Maintenance Of Cell Identity Research Articles

Long noncoding RNAs, emerging players in muscle differentiation and disease

The vast majority of the mammalian genome is transcribed giving rise to many different types of noncoding RNAs. Among them, long noncoding RNAs are the most numerous and functionally versatile class. Indeed, the lncRNA repertoire might be as rich as the proteome. LncRNAs have emerged as key regulators of gene expression at multiple levels. They play important roles in the regulation of development, differentiation and maintenance of cell identity and they also contribute to disease. In this review, we present recent advances in the biology of lncRNAs in muscle development and differentiation. We will also discuss the contribution of lncRNAs to muscle disease with a particular focus on Duchenne and facioscapulohumeral muscular dystrophies.

Principles of nucleation of H3K27 methylation during embryonic development

During embryonic development, maintenance of cell identity and lineage commitment requires the Polycomb-group PRC2 complex, which catalyzes histone H3 lysine 27 trimethylation (H3K27me3). However, the developmental origins of this regulation are unknown. Here we show that H3K27me3 enrichment increases from blastula stages onward in embryos of the Western clawed frog (Xenopus tropicalis) within constrained domains strictly defined by sequence. Strikingly, although PRC2 also binds widely to active enhancers, H3K27me3 is only deposited at a small subset of these sites. Using a Support Vector Machine algorithm, these sequences can be predicted accurately on the basis of DNA sequence alone, with a sequence signature conserved between humans, frogs, and fish. These regions correspond to the subset of blastula-stage DNA methylation-free domains that are depleted for activating promoter motifs, and enriched for motifs of developmental factors. These results imply a genetic-default model in which a preexisting absence of DNA methylation is the major determinant of H3K27 methylation when not opposed by transcriptional activation. The sequence and motif signatures reveal the hierarchical and genetically inheritable features of epigenetic cross-talk that impose constraints on Polycomb regulation and guide H3K27 methylation during the exit of pluripotency.

NURR1 in Parkinson disease—from pathogenesis to therapeutic potential

In Parkinson disease (PD), affected midbrain dopamine (DA) neurons lose specific dopaminergic properties before the neurons die. How the phenotype of DA neurons is normally established and the ways in which pathology affects the maintenance of cell identity are, therefore, important considerations. Orphan nuclear receptor NURR1 (NURR1, also known as NR4A2) is involved in the differentiation of midbrain DA neurons, but also has an important role in the adult brain. Emerging evidence indicates that impaired NURR1 function might contribute to the pathogenesis of PD: NURR1 and its transcriptional targets are downregulated in midbrain DA neurons that express high levels of the disease-causing protein α-synuclein. Clinical and experimental data indicate that disrupted NURR1 function contributes to induction of DA neuron dysfunction, which is seen in early stages of PD. The likely involvement of NURR1 in the development and progression of PD makes this protein a potentially interesting target for therapeutic intervention.

Deciphering cis -regulatory control in inflammatory cells

In innate immune system cells, such as macrophages and dendritic cells, deployment of inducible gene expression programmes in response to microbes and danger signals requires highly precise regulatory mechanisms. The inflammatory response has to be tailored based on both the triggering stimulus and its dose, and it has to be unfolded in a kinetically complex manner that suits the different phases of the inflammatory process. Genomic characterization of regulatory elements in this context indicated that transcriptional regulators involved in macrophage specification act as pioneer transcription factors (TFs) that generate regions of open chromatin that enable the recruitment of TFs activated in response to external inputs. Therefore, competence for responses to a specific stimulus is programmed at an early stage of differentiation by factors involved in lineage commitment and maintenance of cell identity, which are responsible for the organization of a cell-type-specific cis-regulatory repertoire. The basic functional and organizational principles that regulate inflammatory gene expression in professional cells of the innate immune system provide general paradigms on the interplay between differentiation and environmental responses.

Control of Cell Identity in Pancreas Development and Regeneration

The endocrine and exocrine cells in the adult pancreas are not static, but can change their differentiation state in response to injury or stress. This concept of cells in flux means that there may be ways to generate certain types of cells (such as insulin-producing β-cells) and prevent formation of others (such as transformed neoplastic cells). We review different aspects of cell identity in the pancreas, discussing how cells achieve their identity during embryonic development and maturation, and how this identity remains plastic, even in the adult pancreas.

