Polycomb group protein complexes exchange rapidly in living Drosophila

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo. We determined the translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria

Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle.

Chromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear. To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL), to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3. We show that chromosome regions (bands) of 10-50 Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G1 and G2. They comprise 1-5 Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely. We found little change between cell cycle phases, whether compared by 5 Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains. Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in G2M, possibly because of ongoing transcription. In conclusion, modified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1 Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses

Transition to naïve human pluripotency mirrors pan-cancer DNA hypermethylation.

Epigenetic reprogramming is a cancer hallmark, but how it unfolds during early neoplastic events and its role in carcinogenesis and cancer progression is not fully understood. Here we show that resetting from primed to naïve human pluripotency results in acquisition of a DNA methylation landscape mirroring the cancer DNA methylome, with gradual hypermethylation of bivalent developmental genes. We identify a dichotomy between bivalent genes that do and do not become hypermethylated, which is also mirrored in cancer. We find that loss of H3K4me3 at bivalent regions is associated with gain of methylation. Additionally, we observe that promoter CpG island hypermethylation is not restricted solely to emerging naïve cells, suggesting that it is a feature of a heterogeneous intermediate population during resetting. These results indicate that transition to naïve pluripotency and oncogenic transformation share common epigenetic trajectories, which implicates reprogramming and the pluripotency network as a central hub in cancer formation

Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.EAR is a Herchel Smith Fellow. MB and HB are supported by the Centre for Trophoblast Research, MB is a Next Generation Research Fellow. MJB is supported by a BBSRC studentship. The WR lab is supported by BBSRC, MRC, the Wellcome Trust, EU EpiGeneSys and BLUEPRINT. The SB lab is supported by core funding from Cancer Research UK

Resetting transcription factor control circuitry toward ground-state pluripotency in human.

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.This research was supported by the UK Medical Research Council, the Japan Science and Technology agency (JST, PRESTO), the Genome Biology Unit of the European Molecular Biology Laboratory, European Commission projects PluriMes, BetaCellTherapy, EpiGeneSys, and Blueprint, and the Wellcome Trust. Y.T. was a University of Cambridge Herchel Smith Fellow. A.S. is a Medical Research Council Professor

FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency

SummaryGenome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency

Transient accumulation of 5-carboxylcytosine indicates involvement of active demethylation in lineage specification of neural stem cells

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and in vivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain

Erk5 is a key regulator of naïve-­primed transition and Embryonic Stem Cell identity

Embryonic stem cells (ESCs) can self-renew or differentiate into any cell type, a phenomenon known as pluripotency. Distinct pluripotent states, termed naive and primed pluripotency, have been described. However, the mechanisms that control naive-primed pluripotent transition are poorly understood. Here, we perform a targeted screen for kinase inhibitors, which modulate the naive-primed pluripotent transition. We find that XMD compounds, which selectively inhibit Erk5 kinase and BET bromodomain family proteins, drive ESCs toward primed pluripotency. Using compound selectivity engineering and CRISPR/Cas9 genome editing, we reveal distinct functions for Erk5 and Brd4 in pluripotency regulation. We show that Erk5 signaling maintains ESCs in the naive state and suppresses progression toward primed pluripotency and neuroectoderm differentiation. Additionally, we identify a specialized role for Erk5 in defining ESC lineage selection, whereby Erk5 inhibits a cardiomyocyte-specific differentiation program. Our data therefore reveal multiple critical functions for Erk5 in controlling ESC identity

Analisis Pengaruh Budaya Organisasi Dan Kompensasi Terhadap Kinerja Karyawan Dengan Motivasi Sebagai Variabel Intervening (Studi Kasus Pada PT. Lg Bagian Penjualan Indonesia Semarang)

The problems that occurred in the employee portion of sales LG Indonesia Semarang is adecline in performance is indicated by not achieving the target for 2015. The employeeperformance and motivation is also thought to be influenced by factors of organizationalculture and also compensation deemed not feasible by most employees. This study aimedto analyze the influence of organizational culture on the motivation and compensationand employee performance parts sales LG Indonesia Semarang. The population used inthis study were all employees of LG Indonesia Semarang. The sampling technique usedwas purposive sampling. Criteria samples taken were all employees of the salesdepartment LG Indonesia Semarang who have worked more than two years are 71nurses. The method of collecting the data in this study using questionnaires andinterviews. Methods of data analysis using path analysis. Based on the research,organizational culture and compensation have a positive effect on motivation andperformance, while motivation is also a positive effect on performance. Based on theresults Sobel Test to determine whether there is mediating the relationship between theindependent and dependent variables, it is known that motivation mediates the effect ofcompensation and organizational culture on performance