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 1β (HP1β) 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 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1aα, 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 HP1β 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 HP1β in ESCs. The minor fraction of HP1β 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 HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

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-1β, 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

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-1β, 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

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

Residual Expression of Reprogramming Factors Affects the Transcriptional Program and Epigenetic Signatures of Induced Pluripotent Stem Cells

<div><p>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 <em>in vivo</em> and <em>in vitro</em>. 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 <em>Gtl2</em>. 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.</p> </div

Chad Tillberg READ Poster

Chad Tillberg, Assistant Professor of Biology, reading Cockroaches: Ecology, Behavior, and Natural History, by William J. Bell, Louis M. Roth, and Christine A. Nalepa.https://digitalcommons.linfield.edu/libraries_read/1067/thumbnail.jp

Data from: Heterochromatin Protein 1β (HP1β) 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 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, 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 HP1β 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 HP1β in ESCs. The minor fraction of HP1β 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 HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

Data from: Heterochromatin Protein 1β (HP1β) 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 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, 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 HP1β 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 HP1β in ESCs. The minor fraction of HP1β 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 HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state

Global gene expression differences between iPSCs and ESCs are attenuated following excision of a lentiviral reprogramming cassette.

<p>(A) Schematic representation of the different groups of iPSC/ESC lines subjected to microarray analysis. iPSCs carrying a single copy of a “floxed” STEMCCA vector encoding either three (OKS) or four (OKSM) reprogramming factors were treated with Cre-recombinase to generate transgene-free OKS-Cre and OKSM-Cre iPSC clones. Five subclones of the <i>Sox2</i>-GFP/M2rtTA ESC line were isolated, expanded and included as a control. (B) Principal Components Analysis (PCA) performed on the microarray datasets clearly separates ESCs, transgene-carrying iPSCs (OKS and OKSM) and transgene-free iPSCs (OKS-Cre and OKSM-Cre) into three distinct groups, indicative of similar but distinctive gene expression profiles. Notably, PCA is unable to discriminate iPSCs generated with 3 or 4 factors, both before and after transgene removal. Instead, the presence of the transgenes appears to be a major factor influencing the iPSC transcriptome. (C) qRT-PCR measurement of the residual transcriptional activity of the reprogramming vector demonstrates differences in expression across the iPSC lines that correlate with the degree of gene dysregulation revealed by PCA. (D) Hierarchical clustering of the 2,327 genes significantly different between transgene-carrying iPSCs and transgene-free iPSCs (two-way ANOVA, FDR-adjusted p-value <0.01) reveals distinct patterns of gene expression specific to each of the three groups and increased similarity of iPSCs to ESCs following transgene excision.</p