Epigenomic tomography for probing spatially defined chromatin state in the brain.

Spatially resolved epigenomic profiling is critical for understanding biology in the mammalian brain. Single-cell spatial epigenomic assays were developed recently for this purpose, but they remain costly and labor intensive for examining brain tissues across substantial dimensions and surveying a collection of brain samples. Here, we demonstrate an approach, epigenomic tomography, that maps spatial epigenomes of mouse brain at the scale of centimeters. We individually profiled neuronal and glial fractions of mouse neocortex slices with 0.5 mm thickness. Tri-methylation of histone 3 at lysine 27 (H3K27me3) or acetylation of histone 3 at lysine 27 (H3K27ac) features across these slices were grouped into clusters based on their spatial variation patterns to form epigenomic brain maps. As a proof of principle, our approach reveals striking dynamics in the frontal cortex due to kainic-acid-induced seizure, linked with transmembrane ion transporters, exocytosis of synaptic vesicles, and secretion of neurotransmitters. Epigenomic tomography provides a powerful and cost-effective tool for characterizing brain disorders based on the spatial epigenome.

nMOWChIP-seq: low-input genome-wide mapping of non-histone targets.

Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach produces high-quality binding profiles using 1,000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.

Cell-type-specific epigenomic variations associated with BRCA1 mutation in pre-cancer human breast tissues.

BRCA1 germline mutation carriers are predisposed to breast cancers. Epigenomic regulations have been known to strongly interact with genetic variations and potentially mediate biochemical cascades involved in tumorigenesis. Due to the cell-type specificity of epigenomic features, profiling of individual cell types is critical for understanding the molecular events in various cellular compartments within complex breast tissue. Here, we produced cell-type-specific profiles of genome-wide histone modifications including H3K27ac and H3K4me3 in basal, luminal progenitor, mature luminal and stromal cells extracted from a small pilot cohort of pre-cancer BRCA1 mutation carriers (BRCA1mut/+ ) and non-carriers (BRCA1+/+ ), using a low-input ChIP-seq technology that we developed. We discovered that basal and stromal cells present the most extensive epigenomic differences between mutation carriers (BRCA1mut/+ ) and non-carriers (BRCA1+/+ ), while luminal progenitor and mature luminal cells are relatively unchanged with the mutation. Furthermore, the epigenomic changes in basal cells due to BRCA1 mutation appear to facilitate their transformation into luminal progenitor cells. Taken together, epigenomic regulation plays an important role in the case of BRCA1 mutation for shaping the molecular landscape that facilitates tumorigenesis.

Epigenomic and transcriptomic analyses reveal differences between low-grade inflammation and severe exhaustion in LPS-challenged murine monocytes.

Emerging studies suggest that monocytes can be trained by bacterial endotoxin to adopt distinct memory states ranging from low-grade inflammation to immune exhaustion. While low-grade inflammation may contribute to the pathogenesis of chronic diseases, exhausted monocytes with pathogenic and immune-suppressive characteristics may underlie the pathogenesis of polymicrobial sepsis including COVID-19. However, detailed processes by which the dynamic adaption of monocytes occur remain poorly understood. Here we exposed murine bone-marrow derived monocytes to chronic lipopolysaccharide (LPS) stimulation at low-dose or high-dose, as well as a PBS control. The cells were profiled for genome-wide H3K27ac modification and gene expression. The gene expression of TRAM-deficient and IRAK-M-deficient monocytes with LPS exposure was also analyzed. We discover that low-grade inflammation preferentially utilizes the TRAM-dependent pathway of TLR4 signaling, and induces the expression of interferon response genes. In contrast, high dose LPS uniquely upregulates exhaustion signatures with metabolic and proliferative pathways. The extensive differences in the epigenomic landscape between low-dose and high-dose conditions suggest the importance of epigenetic regulations in driving differential responses. Our data provide potential targets for future mechanistic or therapeutic studies.

Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.

