAs) emerged as a versatile class of modulators that control many steps within the differentiation machinery. Leveraging the naturally varying propensities of different inbred mouse strains for white adipose tissue browning, we identify the nuclear lncRNA Ctcflos as a pivotal orchestrator of thermogenic gene expression during brite adipocyte differentiation. Mechanistically, Ctcflos acts as a pleiotropic regulator, being essential for the transcriptional recruitment of the early core thermogenic regulatory program and the modulation of alternative splicing to drive brite adipogenesis. This is showcased by Ctcflos-regulated gene transcription and splicing of the key browning factor Prdm16 toward the isoform that is specific for the thermogenic gene program. Conclusively, our findings emphasize the mechanistic versatility of lncRNAs acting at several independent levels of gene expression for effective regulation of key differentiation factors to direct cell fate and function.

Systems-Genetics-Based Inference of a Core Regulatory Network Underlying White Fat Browning.

Recruitment of brite/beige cells, known as browning of white adipose tissue (WAT), is an efficient way to turn an energy-storing organ into an energy-dissipating one and may therefore be of therapeutic value in combating obesity. However, a comprehensive understanding of the regulatory mechanisms mediating WAT browning is still lacking. Here, we exploit the large natural variation in WAT browning propensity between inbred mouse strains to gain an inclusive view of the core regulatory network coordinating this cellular process. Combining comparative transcriptomics, perturbation-based validations, and gene network analyses, we present a comprehensive gene regulatory network of inguinal WAT browning, revealing up to four distinct regulatory modules with key roles for uncovered transcriptional factors, while also providing deep insights into the genetic architecture of brite adipogenesis. The presented findings therefore greatly increase our understanding of the molecular drivers mediating the intriguing cellular heterogeneity and plasticity of adipose tissue.

ZFP30 promotes adipogenesis through the KAP1-mediated activation of a retrotransposon-derived Pparg2 enhancer.

Krüppel-associated box zinc finger proteins (KZFPs) constitute the largest family of mammalian transcription factors, but most remain completely uncharacterized. While initially proposed to primarily repress transposable elements, recent reports have revealed that KFZPs contribute to a wide variety of other biological processes. Using murine and human in vitro and in vivo models, we demonstrate here that one poorly studied KZFP, ZFP30, promotes adipogenesis by directly targeting and activating a retrotransposon-derived Pparg2 enhancer. Through mechanistic studies, we further show that ZFP30 recruits the co-regulator KRAB-associated protein 1 (KAP1), which, surprisingly, acts as a ZFP30 co-activator in this adipogenic context. Our findings provide an understanding of both adipogenic and KZFP-KAP1 complex-mediated gene regulation, showing that the KZFP-KAP1 axis can also function in a non-repressive manner.

Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell.

Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

A stromal cell population that inhibits adipogenesis in mammalian fat depots.

Adipocyte development and differentiation have an important role in the aetiology of obesity and its co-morbidities1,2. Although multiple studies have investigated the adipogenic stem and precursor cells that give rise to mature adipocytes3-14, our understanding of their in vivo origin and properties is incomplete2,15,16. This is partially due to the highly heterogeneous and unstructured nature of adipose tissue depots17, which has proven difficult to molecularly dissect using classical approaches such as fluorescence-activated cell sorting and Cre-lox lines based on candidate marker genes16,18. Here, using the resolving power of single-cell transcriptomics19 in a mouse model, we reveal distinct subpopulations of adipose stem and precursor cells in the stromal vascular fraction of subcutaneous adipose tissue. We identify one of these subpopulations as CD142+ adipogenesis-regulatory cells, which can suppress adipocyte formation in vivo and in vitro in a paracrine manner. We show that adipogenesis-regulatory cells are refractory to adipogenesis and that they are functionally conserved in humans. Our findings point to a potentially critical role for adipogenesis-regulatory cells in modulating adipose tissue plasticity, which is linked to metabolic control, differential insulin sensitivity and type 2 diabetes.

Reversible De-differentiation of Mature White Adipocytes into Preadipocyte-like Precursors during Lactation.

Adipose tissue in the mammary gland undergoes dramatic remodeling during reproduction. Adipocytes are replaced by mammary alveolar structures during pregnancy and lactation, then reappear upon weaning. The fate of the original adipocytes during lactation and the developmental origin of the re-appearing adipocyte post involution are unclear. Here, we reveal that adipocytes in the mammary gland de-differentiate into Pdgfrα+ preadipocyte- and fibroblast-like cells during pregnancy and remain de-differentiated during lactation. Upon weaning, de-differentiated fibroblasts proliferate and re-differentiate into adipocytes. This cycle occurs over multiple pregnancies. These observations reveal the potential of terminally differentiated adipocytes to undergo repeated cycles of de-differentiation and re-differentiation in a physiological setting.

Cross-Tissue Identification of Somatic Stem and Progenitor Cells Using a Single-Cell RNA-Sequencing Derived Gene Signature.

A long-standing question in biology is whether multipotent somatic stem and progenitor cells (SSPCs) feature molecular properties that could guide their system-independent identification. Population-based transcriptomic studies have so far not been able to provide a definite answer, given the rarity and heterogeneous nature of these cells. Here, we exploited the resolving power of single-cell RNA-sequencing to develop a computational model that is able to accurately distinguish SSPCs from differentiated cells across tissues. The resulting classifier is based on the combined expression of 23 genes including known players in multipotency, proliferation, and tumorigenesis, as well as novel ones, such as Lcp1 and Vgll4 that we functionally validate in intestinal organoids. We show how this approach enables the identification of stem-like cells in still ambiguous systems such as the pancreas and the epidermis as well as the exploration of lineage commitment hierarchies, thus facilitating the study of biological processes such as cellular differentiation, tissue regeneration, and cancer. Stem Cells 2017;35:2390-2402.

