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1.
FEBS Lett ; 597(14): 1805-1817, 2023 07.
Article in English | MEDLINE | ID: mdl-37343149

ABSTRACT

DNA methylation (5mC) is an essential epigenetic mark associated with transcriptional silencing. The role of 5mC in transcriptional repression is well established for a few hundred genes through methylation of their promoters. Yet, whether 5mC contributes more broadly to gene expression is an important open question. 5mC removal has recently been associated with the activation of enhancers, opening the possibility that 5mC may globally contribute to the expression of genes defining cell identities. Here, we will review the evidence and molecular mechanisms that link 5mC with the activity of enhancers. We will discuss the spread and amplitude of the potential gene expression changes controlled by 5mC at enhancers, and how these may contribute to the determination of cell identities during development.


Subject(s)
DNA Methylation , Epigenesis, Genetic , Gene Expression , Promoter Regions, Genetic , Epigenomics
2.
Mol Cell ; 83(5): 787-802.e9, 2023 03 02.
Article in English | MEDLINE | ID: mdl-36758546

ABSTRACT

Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA demethylation. However, whether this epigenetic remodeling is necessary for enhancer activation is unknown. Here, we adapted single-molecule footprinting to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their chromatin accessibility in multiple cell lineages. Although reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where DNA methylation antagonizes the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding require active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at active enhancers.


Subject(s)
DNA Methylation , Enhancer Elements, Genetic , Animals , Chromatin , Transcription Factors/metabolism , Gene Expression Regulation , Mammals/metabolism
3.
Nat Genet ; 54(11): 1702-1710, 2022 11.
Article in English | MEDLINE | ID: mdl-36333500

ABSTRACT

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.


Subject(s)
DNA Methylation , Genomic Imprinting , Mice , Animals , Base Sequence , DNA Methylation/genetics , Epigenomics , Chromatin/genetics , Repressor Proteins/genetics
4.
Trends Biochem Sci ; 47(12): 993-995, 2022 12.
Article in English | MEDLINE | ID: mdl-35970663

ABSTRACT

Cofactors are essential effectors of the transcription control machinery. How this functionally diverse group of factors is used in the genome remains elusive. A recent study by Neumayr, Haberle et al. sheds light on this question, showing that enhancers depend on defined combinations of cofactors for their activation.


Subject(s)
Enhancer Elements, Genetic , Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Gene Expression Regulation , Promoter Regions, Genetic
5.
Nat Protoc ; 16(12): 5673-5706, 2021 12.
Article in English | MEDLINE | ID: mdl-34773120

ABSTRACT

Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to simultaneously resolve the occupancy of multiple proteins including transcription factors, RNA polymerase II and nucleosomes on single DNA molecules genome-wide. The technique combines the use of cytosine methyltransferases to footprint the genome with bisulfite sequencing to resolve transcription factor binding patterns at cis-regulatory elements. DNA footprinting is performed by incubating permeabilized nuclei with recombinant methyltransferases. Upon DNA extraction, whole-genome or targeted bisulfite libraries are prepared and loaded on Illumina sequencers. The protocol can be completed in 4-5 d in any laboratory with access to high-throughput sequencing. Analysis can be performed in 2 d using a dedicated R package and requires access to a high-performance computing system. Our method can be used to analyze how transcription factors cooperate and antagonize to regulate transcription.


Subject(s)
DNA Footprinting/methods , DNA Modification Methylases/metabolism , DNA/metabolism , Genome , Single Molecule Imaging/methods , Transcription Factors/metabolism , Animals , Cell Nucleus/metabolism , DNA/genetics , DNA Modification Methylases/genetics , Gene Expression Regulation , Gene Library , High-Throughput Nucleotide Sequencing , Humans , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Nucleosomes/chemistry , Nucleosomes/metabolism , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Sequence Analysis, DNA/statistics & numerical data , Software , Transcription Factors/genetics
6.
Nature ; 596(7870): 133-137, 2021 08.
Article in English | MEDLINE | ID: mdl-34234345

ABSTRACT

The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.


Subject(s)
Cell Cycle Proteins/metabolism , Chromatin Assembly and Disassembly , Chromatin/genetics , Chromatin/metabolism , CpG Islands/genetics , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Base Sequence , Cell Line, Tumor , Chromatin/chemistry , Chromatin Assembly and Disassembly/genetics , DNA Methylation , Gene Expression Regulation , Genes, Essential , Humans , Mice , Single Molecule Imaging
7.
Trends Genet ; 37(9): 798-806, 2021 09.
Article in English | MEDLINE | ID: mdl-33892959

ABSTRACT

About 7% of the human genome encodes cis-regulatory elements (CREs) that function as regulatory switches to modulate the expression of genes. These short genetic sequences control the complex transcriptional changes necessary for organismal development. A topical challenge in the field is to understand how transcription factors (TFs) read and translate this information into gene expression patterns. Here, I review how the development of single-molecule footprinting (SMF) that resolves the genome occupancy of TFs on individual DNA molecules resolution contributes to our ability to establish how the regulatory genetic information is interpreted at the mechanistic level. I further discuss how future developments in the nascent field of single-molecule genomics (SMG) could impact our understanding of gene regulation mechanisms.