Clinical evidence suggests that rapid and sustained antidepressant action can be attained with a single exposure to psychedelics. However, the biological substrates and key mediators of psychedelics' enduring action remain unknown. Here, we show that a single administration of the psychedelic DOI produces fast-acting effects on frontal cortex dendritic spine structure and acceleration of fear extinction via the 5-HT2A receptor. Additionally, a single dose of DOI leads to changes in chromatin organization, particularly at enhancer regions of genes involved in synaptic assembly that stretch for days after the psychedelic exposure. These DOI-induced alterations in the neuronal epigenome overlap with genetic loci associated with schizophrenia, depression, and attention deficit hyperactivity disorder. Together, these data support that epigenomic-driven changes in synaptic plasticity sustain psychedelics' long-lasting antidepressant action but also warn about potential substrate overlap with genetic risks for certain psychiatric conditions.

Multiplexed and Ultralow-Input ChIP-seq Enabled by Tagmentation-Based Indexing and Facile Microfluidics.

Epigenome constitutes an important layer that regulates gene expression and dynamics during development and diseases. Extensive efforts have been made to develop epigenome profiling methods using a low number of cells and with high throughput. Chromatin immunoprecipitation (ChIP) is the most important approach for profiling genome-wide epigenetic changes such as histone modifications. In this report, we demonstrate microfluidic ChIPmentation (mu-CM), a microfluidic technology that enables profiling cell samples that individually do not generate enough ChIP DNA for sequencing library preparation. We used a simple microfluidic device to allow eight samples to be processed simultaneously. The samples were indexed differently using a tagmentation-based approach (ChIPmentation) and then merged for library preparation. A histone modification profile for each individual sample was obtained by demultiplexing the sequencing reads based on the indexes. Our technology allowed profiling 20 cells and is well suited for cell-type-specific studies using low-abundance tissues.

Microfluidic Platform for Next-Generation Sequencing Library Preparation with Low-Input Samples.

Advances in next-generation sequencing (NGS) have made available a wealth of information that had previously been inaccessible to researchers and clinicians. NGS has been applied to understand genomic, transcriptomic, and epigenomic changes and gained traction as a significant tool capable of accelerating diagnosis, prognosis, and biomarker discovery. However, these NGS assays have yet to be practical methods for patient stratification or diagnosis because of the gap between the tiny quantities of biomaterials provided by a clinical sample and the large DNA input required by most of these assays. Current library preparation methodologies typically require large input amounts of DNA and a long and complicated manual process. Here, we present a microfluidic droplet-based system for NGS library preparation, capable of reducing the number of pipetting steps significantly, reducing reagent consumption by 10×, and automating much of the process, while supporting an extremely low DNA input requirement (10 pg per library). This semiautomated technology will allow for low-input preparations of 8 libraries simultaneously while reducing batch-to-batch variation and operator hands-on time.

Effects of culture condition on epigenomic profiles of brain tumor cells.

Personalized cancer medicine offers the promise of more effective treatments that are tailored to an individual's own dynamic cancer phenotype. Meanwhile, tissue-engineering approaches to modeling tumors may complement these advances by providing a powerful new approach to understanding the adaptation dynamics occurring during treatment. However, in both of these areas new tools will be required to gain a full picture of the genetic and epigenetic regulators of phenotype dynamics occurring in the small populations of cells that drive resistance. In this study, we perform epigenomic analysis of brain tumor cells that are collected from micro-engineered three-dimensional tumor models, overcoming the challenges associated with the small numbers of cells contained within these micro-tissue niches, in this case collecting ~1,000 cells per sample. Specifically, we use a high-resolution epigenomic analysis method known as microfluidic-oscillatory-washing-based chromatin immunoprecipitation with sequencing (MOWChIP-seq) to analyze histone methylation patterns (H3K4me3). We identified gene loci that are associated with the H3K4me3 modification, which is generally a mark of active transcription. We compared methylation patterns in standard 2D cultures and 3D cultures based on type I collagen hydrogels, under both normoxic and hypoxic conditions. We found that culture dimensionality drastically impacted the H3k4me3 profile and resulted in differential modifications in response to hypoxic stress. Differentially H3K4me3-marked regions under the culture conditions used in this study have important implications for gene expression differences that have been previously observed. In total, our work illustrates a direct connection between cell culture or tissue niche condition and genome-wide alterations in histone modifications, providing the first steps towards analyzing the spatiotemporal variations in epigenetic regulation of cancer cell phenotypes. This study, to our knowledge, also represents the first time broad-spectrum epigenomic analysis has been applied to small cell samples collected from engineered micro-tissues.