Roles of repressive epigenetic machinery in lineage decision of T cells

DNA methylation and histone modifications are central to epigenetic gene regulation, which has been shown to play a crucial role in development. Epigenetics has often been discussed in the context of the maintenance of cell identity because of the heritable nature of gene expression status. Indeed, crucial roles of the epigenetic machinery in establishment and maintenance of particular lineages during early development have been well documented. However, unexpected observation of a developmental plasticity retained in mature T lymphocytes, in particular in CD4(+) T-cell subsets, by recent studies is accelerating studies that focus on roles of each epigenetic pathway in cell fate decisions of T lymphocytes. Here, we focus on the repressive epigenetic machinery, i.e. DNA methylation, histone deacetylation, H3K9 methylation and Polycomb repressive complexes, and briefly review the studies examining the role of these mechanisms during T-lymphocyte differentiation. We also discuss the current challenges faced when analysing the function of the epigenetic machinery and potential directions to overcome the problems.

The emerging role of Polycomb repressors in the response to DNA damage

Polycomb group (PcG) genes encode chromatin modifiers that are involved in the maintenance of cell identity and in proliferation, processes that are often deregulated in cancer. Interestingly, besides a role in epigenetic gene silencing, recent studies have begun to uncover a function for PcG proteins in the cellular response to DNA damage. In particular, PcG proteins have been shown to accumulate at sites of DNA double-strand breaks (DSBs). Several signaling pathways contribute to the recruitment of PcG proteins to DSBs, where they catalyze the ubiquitylation of histone H2A. The relevance of these findings is supported by the fact that loss of PcG genes decreases the efficiency of cells to repair DSBs and renders them sensitive to ionizing radiation. The recruitment of PcG proteins to DNA breaks suggests that they have a function in coordinating gene silencing and DNA repair at the chromatin flanking DNA lesions. In this Commentary, we discuss the current knowledge of the mechanisms that allow PcG proteins to exert their positive functions in genome maintenance.

Differentiation of postnatal cerebellar glial progenitors is controlled by Bmi1 through BMP pathway inhibition

Bmi1 is a polycomb group (Pc-G) protein involved in heritable gene repression, maintenance of cell identity, and proliferation. During the development of the central nervous system, Bmi1 is crucial for self-renewal of neural stem cells and for proliferation of neuronal (granule cell) progenitors of the cerebellum. Here, we use loss of function mouse models and in vitro assays--granule cell cultures and glial-neuronal co-cultures--to show that Bmi1 plays a crucial role in specification of glial progenitors during postnatal cerebellar development. Moreover, we demonstrate in in vitro assays that Bmi1 exerts this novel function through repression of BMP pathway and that this is independent of its known role in mediating the cellular response to Shh signaling. Thus modulation of Bmi1 expression in glial progenitors may represent a key event in determining the differentiation potential of these cells.

Regulated expression of microRNAs-126/126* inhibits erythropoiesis from human embryonic stem cells

AbstractMicroRNAs (miRs) play an important role in cell differentiation and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage differentiation. Human embryonic stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34+ EB cells. miRs-126/126* were the most enriched of the 7 miRs that were up-regulated in CD34+ cells, and their expression paralleled the kinetics of hematopoietic transcription factors RUNX1, SCL, and PU.1. To define the role of miRs-126/126* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-126/126* and analyzed their hematopoietic differentiation. Induction of miRs-126/126* during both EB differentiation and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-126/126* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for growth and expansion of erythroid cells, is one target of miR-126. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-126/126*. Our results define an important function of miRs-126/126* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic differentiation of hESCs.

EZH2 silencing by RNA interference inhibits proliferation in bladder cancer cell lines

The Enhancer of Zeste homologue2 gene (EZH2) is frequently expressed at high levels in malignant tumours, including bladder cancer. It functions as a transcriptional regulator to the maintenance of cell identity, cell cycle regulation and oncogenesis. In the study, we detected EZH2 expression in bladder cancer tissues. These results showed EZH2 high expression in bladder cancer tissue at level of transcript and protein compared with normal bladder tissue and EZH2 expression correlated positively with tumour stage and grade. Then, we used RNA interference to inhibit EZH2 expression in bladder cancer EJ cell line. Efficient downregulation of EZH2 resulted in significantly decreased cell proliferation in EJ cells and retarded transition of G(1) phase to S phase. Our data suggest that EZH2 is involved in the tumourigenesis of bladder cancer and EZH2 downregulation contributes to inhibiting malignant growth by retarding cell entrance to S phase.

Hyperphosphorylation by Cyclin B/CDK1 in Mitosis Resets CUX1 DNA Binding Clock at Each Cell Cycle*

The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).

Origin of the polycomb repressive complex 2 and gene silencing by an E(z) homolog in the unicellular alga Chlamydomonas

Polycomb group proteins play an essential role in the maintenance of cell identity and the regulation of development in both animals and plants. The Polycomb Repressive Complex 2 (PRC2) is involved in the establishment of transcriptionally silent chromatin states, in part through its ability to methylate lysine 27 of histone H3 by the Enhancer of zeste [E(z)] subunit. The absence of PRC2 in unicellular model fungi and its function in the repression of genes vital for the development of higher eukaryotes led to the proposal that this complex may have evolved together with the emergence of multicellularity. However, we report here on the widespread presence of PRC2 core subunits in unicellular eukaryotes from the Opisthokonta, Chromalveolata and Archaeplastida supergroups. To gain insight on the role of PRC2 in single celled organisms, we characterized an E(z) homolog, EZH, in the green alga Chlamydomonas reinhardtii. RNAi-mediated suppression of EZH led to defects in the silencing of transgenes and retrotransposons as well as to a global increase in histone post-translational modifications associated with transcriptional activity, such as trimethylation of histone H3 lysine 4 and acetylation of histone H4. On the basis of the parsimony principle, our findings suggest that PRC2 appeared early in eukaryotic evolution, even perhaps in the last unicellular common ancestor of eukaryotes. One of the ancestral roles of PCR2 may have been in defense responses against intragenomic parasites such as transposable elements, prior to being co-opted for lineage specific functions like developmental regulation in multicellular eukaryotes.

Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing

To examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of hESCs into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation-N1 (early initiation), N2 (neural progenitor), and N3 (early glial-like)-were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call isoform specialization. During neural differentiation, we observed differential expression of many types of genes, including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, neural progenitor cell identity maintenance, and the transition from a predominantly neuronal state into one with increased gliogenic potential.

ConteXt of change—X inactivation and disease

Epigenetic regulation is important for stable maintenance of cell identity. For continued function of organs and tissues, illegitimate changes in cell identity must be avoided. Failure to do so can trigger tumour development and disease. How epigenetic patterns are established during cell differentiation has been explored by studying model systems such as X inactivation. Mammals balance the X-linked gene dosage between the sexes by silencing of one of the two X chromosomes in females. This is initiated by expression of the non-coding X-inactive specific transcript (Xist) RNA and depends on specific cellular contexts, in which essential silencing factors are expressed. Normally X inactivation is initiated in early embryogenesis, but recent reports identified instances where Xist is expressed and can initiate gene repression. Here we describe the features that characterize the cellular permissivity to initiation of X inactivation and note that these can also occur in cancer cells and in specific haematopoietic progenitors. We propose that embryonic pathways for epigenetic regulation are re-established in adult progenitor cells and tumour cells. Understanding their reactivation will deepen our understanding of tumourigenesis and may be exploited for cancer therapy.

Mechanisms of transcriptional repression by histone lysine methylation

During development, covalent modification of both, histones and DNA contribute to the specification and maintenance of cell identity. Repressive modifications are thought to stabilize cell type specific gene expression patterns, reducing the likelihood of reactivation of lineage-unrelated genes. In this report, we review the recent literature to deduce mechanisms underlying Polycomb and H3K9 methylation mediated repression, and describe the functional interplay with activating H3K4 methylation. We summarize recent data that indicate a close relationship between GC density of promoter sequences, transcription factor binding and the antagonizing activities of distinct epigenetic regulators such as histone methyltransferases (HMTs) and histone demethylases (HDMs). Subsequently, we compare chromatin signatures associated with different types of transcriptional outcomes from stable repression to highly dynamic regulated genes, strongly suggesting that the interplay of different epigenetic pathways is essential in defining specific types of heritable chromatin and associated transcriptional states.

Polycomb-Group Oncogenes EZH2, BMI1, and RING1 Are Overexpressed in Prostate Cancer With Adverse Pathologic and Clinical Features

ObjectivesPolycomb group (PcG) proteins are involved in maintenance of cell identity and proliferation. The protein EZH2 is overexpressed in disseminated prostate cancer, implicating a role of PcG complexes in tumor progression. In this study, we evaluated the expression of eight members of both PcG complexes in clinicopathologically defined prostate cancer. MethodsComponents of both PcG protein complexes PRC2 (EZH2, EED, YY1) and PRC1 (BMI1, RING1, HPH1, HPC1, HPC2) were immunohistochemically identified in tissue microarrays of 114 prostate cancer patients. Protein expression was semi-quantitatively scored and correlated with pathologic parameters and recurrence of prostate-specific antigen (PSA). ResultsWhereas BMI1, RING1, HPC1 and HPH1 were all abundantly present in normal and malignant prostate epithelium, expression of EZH2 occurred in only <10% of cells. Expression of EZH2, BMI1 and RING1 were all significantly enhanced in tumours with Gleason score (GS) ≥ 8, extraprostatic extension, positive surgical margins, and PSA recurrence. When only the subgroup of GS ≤ 6 was considered, representing the tumour grade in the majority of needle biopsies, EZH2 and BMI1 were also predictive for PSA recurrence. In a multivariable analysis, BMI1 was the only PcG protein with an independent prognostic value. ConclusionsPcG proteins EZH2, BMI1, and RING1 are associated with adverse pathologic features and clinical PSA recurrence of prostate cancer. Whereas BMI1 and RING1 are abundantly present in prostate cancer, EZH2 is expressed at relatively low levels, making it a less obvious target for therapy.

Micro-RNA Profiling in CD4+ T Cell Differentiation.

Micro-RNAs comprise a class of small noncoding RNAs which have been found to be important regulators of cellular differentiation in multiple species. Previous analysis of micro-RNA expression in the murine hematopoietic system has suggested a role in cell differentiation and the maintenance of cell identity. Naïve progenitor CD4+ T cells respond to a combination of appropriate antigen and other specific signals by undergoing proliferation and further differentiation into one of at least two subsets. T helper 1 (TH1) cells produce high levels of the cytokine IFN-γ and T helper 2 (TH2) cells produce high levels of IL-4, optimizing them for control of intracellular and extracellular pathogens, respectively. It is currently not known whether micro-RNA molecules influence CD4+ T cell differentiation. We have used oligonucleotide arrays to analyze micro-RNA expression profiles of freshly isolated murine CD4+ T cells compared to cells differentiating into TH1 and TH2 subsets. Expression profiles were found to differ significantly between naïve and stimulated CD4+ cells, with fewer differences between TH1 and TH2 subsets. Promising candidate micro-RNAs are being further evaluated by northern blot and genetic studies. Micro-RNA-155 is upregulated on stimulation of CD4+ T cells in multiple oligonucleotide array assays. Micro-RNA-155 is encoded by the BIC oncogene and has been implicated in lymphomagenesis as well as in other malignancies. We have verified the induction of micro-RNA-155 in stimulated helper T cells by northern blot and are studying the effects of this micro-RNA on CD4+ T cell differentiation. Our observations support a role for micro-RNAs in helper T cell differentiation during the immune response.

Steroid hormone-dependent transformation ofpolyhomeoticmutant neurons in theDrosophilabrain

Polyhomeotic (Ph), which forms complexes with other Polycomb-group (PcG) proteins, is widely required for maintenance of cell identity by ensuring differential gene expression patterns in distinct types of cells. Genetic mosaic screens in adult fly brains allow for recovery of a mutation that simultaneously disrupts the tandemly duplicated Drosophila ph transcriptional units. Distinct clones of neurons normally acquire different characteristic projection patterns and can be differentially labeled using various subtype-specific drivers in mosaic brains. Such neuronal diversity is lost without Ph. In response to ecdysone, ph mutant neurons are transformed into cells with unidentifiable projection patterns and indistinguishable gene expression profiles during early metamorphosis. Some subtype-specific neuronal drivers become constitutively activated, while others are constantly suppressed. By contrast, loss of other PcG proteins, including Pc and E(z), causes different neuronal developmental defects; and, consistent with these phenomena, distinct Hox genes are differentially misexpressed in different PcG mutant clones. Taken together, Drosophila Ph is essential for governing neuronal diversity, especially during steroid hormone signaling.

The Elegance of the MicroRNAs: A Neuronal Perspective

As knowledge of microRNAs (miRNA) grows from a compendium of sequences to annotated functional data it has become increasingly clear that a highly significant segment of regulatory biology depends on these approximately 22 nucleotide noncoding transcripts. The expression of many miRNAs in the nervous system, some with a high degree of temporal and spatial specificity, suggests that understanding miRNAs in the nervous system will yield rewarding neurobiological insights. High on the list of insights that microRNAs promise is a deeper understanding of the remarkable cellular diversity found among neurons. This review examines the interface between an emerging biology of miRNAs and their role in nervous systems.

Of Mice, Flies, and Man: The Emerging Role of Polycomb-Group Genes in Human Malignant Lymphomas

Genes belonging to the Polycomb group (PcG) are responsible for the maintenance of cell identity and are directly involved in epigenetic gene silencing. They perform a vital role in the regulation of embryogenesis but also contribute to various adult processes, including regulation of the cell cycle and lymphopoiesis. Experimental model systems have demonstrated that enhanced expression of individual PcG genes, such as Bmi1, results in the development of B-cell and T-cell lymphomas. In humans, a growing body of work has now linked human PcG genes to various hematologic and epithelial cancers. This review focuses on the emerging role of PcG genes in the development of human malignant lymphomas.