Dissection of gene regulatory networks in embryonic stem cells by means of high-throughput sequencing

Transcription factor regulation of gene expression and chromatin-controlled epigenetic memory systems are closely cooperating in establishing the pluripotent state of embryonic stem (ES) cells and maintaining cell fate decisions throughout development of an organism. A thorough understanding of the regulatory transcriptional circuitry that rules the underlying plastic yet heritable gene expression programs in ES cells is of great importance. With the advent of next-generation sequencing technologies facilitating the quantitative assessment of functional genomics assays it is now feasible to interrogate transcription networks at a genome-wide scale. Here, we discuss the application of next-generation sequencing in elucidating the molecular mechanisms underlying ES cell functio

Introduction to Epigenetics

This open access textbook leads the reader from basic concepts of chromatin structure and function and RNA mechanisms to the understanding of epigenetics, imprinting, regeneration and reprogramming. The textbook treats epigenetic phenomena in animals, as well as plants. Written by four internationally known experts and senior lecturers in this field, it provides a valuable tool for Master- and PhD- students who need to comprehend the principles of epigenetics, or wish to gain a deeper knowledge in this field. After reading this book, the student will: Have an understanding of the basic toolbox of epigenetic regulation Know how genetic and epigenetic information layers are interconnected Be able to explain complex epigenetic phenomena by understanding the structures and principles of the underlying molecular mechanisms Understand how misregulated epigenetic mechanisms can lead to diseas

Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data

The Photo-Activatable Ribonucleoside-enhanced CrossLinking and ImmunoPrecipitation (PAR-CLIP) method was recently developed for global identification of RNAs interacting with proteins. The strength of this versatile method results from induction of specific T to C transitions at sites of interaction. However, current analytical tools do not distinguish between non-experimentally and experimentally induced transitions. Furthermore, geometric properties at potential binding sites are not taken into account. To surmount these shortcomings, we developed a two-step algorithm consisting of a non-parametric two-component mixture model and a wavelet-based peak calling procedure. Our algorithm can reduce the number of false positives up to 24% thereby identifying high confidence interaction sites. We successfully employed this approach in conjunction with a modified PAR-CLIP protocol to study the functional role of nuclear Moloney leukemia virus 10, a putative RNA helicase interacting with Argonaute2 and Polycomb. Our method, available as the R package wavClusteR, is generally applicable to any substitution-based inference problem in genomic

An efficient strategy for TALEN-mediated genome engineering in Drosophila

In reverse genetics, a gene's function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generatio

The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila

BACKGROUND: The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. RESULTS AND DISCUSSION: Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. CONCLUSIONS: Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes

Reference Genes for Expression Studies in Human CD8 + Naïve and Effector Memory T Cells under Resting and Activating Conditions

Abstract: Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is widely used for mRNA quantification. To accurately measure changing gene transcript levels under different experimental conditions, the use of appropriate reference gene transcripts is instrumental. In T cell immunology, suitable reference genes have been reported for bulk CD4+ and CD8+ T cells. However, many CD4+ and CD8+ T cell subsets have been described in the past. Although they respond differently to given activation stimuli, proper validation of suitable reference genes in these subsets is lacking. In this study, we evaluated twelve commonly used reference gene products in human naïve (NV) and effector memory (EM) CD8+ T cells under non-activated and activated (2 h, 10 h and 20 h) conditions. We used five different statistical approaches for data analysis. Our results show that a number of widely used reference transcripts become differentially expressed under activating conditions. Using them as references markedly alters results as exemplified with IFNG mRNA expression. The only candidate reference gene products that remained stable during the activation process were 18S rRNA and SDHA mRNA, encouraging their usage as reference gene products for RT-qPCR experiments, when quantifying mRNA levels in human NV and EM CD8+ T cells

O fim dos contratos de programa no saneamento: o início de uma nova discussão? / The end of program contracts in sanitation: the beginning of a new discussion?

O problema da universalização do acesso ao serviço de saneamento básico estimulou a alteração legislativa atual, que tem em sua subjacência a pretensão de criar ambiente juridicamente seguro para atrair o capital privado, e assim garantir que quase a totalidade da população brasileira tenha acesso à água potável e ao esgotamento sanitário até 2033. Ocorre que, com esse objetivo precípuo de deslocar o Estado da função de prestador de serviço, as mudanças acabaram por limitar a possibilidade de os Municípios, valendo-se de sua autonomia, realizarem a gestão associada mediante contratos de programa firmados com as Companhias Estaduais de Saneamento Básico (CESB), estatais que desde a década de 70 com o PLANASA assumem o protagonismo no setor. Mediante lei, intentou-se romper de forma abrupta com estrutura e arranjo institucional historicamente consolidado, em que se manifesta a dependência de trajetória (path-dependence), com forte resiliência dos atores protagonistas. Com isso, ao revés de criar ambiente de segurança jurídica para atrair investimentos, ao romper sem direito transitório conformador de interesses, inaugurou novo ponto de tensão, enfrentando as CESB que, para defender-se, aponta inconstitucionalidade da vedação dos contratos de programa no atual marco regulatório do saneamento básico. O presente trabalho, neste contexto, preocupar-se-á em identificar a estratégia argumentativa e o interesse das CESBs, a partir de pesquisa documental mediante revisão bibliográfica, e também análise indutiva, por meio de estudo de caso, debruçando-se sobre a Ação Direta de Inconstitucionalidade 6882 ajuizada pela Associação Brasileira das Empresas Estaduais de Saneamento

O fim dos contratos de programa no saneamento: o início de uma nova discussão? / The end of program contracts in sanitation: the beginning of a new discussion?

O problema da universalização do acesso ao serviço de saneamento básico estimulou a alteração legislativa atual, que tem em sua subjacência a pretensão de criar ambiente juridicamente seguro para atrair o capital privado, e assim garantir que quase a totalidade da população brasileira tenha acesso à água potável e ao esgotamento sanitário até 2033. Ocorre que, com esse objetivo precípuo de deslocar o Estado da função de prestador de serviço, as mudanças acabaram por limitar a possibilidade de os Municípios, valendo-se de sua autonomia, realizarem a gestão associada mediante contratos de programa firmados com as Companhias Estaduais de Saneamento Básico (CESB), estatais que desde a década de 70 com o PLANASA assumem o protagonismo no setor. Mediante lei, intentou-se romper de forma abrupta com estrutura e arranjo institucional historicamente consolidado, em que se manifesta a dependência de trajetória (path-dependence), com forte resiliência dos atores protagonistas. Com isso, ao revés de criar ambiente de segurança jurídica para atrair investimentos, ao romper sem direito transitório conformador de interesses, inaugurou novo ponto de tensão, enfrentando as CESB que, para defender-se, aponta inconstitucionalidade da vedação dos contratos de programa no atual marco regulatório do saneamento básico. O presente trabalho, neste contexto, preocupar-se-á em identificar a estratégia argumentativa e o interesse das CESBs, a partir de pesquisa documental mediante revisão bibliográfica, e também análise indutiva, por meio de estudo de caso, debruçando-se sobre a Ação Direta de Inconstitucionalidade 6882 ajuizada pela Associação Brasileira das Empresas Estaduais de Saneamento