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Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications. The authors describe detailed procedures for an epigenomic profiling method suitable for low-input samples that is based on in situ labeling with an oligonucleotide-conjugated antibody.

Key Points O -GlcNAcylation is a nutrient- and stress-responsive post-translational modification (PTM) that involves the attachment of O -linked N -acetylglucosamine moieties to Ser and Thr residues of cytoplasmic, nuclear and mitochondrial proteins. A single pair of enzymes — O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA) — controls the dynamic cycling of this PTM. Potential mechanisms that enable a single OGT enzyme to recognize hundreds of protein substrates include substrate-specific interactions with the tetratricopeptide repeat (TPR) domain of OGT and context-dependent recruitment of OGT to its substrates by a hierarchy of conserved adaptor proteins. Furthermore, in response to cellular stress, O -GlcNAcylation may occur nonspecifically in unstructured regions of unfolded proteins in order to block their aggregation and degradation and facilitate their refolding. O -GlcNAcylation is involved in the spatiotemporal regulation of diverse cellular processes, which include transcription, epigenetic modifications and cell signalling dynamics. O -GlcNAcylation is highly dynamic and often transient, but the mechanisms underlying the temporal control of O -GlcNAc signalling are largely unknown. Nutrient availability regulates cellular O -GlcNAcylation levels not only by determining the abundance of the donor substrate uridine diphosphate GlcNAc (UDP-GlcNAc) but also by modulating the levels of OGT, OGA and their respective adaptor proteins and substrates. Hormones such as insulin, glucagon and ghrelin are secreted in response to systemic metabolic changes and modulate O -GlcNAc signalling in specific cell types and tissues to regulate key response pathways that help maintain metabolic homeostasis. Cellular O -GlcNAcylation levels may be maintained within an 'optimal zone' by a 'buffering system' that is generated by mutual regulation of OGT and OGA at the transcriptional and post-translational levels. Maintenance of O -GlcNAc homeostasis is essential for optimal cellular function, and disruption of the cellular O -GlcNAcylation 'buffer' may contribute to the pathogenesis of various human diseases. O -GlcNAcylation can be viewed as the essential 'grease and glue' of the cell: it acts as a 'grease' by coating target proteins (folded or unfolded, mature or nascent) and preventing unwanted protein aggregation or modification; it also acts as a 'glue' by modulating protein–protein interactions in time and space in response to internal and external cues, thereby affecting the functions of various proteins in the cell. Many cellular proteins are reversibly modified by O -linked N -acetylglucosamine ( O -GlcNAc) moieties on Ser and Thr residues. Studies on the mechanisms and functions of O -GlcNAcylation and its links to metabolism reveal the importance of this modification in the maintenance of cellular and organismal homeostasis. O -GlcNAcylation — the attachment of O -linked N -acetylglucosamine ( O -GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins — is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes — O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA) — controls the dynamic cycling of this protein modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O -GlcNAcylation at levels that range from structural and molecular biology to cell signalling and gene regulation to physiology and disease. New mechanisms and functions of O -GlcNAcylation that are emerging from these recent developments enable us to begin constructing a unified conceptual framework through which the significance of this modification in cellular and organismal physiology can be understood.

Cell walls define the shape of plant cells, controlling the extent and orientation of cell elongation, and hence organ growth. The main load-bearing component of plant cell walls is cellulose, and how plants regulate its biosynthesis during development and in response to various environmental perturbations is a central question in plant biology. Cellulose is synthesized by cellulose synthase (CESA) complexes (CSCs) that are assembled in the Golgi apparatus and then delivered to the plasma membrane (PM), where they actively synthesize cellulose. CSCs travel along cortical microtubule paths that define the orientation of synthesis of the cellulose microfibrils. CSCs recycle between the PM and various intracellular compartments, and this trafficking plays an important role in determining the level of cellulose synthesized. In this review, we summarize recent findings in CESA complex organization, CESA posttranslational modifications and trafficking, and other components that interact with CESAs. We also discuss cell wall integrity maintenance, with a focus on how this impacts cellulose biosynthesis.

Autophagy is a versatile degradation system for maintaining cellular homeostasis whereby cytosolic materials are sequestered in a double-membrane autophagosome and subsequently delivered to lysosomes, where they are broken down. In multicellular organisms, newly formed autophagosomes undergo a process called ‘maturation’, in which they fuse with vesicles originating from endolysosomal compartments, including early/late endosomes and lysosomes, to form amphisomes, which eventually become degradative autolysosomes. This fusion process requires the concerted actions of multiple regulators of membrane dynamics, including SNAREs, tethering proteins and RAB GTPases, and also transport of autophagosomes and late endosomes/lysosomes towards each other. Multiple mechanisms modulate autophagosome maturation, including post-translational modification of key components, spatial distribution of phosphoinositide lipid species on membranes, RAB protein dynamics, and biogenesis and function of lysosomes. Nutrient status and various stresses integrate into the autophagosome maturation machinery to coordinate the progression of autophagic flux. Impaired autophagosome maturation is linked to the pathogenesis of various human diseases, including neurodegenerative disorders, cancer and myopathies. Furthermore, invading pathogens exploit various strategies to block autophagosome maturation, thus evading destruction and even subverting autophagic vacuoles (autophagosomes, amphisomes and autolysosomes) for survival, growth and/or release. Here, we discuss the recent progress in our understanding of the machinery and regulation of autophagosome maturation, the relevance of these mechanisms to human pathophysiology and how they are harnessed by pathogens for their benefit. We also provide perspectives on targeting autophagosome maturation therapeutically.Following their biogenesis, autophagosomes undergo maturation into degradative autolysosomes by fusing with late endosomes/lysosomes. This process — involving an array of molecular regulators of membrane dynamics — is essential for autophagic degradation, and its deregulation can lead to disease, including neurodegeneration, muscle diseases and cancer, and propagation of pathogens.