Subject(s)
Gene Expression Regulation , Genomics/methods , Regulatory Elements, Transcriptional , Transcription Factors/genetics , DNA/genetics , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Single Molecule Imaging , Transcription Factors/metabolism
8.
Nat Commun ; 11(1): 2680, 2020 05 29.
Article in English | MEDLINE | ID: mdl-32471981

ABSTRACT

DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.


Subject(s)
CpG Islands/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA Demethylation , DNA Methylation/genetics , DNA-Binding Proteins/metabolism , Mouse Embryonic Stem Cells/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Binding Sites/genetics , Cell Line , Chromosome Mapping , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methyltransferase 3A , DNA-Binding Proteins/genetics , Dioxygenases/genetics , Dioxygenases/metabolism , Epigenesis, Genetic/genetics , Genome/genetics , Histones/metabolism , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/metabolism , Transcription, Genetic/genetics , DNA Methyltransferase 3B
9.
Nat Neurosci ; 22(8): 1345-1356, 2019 08.
Article in English | MEDLINE | ID: mdl-31285614

ABSTRACT

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.


Subject(s)
Dependovirus/genetics , Gene Targeting/methods , Neuroglia/virology , Neurons/virology , Animals , Gene Transfer Techniques , Humans , Macaca fascicularis , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic/genetics , Retina/virology
10.
Genome Res ; 29(4): 554-563, 2019 04.
Article in English | MEDLINE | ID: mdl-30709850

ABSTRACT

Most mammalian RNA polymerase II initiation events occur at CpG islands, which are rich in CpGs and devoid of DNA methylation. Despite their relevance for gene regulation, it is unknown to what extent the CpG dinucleotide itself actually contributes to promoter activity. To address this question, we determined the transcriptional activity of a large number of chromosomally integrated promoter constructs and monitored binding of transcription factors assumed to play a role in CpG island activity. This revealed that CpG density significantly improves motif-based prediction of transcription factor binding. Our experiments also show that high CpG density alone is insufficient for transcriptional activity, yet results in increased transcriptional output when combined with particular transcription factor motifs. However, this CpG contribution to promoter activity is independent of DNA methyltransferase activity. Together, this refines our understanding of mammalian promoter regulation as it shows that high CpG density within CpG islands directly contributes to an environment permissive for full transcriptional activity.


Subject(s)
CpG Islands , DNA Methylation , Promoter Regions, Genetic , Transcriptional Activation , Animals , Cell Line , Cells, Cultured , Mice , Protein Binding , Transcription Factors/metabolism
11.
Nucleic Acids Res ; 45(20): 11607-11621, 2017 Nov 16.
Article in English | MEDLINE | ID: mdl-29059322

ABSTRACT

The retina is composed of ∼50 cell-types with specific functions for the process of vision. Identification of the cis-regulatory elements active in retinal cell-types is key to elucidate the networks controlling this diversity. Here, we combined transcriptome and epigenome profiling to map the regulatory landscape of four cell-types isolated from mouse retinas including rod and cone photoreceptors as well as rare inter-neuron populations such as horizontal and starburst amacrine cells. Integration of this information reveals sequence determinants and candidate transcription factors for controlling cellular specialization. Additionally, we refined parallel reporter assays to enable studying the transcriptional activity of large collection of sequences in individual cell-types isolated from a tissue. We provide proof of concept for this approach and its scalability by characterizing the transcriptional capacity of several hundred putative regulatory sequences within individual retinal cell-types. This generates a catalogue of cis-regulatory regions active in retinal cell types and we further demonstrate their utility as potential resource for cellular tagging and manipulation.


Subject(s)
Regulatory Elements, Transcriptional/genetics , Regulatory Sequences, Nucleic Acid/genetics , Retina/cytology , Retinal Cone Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/cytology , Animals , DNA Methylation/genetics , Female , Mice , Mice, Inbred C57BL , Mice, Transgenic , Retina/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Transcriptome/genetics
12.
Mol Cell ; 67(3): 411-422.e4, 2017 Aug 03.
Article in English | MEDLINE | ID: mdl-28735898

ABSTRACT

Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.


Subject(s)
DNA/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Promoter Regions, Genetic , RNA Polymerase II/metabolism , RNA/biosynthesis , Single Molecule Imaging , Transcription, Genetic , Animals , Binding Sites , DNA/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Genome-Wide Association Study , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Half-Life , Kinetics , Protein Binding , Protein Stability , Proteolysis , RNA/genetics , RNA Polymerase II/genetics , TATA Box , Transcription Initiation Site , Transcription Initiation, Genetic , Transcription Termination, Genetic
13.
Nature ; 520(7546): 243-7, 2015 Apr 09.
Article in English | MEDLINE | ID: mdl-25607372

ABSTRACT

DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.


Subject(s)
DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation/genetics , Epigenesis, Genetic/genetics , Genome/genetics , Animals , Cell Line , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , CpG Islands/genetics , DNA (Cytosine-5-)-Methyltransferases/chemistry , DNA Methyltransferase 3A , Embryonic Stem Cells/enzymology , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic/genetics , Genomics , Histone-Lysine N-Methyltransferase/deficiency , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/chemistry , Histones/metabolism , Lysine/metabolism , Mice , Promoter Regions, Genetic/genetics , Protein Binding , Protein Structure, Tertiary , Protein Transport , Transcription, Genetic/genetics , DNA Methyltransferase 3B
14.
Elife ; 3: e04094, 2014 Sep 26.
Article in English | MEDLINE | ID: mdl-25259795

ABSTRACT

The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.


Subject(s)
CpG Islands/genetics , DNA Methylation/genetics , Genetic Engineering/methods , Genome/genetics , High-Throughput Nucleotide Sequencing , Animals , Base Sequence , Cell Differentiation/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Library , Humans , Mammals/genetics , Mice , Models, Genetic , Protein Binding , Recombinases/metabolism , Transcription Factors/metabolism
15.
Methods Mol Biol ; 1150: 141-52, 2014.
Article in English | MEDLINE | ID: mdl-24743995

ABSTRACT

Chromatin immunoprecipitation coupled high-throughput sequencing (ChIP-seq) is a common method to study in vivo protein-DNA interactions at the genome-wide level. The processing, analysis, and biological interpretation of gigabyte datasets, generated by several ChIP-seq runs, is a challenging task for biologists. The seqMINER platform has been designed to handle, compare, and visualize different sequencing datasets in a user-friendly way. Different analysis methods are applied to understand common and specific binding patterns of single or multiple datasets to answer complex biological questions. Here, we give a detailed protocol about the different analysis modules implemented in the recent version of seqMINER.


Subject(s)
Biostatistics/methods , Chromatin Immunoprecipitation/methods , Computer Graphics , High-Throughput Nucleotide Sequencing/methods , Software , Cluster Analysis
16.
Nat Genet ; 44(11): 1173-4, 2012 Nov.
Article in English | MEDLINE | ID: mdl-23104061

ABSTRACT

Cellular transformation in cancer has long been associated with aberrant DNA methylation, most notably, hypermethylation of promoter sequences. A new study uses a clever approach of selective high-resolution profiling to follow DNA methylation over a time course of cellular transformation and challenges the notion that hypermethylation in cancer arises in an orchestrated fashion.


Subject(s)
DNA Methylation/genetics , Epigenesis, Genetic , Repressor Proteins/genetics , CCCTC-Binding Factor , Humans
17.
BMC Genomics ; 13: 424, 2012 Aug 24.
Article in English | MEDLINE | ID: mdl-22920947

ABSTRACT

BACKGROUND: Transcription regulation in pluripotent embryonic stem (ES) cells is a complex process that involves multitude of regulatory layers, one of which is post-translational modification of histones. Acetylation of specific lysine residues of histones plays a key role in regulating gene expression. RESULTS: Here we have investigated the genome-wide occurrence of two histone marks, acetylation of histone H3K9 and K14 (H3K9ac and H3K14ac), in mouse embryonic stem (mES) cells. Genome-wide H3K9ac and H3K14ac show very high correlation between each other as well as with other histone marks (such as H3K4me3) suggesting a coordinated regulation of active histone marks. Moreover, the levels of H3K9ac and H3K14ac directly correlate with the CpG content of the promoters attesting the importance of sequences underlying the specifically modified nucleosomes. Our data provide evidence that H3K9ac and H3K14ac are also present over the previously described bivalent promoters, along with H3K4me3 and H3K27me3. Furthermore, like H3K27ac, H3K9ac and H3K14ac can also differentiate active enhancers from inactive ones. Although, H3K9ac and H3K14ac, a hallmark of gene activation exhibit remarkable correlation over active and bivalent promoters as well as distal regulatory elements, a subset of inactive promoters is selectively enriched for H3K14ac. CONCLUSIONS: Our study suggests that chromatin modifications, such as H3K9ac and H3K14ac, are part of the active promoter state, are present over bivalent promoters and active enhancers and that the extent of H3K9 and H3K14 acetylation could be driven by cis regulatory elements such as CpG content at promoters. Our study also suggests that a subset of inactive promoters is selectively and specifically enriched for H3K14ac. This observation suggests that histone acetyl transferases (HATs) prime inactive genes by H3K14ac for stimuli dependent activation. In conclusion our study demonstrates a wider role for H3K9ac and H3K14ac in gene regulation than originally thought.


Subject(s)
Embryonic Stem Cells/metabolism , Histones/metabolism , Promoter Regions, Genetic/genetics , Regulatory Elements, Transcriptional/genetics , Acetylation , Animals , CpG Islands/genetics , Epigenomics , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Mice
18.
Mol Cell ; 44(3): 410-423, 2011 Nov 04.
Article in English | MEDLINE | ID: mdl-22055187

ABSTRACT

Histone acetyltransferase (HAT) complexes are coactivators that are important for transcriptional activation by modifying chromatin. Metazoan SAGA and ATAC are distinct multisubunits complexes that share the same catalytic HAT subunit (GCN5 or PCAF). Here, we show that these human HAT complexes are targeted to different genomic loci representing functionally distinct regulatory elements both at broadly expressed and tissue-specific genes. While SAGA can principally be found at promoters, ATAC is recruited to promoters and enhancers, yet only its enhancer binding is cell-type specific. Furthermore, we show that ATAC functions at a set of enhancers that are not bound by p300, revealing a class of enhancers not yet identified. These findings demonstrate important functional differences between SAGA and ATAC coactivator complexes at the level of the genome and define a role for the ATAC complex in the regulation of a set of enhancers.


Subject(s)
Gene Expression Regulation, Neoplastic , Histone Acetyltransferases/metabolism , p300-CBP Transcription Factors/metabolism , Binding Sites , DNA Polymerase II/metabolism , Enhancer Elements, Genetic , HeLa Cells , Histone Acetyltransferases/genetics , Humans , Multiprotein Complexes , Promoter Regions, Genetic , RNA Interference , Transcription, Genetic , Transfection , p300-CBP Transcription Factors/genetics
19.
Nucleic Acids Res ; 39(6): e35, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21177645

ABSTRACT

In a single experiment, chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq) provides genome-wide information about a given covalent histone modification or transcription factor occupancy. However, time efficient bioinformatics resources for extracting biological meaning out of these gigabyte-scale datasets are often a limiting factor for data interpretation by biologists. We created an integrated portable ChIP-seq data interpretation platform called seqMINER, with optimized performances for efficient handling of multiple genome-wide datasets. seqMINER allows comparison and integration of multiple ChIP-seq datasets and extraction of qualitative as well as quantitative information. seqMINER can handle the biological complexity of most experimental situations and proposes methods to the user for data classification according to the analysed features. In addition, through multiple graphical representations, seqMINER allows visualization and modelling of general as well as specific patterns in a given dataset. To demonstrate the efficiency of seqMINER, we have carried out a comprehensive analysis of genome-wide chromatin modification data in mouse embryonic stem cells to understand the global epigenetic landscape and its change through cellular differentiation.


Subject(s)
Chromatin Immunoprecipitation , Genomics/methods , High-Throughput Nucleotide Sequencing , Algorithms , Animals , Brain/metabolism , Chromatin/metabolism , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Histones/metabolism , Mice , Promoter Regions, Genetic , Software
20.
Epigenetics Chromatin ; 3(1): 18, 2010 Oct 20.
Article in English | MEDLINE | ID: mdl-20961410

ABSTRACT

Histone acetylation is one of the key regulatory mechanisms controlling transcriptional activity in eukaryotic cells. In higher eukaryotes, a number of nuclear histone acetyltransferase (HAT) enzymes have been identified, most of which are part of a large multisubunit complex. This diversity, combined with the large number of potentially acetylable lysines on histones, suggested the existence of a specific regulatory mechanism based on the substrate specificity of HATs. Over the past decade, intensive characterisations of the HAT complexes have been carried out. However, the precise mode of action of HATs, and particularly the functional differences amongst these complexes, remains elusive. Here we review current insights into the functional role of HATs, focusing on the specificity of their action. Studies based on biochemical as well as genetic approaches suggested that HATs exert a high degree of specificity in their acetylation spectra and in the cellular processes they regulate. However, a different view emerged recently from genomic approaches that provided genome-wide maps of HAT recruitments. The careful analysis of genomic data suggests that all HAT complexes would be simultaneously recruited to a similar set of loci in the genome, arguing for a low specificity in their function. In this review, we discuss the significance of these apparent contradictions and suggest a new model that integrates biochemical, genetic and genome-wide data to better describe the functional specificity of HAT complexes.

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