Alva Biran

Global epigenetic changes during somatic cell reprogramming to iPS cells

Embryonic stem cells (ESCs) exhibit unique chromatin features, including a permissive transcriptional program and an open, decondensed chromatin state. Induced pluripotent stem cells (iPSCs), which are very similar to ESCs, hold great promise for therapy and basic research. However, the mechanisms by which reprogramming occurs and the chromatin organization that underlies the reprogramming process are largely unknown. Here we characterize and compare the epigenetic landscapes of partially and fully reprogrammed iPSCs to mouse embryonic fibroblasts (MEFs) and ESCs, which serves as a standard for pluripotency. Using immunofluorescence and biochemical fractionations, we analyzed the levels and distribution of a battery of histone modifications (H3ac, H4ac, H4K5ac, H3K9ac, H3K27ac, H3K4me3, H3K36me2, H3K9me3, H3K27me3, and γH2AX), as well as HP1α and lamin A. We find that fully reprogrammed iPSCs are epigenetically identical to ESCs, and that partially reprogrammed iPSCs are closer to MEFs. Intriguingly, combining both time-course reprogramming experiments and data from the partially reprogrammed iPSCs, we find that heterochromatin reorganization precedes Nanog expression and active histone marking. Together, these data delineate the global epigenetic state of iPSCs in conjunction with their pluripotent state, and demonstrate that heterochromatin precedes euchromatin in reorganization during reprogramming.

H3K9 histone acetylation predicts pluripotency and reprogramming capacity of ES cells

The pluripotent genome is characterized by unique epigenetic features and a decondensed chromatin conformation. However, the relationship between epigenetic regulation and pluripotency is not altogether clear. Here, using an enhanced MEF/ESC fusion protocol, we compared the reprogramming potency and histone modifications of different embryonic stem cell (ESC) lines (R1, J1, E14, C57BL/6) and found that E14 ESCs are significantly less potent, with significantly reduced H3K9ac levels. Treatment of E14 ESCs with histone deacetylase (HDAC) inhibitors (HDACi) increased H3K9ac levels and restored their reprogramming capacity. Microarray and H3K9ac ChIP-seq analyses, suggested increased extracellular matrix (ECM) activity following HDACi treatment in E14 ESCs. These data suggest that H3K9ac may predict pluripotency and that increasing pluripotency by HDAC inhibition acts through H3K9ac to enhance the activity of target genes involved in ECM production to support pluripotency.

Bacterial genotoxicity bioreporters.

Ever since the introduction of the Salmonella typhimurium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotoxicity assays based upon the use of genetically engineered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose‐dependent signal in the presence of potential DNA damaging compounds and the induction of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu‐test, has been fully validated and ISO‐ and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial‐based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual genotoxicity testing devices.