Neutrophil extracellular traps (NET) formation is part of the neutrophil response to infections, but excessive or inappropriate NETosis may trigger the production of autoantibodies and cause organ damage in autoimmune disorders. Spontaneously netting neutrophils are not frequent and induction of NET in vitro by selected stimuli is necessary to investigate their structure. In the present work, the protein composition and post-translational modifications of NET produced under different stimuli have been studied by means of proteomic analysis. Neutrophils from healthy donors were stimulated by PMA, A23187, Escherichia coli LPS or untreated; after three hours, cells were washed, treated with DNase and supernatants collected for mass spectrometry. Data were analyzed by unsupervised hierarchical clustering analyses. We identified proteins contained in NETs of any source or exclusive of one stimulus: LPS-induced and spontaneous NET diverge in protein composition, while PMA- and A23187-induced NET appear more similar. Among the post-translational modifications we examined, methionine sulfoxidation is frequent especially in PMA- and LPS-induced NETs. Myeloperoxidase is the protein more extensively modified. Thus, proteomic analysis indicates that NETs induced by different stimuli are heterogeneous in terms of both protein composition and post-translational modifications, suggesting that NET induced in different conditions may have different biological effects.

A diverse catalog of long noncoding RNAs (lncRNAs), which lack protein-coding potential, are transcribed from the mammalian genome. They are emerging as important regulators in gene expression networks by controlling nuclear architecture and transcription in the nucleus and by modulating mRNA stability, translation and post-translational modifications in the cytoplasm. In this Review, we highlight recent progress in cellular functions of lncRNAs at the molecular level in mammalian cells. Yao et al. review functions of lncRNAs in controlling chromatin architecture, transcription and nuclear bodies in the nucleus and in modulating mRNA stability, translation and protein modifications in the cytoplasm.

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that participate in a majority of biological processes via regulating target gene expression. The post-transcriptional repression through miRNA seed region binding to 3′ UTR of target mRNA is considered as the canonical mode of miRNA-mediated gene regulation. However, emerging evidence suggests that other regulatory modes exist beyond the canonical mechanism. In particular, the function of intranuclear miRNA in gene transcriptional regulation is gradually revealed, with evidence showing their contribution to gene silencing or activating. Therefore, miRNA-mediated regulation of gene transcription not only expands our understanding of the molecular mechanism underlying miRNA regulatory function, but also provides new evidence to explain its ability in the sophisticated regulation of many bioprocesses. In this review, mechanisms of miRNA-mediated gene transcriptional and post-transcriptional regulation are summarized, and the synergistic effects among these actions which form a regulatory network of a miRNA on its target are particularly elaborated. With these discussions, we aim to emphasize the importance of miRNA regulatory network on target gene regulation and further highlight the potential application of the network mode in the achievement of a more effective and stable modulation of the target gene expression.

Histone-modifying enzymes are implicated in the control of diverse DNA-templated processes including gene expression. Here, we outline historical and current thinking regarding the functions of histone modifications and their associated enzymes. One current viewpoint, based largely on correlative evidence, posits that histone modifications are instructive for transcriptional regulation and represent an epigenetic ‘code’. Recent studies have challenged this model and suggest that histone marks previously associated with active genes do not directly cause transcriptional activation. Additionally, many histone-modifying proteins possess non-catalytic functions that overshadow their enzymatic activities. Given that much remains unknown regarding the functions of these proteins, the field should be cautious in interpreting loss-of-function phenotypes and must consider both cellular and developmental context. In this Perspective, we focus on recent progress relating to the catalytic and non-catalytic functions of the Trithorax–COMPASS complexes, Polycomb repressive complexes and Clr4/Suv39 histone-modifying machineries. Recent progress relating to the catalytic and non-catalytic functions of histone modifying complexes warrants a fresh look at the role of histone modifications and the “histone code” model.

Since their serendipitous discovery in nematodes, microRNAs (miRNAs) have emerged as key regulators of biological processes in animals. These small RNAs form complex networks that regulate cell differentiation, development and homeostasis. Deregulation of miRNA function is associated with an increasing number of human diseases, particularly cancer. Recent discoveries have expanded our understanding of the control of miRNA function. Here, we review the mechanisms that modulate miRNA activity, stability and cellular localization through alternative processing and maturation, sequence editing, post-translational modifications of Argonaute proteins, viral factors, transport from the cytoplasm and regulation of miRNA–target interactions. We conclude by discussing intriguing, unresolved research questions.

Proper cellular functionality and homeostasis are maintained by the convergent integration of various signaling cascades, which enable cells to respond to internal and external changes. The Dbf2-related kinases LATS1 and LATS2 (LATS) have emerged as central regulators of cell fate, by modulating the functions of numerous oncogenic or tumor suppressive effectors, including the canonical Hippo effectors YAP/TAZ, the Aurora mitotic kinase family, estrogen signaling and the tumor suppressive transcription factor p53. While the basic functions of the LATS kinase module are strongly conserved over evolution, the genomic duplication event leading to the emergence of two closely related kinases in higher organisms has increased the complexity of this signaling network. Here, we review the LATS1 and LATS2 intrinsic features as well as their reported cellular activities, emphasizing unique characteristics of each kinase. While differential activities between the two paralogous kinases have been reported, many converge to similar pathways and outcomes. Interestingly, the regulatory networks controlling the mRNA expression pattern of LATS1 and LATS2 differ strongly, and may contribute to the differences in protein binding partners of each kinase and in the subcellular locations in which each kinase exerts its functions.