MOWChIP-seq for low-input and multiplexed profiling of genome-wide histone modifications.

Epigenetic mechanisms such as histone modifications play critical roles in adaptive tuning of chromatin structures. Profiling of various histone modifications at the genome scale using tissues from animal and human samples is an important step for functional studies of epigenomes and epigenomics-based precision medicine. Because the profile of a histone mark is highly specific to a cell type, cell isolation from tissues is often necessary to generate a homogeneous cell population, and such operations tend to yield a low number of cells. In addition, high-throughput processing is often desirable because of the multiplexity of histone marks of interest and the large quantity of samples in a hospital setting. In this protocol, we provide detailed instructions for device fabrication, setup, and operation of microfluidic oscillatory washing-based chromatin immunoprecipitation followed by sequencing (MOWChIP-seq) for profiling of histone modifications using as few as 100 cells per assay with a throughput as high as eight assays in one run. MOWChIP-seq operation involves flowing of chromatin fragments through a packed bed of antibody-coated beads, followed by vigorous microfluidic oscillatory washing. Our process is semi-automated to reduce labor and improve reproducibility. Using one eight-unit device, it takes 2 d to produce eight sequencing libraries from chromatin samples. The technology is scalable. We used the protocol to study a number of histone modifications in various types of mouse and human tissues. The protocol can be conducted by a user who is familiar with molecular biology procedures and has basic engineering skills.

Microfluidic epigenomic mapping technologies for precision medicine.

Epigenomic mapping of tissue samples generates critical insights into genome-wide regulations of gene activities and expressions during normal development and disease processes. Epigenomic profiling using a low number of cells produced by patient and mouse samples presents new challenges to biotechnologists. In this review, we first discuss the rationale and premise behind profiling epigenomes for precision medicine. We then examine the existing literature on applying microfluidics to facilitate low-input and high-throughput epigenomic profiling, with emphasis on technologies enabling interfacing with next-generation sequencing. We detail assays on studies of histone modifications, DNA methylation, 3D chromatin structures and non-coding RNAs. Finally, we discuss what the future may hold in terms of method development and translational potential.

On-chip manufacturing of synthetic proteins for point-of-care therapeutics.

Therapeutic proteins have recently received increasing attention because of their clinical potential. Currently, most therapeutic proteins are produced on a large scale using various cell culture systems. However, storing and transporting these therapeutic proteins at low temperatures makes their distribution expensive and problematic, especially for applications in remote locations. To this end, an emerging solution is to use point-of-care technologies that enable immediate and accessible protein production at or near the patient's bedside. Here we present the development of "Therapeutics-On-a-Chip (TOC)", an integrated microfluidic platform that enables point-of-care synthesis and purification of therapeutic proteins. We used fresh and lyophilized materials for cell-free synthesis of therapeutic proteins on microfluidic chips and applied immunoprecipitation for highly efficient, on-chip protein purification. We first demonstrated this approach by expressing and purifying a reporter protein, green fluorescent protein. Next, we used TOC to produce cecropin B, an antimicrobial peptide that is widely used to control biofilm-associated diseases. We successfully synthesized and purified cecropin B at 63 ng/µl within 6 h with a 92% purity, followed by confirming its antimicrobial functionality using a growth inhibition assay. Our TOC technology provides a new platform for point-of-care production of therapeutic proteins at a clinically relevant quantity.

Recent advances in the use of microfluidic technologies for single cell analysis.

The inherent heterogeneity in cell populations has become of great interest and importance as analytical techniques have improved over the past decades. With the advent of personalized medicine, understanding the impact of this heterogeneity has become an important challenge for the research community. Many different microfluidic approaches with varying levels of throughput and resolution exist to study single cell activity. In this review, we take a broad view of the recent microfluidic developments in single cell analysis based on microwell, microchamber, and droplet platforms. We cover physical, chemical, and molecular biology approaches for cellular and molecular analysis including newly emerging genome-wide analysis.