The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity

INTRODUCTION Cancer arises from clonal expansion of a single cell. Yet, most human cancers are characterized by extensive intratumor heterogeneity and comprise various subpopulations of cells with distinct phenotypes and biological properties. Intratumor heterogeneity poses major challenges in understanding cancers, managing patients, and designing effective treatment strategies. Functional heterogeneity within individual tumors is partly due to the presence of genetically distinct subclonal cell populations. Furthermore, interactions between cancer cells and the tumor microenvironment can alter the phenotype of cancer cells via nongenetic mechanisms. The combination of cell-intrinsic and cell-extrinsic changes occurring during tumor growth generates functionally distinct subsets of cells that differentially contribute to tumor maintenance. RATIONALE In many cancers, phenotypic and functional heterogeneity can be mapped to distinct differentiation states, suggesting that cellular hierarchies established during tumor growth may affect the long-term proliferative potential of cancer cells. To shed light on the mechanisms responsible for the generation of these hierarchies, we searched for epigenetic mechanisms that determine which cancer cells can preserve unlimited proliferative potential, and thus the ability to drive long-term tumor growth, and which cells lose this ability through a differentiation process. RESULTS We found that, in several cancer types, individual tumors exhibit high heterogeneity of the major chromatin protein linker histone H1.0, showing strongly reduced H1.0 levels in cells characterized by long-term self-renewal ability and tumorigenic potential and higher levels in nontumorigenic cells. Combined analysis of pan-cancer patient data sets and experimental alteration of the H1F0 locus in tumor cells revealed that heterogeneous H1.0 expression patterns are partly due to differential methylation of an enhancer region that dynamically modulates H1.0 expression within tumors. Using a controlled system to model functional intratumor heterogeneity, we showed that maintenance of cell tumorigenic potential required silencing of H1.0 to avoid loss of unlimited proliferative capacity through differentiation. Mechanistically, absence of H1.0 led to destabilization of nucleosome-DNA interactions in AT-rich genomic regions and coordinated derepression of large sets of neighboring genes, resulting in activation of transcriptional programs that support cancer cell self-renewal. Gene expression changes induced by H1.0 loss were reversible, and epigenetic states restricting cell proliferative potential were reestablished upon H1.0 reexpression. In multiple cancer types, in agreement with the observed inhibition of cancer cell self-renewal by H1.0, patients expressing overall strongly reduced levels of H1.0 showed a significantly worse outcome than patients expressing higher H1.0 levels. CONCLUSION Intratumor heterogeneity has emerged as a general feature of cancer, but the molecular features underlying functionally diverse cellular phenotypes have been elusive. Our results uncover epigenetic determinants of tumor-maintaining cells and identify an integral component of chromatin as an important regulator of cell differentiation states within tumors. We propose that only cells insensitive to extracellular differentiation cues, capable of permanently silencing H1.0, can act as self-renewing tumor-maintaining cells and that such a mechanism supports maintenance of several types of cancer. Our results suggest that intervention aimed at restoring high levels of H1.0 in all cancer cells may enhance the differentiation process that naturally occurs during tumor growth and may be beneficial for therapeutic purposes. Epigenetic heterogeneity within tumors. In many cancer types, self-renewing and differentiated epigenetic states coexist in individual tumors. (Left) Image of a breast cancer section showing heterogeneous levels of the linker histone H1.0 (red). (Right) Schematic depiction of the chromatin status of cancer cells in which H1.0 is down-regulated (blue) or expressed at high levels (red). Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells’ long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells.

Concise Review: Chromatin and Genome Organization in Reprogramming†‡§

The ability to reprogram somatic cells to pluripotency is continuingly attracting increasing amounts of attention, providing both potential opportunities for regenerative medicine, as well as an intriguing model to study basic mechanisms of developmental reversal and epigenetic erasure. Currently, nuclear reprogramming is an inefficient process and a better understanding of its components and the underlying mechanisms will no doubt enable us to increase its robustness and to gain a deeper understanding of its regulation. Here we focus on the reprogramming process from the chromatin and genome organization perspective, describing the chromatin changes that occur both globally and locally. At the global level, chromatin decondenses toward the characteristic ‘open’ state, while locally, chromatin reorganization supports the silencing of lineage‐specific genes and the activation of pluripotency‐related genes. Importantly, the proteins that regulate this process are being identified, revealing different layers of chromatin regulation, including histone modifications, histone variants, chromatin remodeling and genomic DNA methylation. The emerging theme is that chromatin and genome organization are not only altered during the transition from a somatic to a pluripotent state, but also play active, regulatory roles during the reprogramming process. Stem Cells2012;30:1793–1799

Evaluation of chrono-amperometric signal detection for the analysis of genotoxicity by a whole cell biosensor

Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.

Genetically Engineered Bacteria for Genotoxicity Assessment

Ever since the introduction of the Salmonella typhimurium mammalian microsome mutagenicity assay (the Ames test ) over three decades ago, there has been a constant development of additional genotoxicity assays based upon the use of genetically engineered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test or on promoter-reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induction of repair mechanisms. While some of these assays were only briefly described in the scientific literature, others have been developed all the way to commercial products. Out of these, only one, the umu -test has been fully validated and ISO- and OECD-standardized. Herein we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial meta- bolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual genotoxicity testing devices.

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

Summary 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, SETβ, 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 SETs 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.

Electronically Directed Integration of Whole-Cell Biosensors on Bio-Chips

Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel This paper presents a whole-cell bio-chip system where viable, functioning cells are deposited onto solid surfaces that are a part of a micro-machined system. The development of such novel hybrid functional sensors depends on the cell deposition methods; in this work new approach integrating live bacterial cells on a bio-chip using electrophoretic deposition is presented. The bio-material deposition technique was characterized under various driving potential and chamber configurations. The deposited bio-mass included genetically engineered bacterial cells generating electrochemically active byproduct upon exposure to toxic materials in the aqueous solution. In this paper we present the deposition apparatus and methods, as well as the characterization results, e.g. signal vs. time and induction factor, of such chips and discussing the highlight and problems of the new deposition method.