Moringa isothiocyanate-1 inhibits LPS-induced inflammation in mouse myoblasts and skeletal muscle.

This study aims to investigate the anti-inflammatory effects of moringa isothiocyanate-1 (MIC-1) extracted from seeds of Moringa oleifera Lam. in lipopolysaccharide (LPS)-induced inflammation models. MIC-1 decreased nitric oxide production and reduced the expression of pro-inflammatory markers (TNF-α, Ifn-α, IL-1ß, IL-6) in C2C12 myoblasts. The daily oral treatment of MIC-1 (80 mg/kg) for three days significantly reduced the expression of pro-inflammatory markers in gastrocnemius muscle tissue of LPS-treated C57BL/6 male mice. Transcriptomic analysis provided further insights into the inhibitory effects of MIC-1 on the LPS-induced inflammation, which suggested that MIC-1 affects inflammation and immunity-related genes in myoblasts and skeletal muscle tissue. MIC-1 inhibited the nuclear accumulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-treated myoblasts. Our data support the hypothesis that the MIC-1's effects in the muscle cells are mediated through the inhibition of the NF-κB translocation in the nucleus, which, in turn, results in immunomodulating and anti-inflammatory responses at the gene expression levels.

Moringa isothiocyanate-1 regulates Nrf2 and NF-κB pathway in response to LPS-driven sepsis and inflammation.

This study aims to document the dual mode of pharmacological action of moringa isothiocyanate-1 (MIC-1) derived from seeds of Moringa oleifera Lam. Oral administration of chemically stable MIC-1 (80 mg/kg) significantly reduced the expression of inflammatory markers (Tnf-α, Ifn-α, IL-1ß, IL-6) in the liver, kidney, spleen, and colon and decreased spleen weight in the lipopolysaccharide (LPS)-induced sepsis / acute inflammation model in mice. Transcriptomic analysis of the effect of MIC-1 on the liver and in the LPS-induced RAW264.7 murine macrophage showed that MIC-1 decreases inflammation via inflammation, immunity, and oxidative stress pathways. These results are supported by the immunocytochemical observations that MIC-1 increased the nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor and decreased the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-induced macrophages. Transcriptional activation of antioxidant genes by MIC-1 translated into a reduction of reactive oxygen species (ROS) in the cytoplasm, decrease of mitochondrial superoxide content, and restoration of the mitochondrial membrane potential in the LPS-induced macrophages. Our data indicate that MIC-1 affects inflammation and oxidative stress, two key processes involved in the etiology of many chronic diseases. These effects involve upstream regulation of two key transcriptional factors regulating responses to these processes at a gene expression level.

PARP1-dependent eviction of the linker histone H1 mediates immediate early gene expression during neuronal activation.

Neuronal stimulation leads to immediate early gene (IEG) expression through calcium-dependent mechanisms. In recent years, considerable attention has been devoted to the transcriptional responses after neuronal stimulation, but relatively little is known about the changes in chromatin dynamics that follow neuronal activation. Here, we use fluorescence recovery after photobleaching, biochemical fractionations, and chromatin immunoprecipitation to show that KCl-induced depolarization in primary cultured cortical neurons causes a rapid release of the linker histone H1 from chromatin, concomitant with IEG expression. H1 release is repressed by PARP inhibition, PARP1 deletion, a non-PARylatable H1, as well as phosphorylation inhibitions and a nonphosphorylatable H1, leading to hindered IEG expression. Further, H1 is replaced by PARP1 on IEG promoters after neuronal stimulation, and PARP inhibition blocks this reciprocal binding response. Our results demonstrate the relationship between neuronal excitation and chromatin plasticity by identifying the roles of polyadenosine diphosphate ribosylation and phosphorylation of H1 in regulating H1 chromatin eviction and IEG expression in stimulated neurons.

Alternative SET/TAFI Promoters Regulate Embryonic Stem Cell Differentiation.

Embryonic stem cells (ESCs) are regulated by pluripotency-related transcription factors in concert with chromatin regulators. To identify additional stem cell regulators, we screened a library of endogenously labeled fluorescent fusion proteins in mouse ESCs for fluorescence loss during differentiation. We identified SET, which displayed a rapid isoform shift during early differentiation from the predominant isoform in ESCs, SETα, to the primary isoform in differentiated cells, SETß, through alternative promoters. SETα is selectively bound and regulated by pluripotency factors. SET depletion causes proliferation slowdown and perturbed neuronal differentiation in vitro and developmental arrest in vivo, and photobleaching methods demonstrate SET's role in maintaining a dynamic chromatin state in ESCs. This work identifies an important regulator of pluripotency and early differentiation, which is controlled by alternative promoter usage.

The regulatory niche of intestinal stem cells.

The niche constitutes a unique category of cells that support the microenvironment for the maintenance and self-renewal of stem cells. Intestinal stem cells reside at the base of the crypt, which contains adjacent epithelial cells, stromal cells and smooth muscle cells, and soluble and cell-associated growth and differentiation factors. We summarize here recent advances in our understanding of the crucial role of the niche in regulating stem cells. The stem cell niche maintains a balance among quiescence, proliferation and regeneration of intestinal stem cells after injury. Mesenchymal cells, Paneth cells, immune cells, endothelial cells and neural cells are important regulatory components that secrete niche ligands, growth factors and cytokines. Intestinal homeostasis is regulated by niche signalling pathways, specifically Wnt, bone morphogenetic protein, Notch and epidermal growth factor. These insights into the regulatory stem cell niche during homeostasis and post-injury regeneration offer the potential to accelerate development of therapies for intestine-related disorders.

Stem Cells Matter in Response to Fasting.

The molecular processes underlying intestinal adaptation to fasting and re-feeding remain largely uncharacterized. In this issue of Cell Reports, Richmond et al. report that dormant intestinal stem cells are regulated by PTEN and nutritional status.

Heterochromatin Protein 1ß (HP1ß) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells.

BACKGROUND: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. RESULTS: Here we identify Heterochromatin Protein 1ß (HP1ß) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1ß is differentially localized and differentially associated with chromatin. Deletion of HP1ß, but not HP1α, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1ß has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1ß in ESCs. The minor fraction of HP1ß that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. CONCLUSIONS: We demonstrate an unexpected duality in the role of HP1ß: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1ß function both depends on, and regulates, the pluripotent state.

Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.

Multilayered chromatin analysis reveals E2f, Smad and Zfx as transcriptional regulators of histones.

Histones, the building blocks of eukaryotic chromatin, are essential for genome packaging, function and regulation. However, little is known about their transcriptional regulation. Here we conducted a comprehensive computational analysis, based on chromatin immunoprecipitation-sequencing and -microarray analysis (ChIP-seq and ChIP-chip) data of over 50 transcription factors and histone modifications in mouse embryonic stem cells. Enrichment scores supported by gene expression data from gene knockout studies identified E2f1 and E2f4 as master regulators of histone genes, CTCF and Zfx as repressors of core and linker histones, respectively, and Smad1, Smad2, YY1 and Ep300 as restricted or cell type-specific regulators. We propose that histone gene regulation is substantially more complex than previously thought, and that a combination of factors orchestrate histone gene regulation, from strict synchronization with S phase to targeted regulation of specific histone subtypes.

Stress-induced epigenetic transcriptional memory of acetylcholinesterase by HDAC4.

Stress induces long-lasting changes in neuronal gene expression and cholinergic neurotransmission, but the underlying mechanism(s) are incompletely understood. Here, we report that chromatin structure and histone modifications are causally involved in this transcriptional memory. Specifically, the AChE gene encoding the acetylcholine-hydrolyzing enzyme acetylcholinesterase is known to undergo long-lasting transcriptional and alternative splicing changes after stress. In mice subjected to stress, we identified two alternative 5' exons that were down-regulated after stress in the hippocampus, accompanied by reduced acetylation and elevated trimethylation of H3K9 at the corresponding promoter. These effects were reversed completely by daily administration of the histone deacetylase (HDAC) inhibitor sodium butyrate for 1 wk after stress. H3K9 hypoacetylation was associated with a selective, sodium butyrate-reversible promoter accumulation of HDAC4. Hippocampal suppression of HDAC4 in vivo completely abolished the long-lasting AChE-related and behavioral stress effects. Our findings demonstrate long-lasting stress-inducible changes in AChE's promoter choices, identify the chromatin changes that support this long-term transcriptional memory, and reveal HDAC4 as a mediator of these effects in the hippocampus.

Residual expression of reprogramming factors affects the transcriptional program and epigenetic signatures of induced pluripotent stem cells.

Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating viral vectors has been widely employed to generate induced pluripotent stem cell (iPSC) lines from both normal and disease-specific somatic tissues, providing an invaluable resource for medical research and drug development. Residual reprogramming transgene expression from integrated viruses nevertheless alters the biological properties of iPSCs and has been associated with a reduced developmental competence both in vivo and in vitro. We performed transcriptional profiling of mouse iPSC lines before and after excision of a polycistronic lentiviral reprogramming vector to systematically define the overall impact of persistent transgene expression on the molecular features of iPSCs. We demonstrate that residual expression of the Yamanaka factors prevents iPSCs from acquiring the transcriptional program exhibited by embryonic stem cells (ESCs) and that the expression profiles of iPSCs generated with and without c-Myc are indistinguishable. After vector excision, we find 36% of iPSC clones show normal methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON) vs Gtl2(OFF) iPSC clones. Our findings provide new insights into the reprogramming process and emphasize the importance of generating iPSCs free of any residual transgene expression.

Chromatin immunoprecipitation in mouse hippocampal cells and tissues.

Chromatin immunoprecipitation (ChIP) has been developed for studying protein-DNA interactions and has been extensively used for mapping the localization of posttranslationally modified histones, histone variants, transcription factors, or chromatin modifying enzymes at a given locus or on a genome-wide scale. ChIP methods have been modified and improved over the years to fit a variety of different cell types and tissues. Here, we present a detailed protocol for hippocampal ChIP, of both minced tissue and enzyme-separated hippocampal cells. This protocol enables to study chromatin-protein interactions in a specified population of hippocampal cells, allowing to study chromatin regulation in the central nervous system in a variety of conditions and disorders. Our assay has been developed for histone modifications but is suited for any chromatin binding protein for which specific ChIP-grade antibodies are available.

Pluripotency-related, valproic acid (VPA)-induced genome-wide histone H3 lysine 9 (H3K9) acetylation patterns in embryonic stem cells.

Embryonic stem cell (ESC) chromatin is characterized by a unique set of histone modifications, including enrichment for H3 lysine 9 acetylation (H3K9ac). Recent studies suggest that histone deacetylase (HDAC) inhibitors promote pluripotency. Here, using H3K9ac ChIP followed by high throughput sequencing analyses and gene expression in E14 mouse ESCs before and after treatment with a low level of the HDAC inhibitor valproic acid, we show that H3K9ac is enriched at gene promoters and is highly correlated with gene expression and with various genomic features, including different active histone marks and pluripotency-related transcription factors. Curiously, it predicts the cellular location of gene products. Treatment of ESCs with valproic acid leads to a pervasive genome-wide and time-dependent increase in H3K9ac, but this increase is selectively suppressed after 4 h in H3K4me3/H3K27me3 bivalent genes. H3K9ac increase is dependent on the promoter's chromatin state and is affected by the binding of P300, various transcription factors, and active histone marks. This study provides insights into the genomic response of ESCs to a low level of HDAC inhibitor, which leads to increased pluripotency. The results suggest that a mild (averaging less than 40%) but global change in the chromatin state is involved in increased pluripotency and that specific mechanisms operate selectively in bivalent genes to maintain constant H3K9ac levels. Our data support the notion that H3K9ac has an important role in ESC biology.

Sanguinarine interacts with chromatin, modulates epigenetic modifications, and transcription in the context of chromatin.

DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.