ASAP: a web-based platform for the analysis and interactive visualization of single-cell RNA-seq data.

MOTIVATION: Single-cell RNA-sequencing (scRNA-seq) allows whole transcriptome profiling of thousands of individual cells, enabling the molecular exploration of tissues at the cellular level. Such analytical capacity is of great interest to many research groups in the world, yet these groups often lack the expertise to handle complex scRNA-seq datasets. RESULTS: We developed a fully integrated, web-based platform aimed at the complete analysis of scRNA-seq data post genome alignment: from the parsing, filtering and normalization of the input count data files, to the visual representation of the data, identification of cell clusters, differentially expressed genes (including cluster-specific marker genes), and functional gene set enrichment. This Automated Single-cell Analysis Pipeline (ASAP) combines a wide range of commonly used algorithms with sophisticated visualization tools. Compared with existing scRNA-seq analysis platforms, researchers (including those lacking computational expertise) are able to interact with the data in a straightforward fashion and in real time. Furthermore, given the overlap between scRNA-seq and bulk RNA-seq analysis workflows, ASAP should conceptually be broadly applicable to any RNA-seq dataset. As a validation, we demonstrate how we can use ASAP to simply reproduce the results from a single-cell study of 91 mouse cells involving five distinct cell types. AVAILABILITY AND IMPLEMENTATION: The tool is freely available at asap.epfl.ch and R/Python scripts are available at github.com/DeplanckeLab/ASAP. CONTACT: bart.deplancke@epfl.ch. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dissecting the brown adipogenic regulatory network using integrative genomics.

Brown adipocytes regulate energy expenditure via mitochondrial uncoupling, which makes them attractive therapeutic targets to tackle obesity. However, the regulatory mechanisms underlying brown adipogenesis are still poorly understood. To address this, we profiled the transcriptome and chromatin state during mouse brown fat cell differentiation, revealing extensive gene expression changes and chromatin remodeling, especially during the first day post-differentiation. To identify putatively causal regulators, we performed transcription factor binding site overrepresentation analyses in active chromatin regions and prioritized factors based on their expression correlation with the bona-fide brown adipogenic marker Ucp1 across multiple mouse and human datasets. Using loss-of-function assays, we evaluated both the phenotypic effect as well as the transcriptomic impact of several putative regulators on the differentiation process, uncovering ZFP467, HOXA4 and Nuclear Factor I A (NFIA) as novel transcriptional regulators. Of these, NFIA emerged as the regulator yielding the strongest molecular and cellular phenotypes. To examine its regulatory function, we profiled the genomic localization of NFIA, identifying it as a key early regulator of terminal brown fat cell differentiation.

Topoisomerase II beta interacts with cohesin and CTCF at topological domain borders.

BACKGROUND: Type II DNA topoisomerases (TOP2) regulate DNA topology by generating transient double stranded breaks during replication and transcription. Topoisomerase II beta (TOP2B) facilitates rapid gene expression and functions at the later stages of development and differentiation. To gain new insight into the genome biology of TOP2B, we used proteomics (BioID), chromatin immunoprecipitation, and high-throughput chromosome conformation capture (Hi-C) to identify novel proximal TOP2B protein interactions and characterize the genomic landscape of TOP2B binding at base pair resolution. RESULTS: Our human TOP2B proximal protein interaction network included members of the cohesin complex and nucleolar proteins associated with rDNA biology. TOP2B associates with DNase I hypersensitivity sites, allele-specific transcription factor (TF) binding, and evolutionarily conserved TF binding sites on the mouse genome. Approximately half of all CTCF/cohesion-bound regions coincided with TOP2B binding. Base pair resolution ChIP-exo mapping of TOP2B, CTCF, and cohesin sites revealed a striking structural ordering of these proteins along the genome relative to the CTCF motif. These ordered TOP2B-CTCF-cohesin sites flank the boundaries of topologically associating domains (TADs) with TOP2B positioned externally and cohesin internally to the domain loop. CONCLUSIONS: TOP2B is positioned to solve topological problems at diverse cis-regulatory elements and its occupancy is a highly ordered and prevalent feature of CTCF/cohesin binding sites that flank TADs.

GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer.

BACKGROUND: Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have undergone an EMT. RESULTS: Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3 expression correlates with EMT markers and is prognostic of poor overall survival. CONCLUSIONS: Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy for the treatment of patients with poorly differentiated tumors.

Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network.

Adipose tissue is a key determinant of whole body metabolism and energy homeostasis. Unraveling the regulatory mechanisms underlying adipogenesis is therefore highly relevant from a biomedical perspective. Our current understanding of fat cell differentiation is centered on the transcriptional cascades driven by the C/EBP protein family and the master regulator PPARγ. To elucidate further components of the adipogenic gene regulatory network, we performed a large-scale transcription factor (TF) screen overexpressing 734 TFs in mouse pre-adipocytes and probed their effect on differentiation. We identified 22 novel pro-adipogenic TFs and characterized the top ranking TF, ZEB1, as being essential for adipogenesis both in vitro and in vivo. Moreover, its expression levels correlate with fat cell differentiation potential in humans. Genomic profiling further revealed that this TF directly targets and controls the expression of most early and late adipogenic regulators, identifying ZEB1 as a central transcriptional component of fat cell differentiation.

CAST-ChIP maps cell-type-specific chromatin states in the Drosophila central nervous system.

Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP), a broadly applicable biochemical procedure. RNA polymerase II (Pol II) CAST-ChIP identifies ~1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophila brain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain.