[show abstract][hide abstract] ABSTRACT: Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells in vivo. Using targeted chromatin purification (TChP), a double-pull-down strategy with a tetracycline-sensitive "hook" bound to a specific promoter, we identify transcription factors bound to the repressed γ-globin gene-associated regulatory regions. After validation of the binding, we show that, in human primary erythroid cells, knockdown of a number of these transcription factors induces γ-globin gene expression. Reactivation of γ-globin gene expression ameliorates the symptoms of β-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients.
[show abstract][hide abstract] ABSTRACT: The coupling of chromosome conformation capture (3C) with next-generation sequencing technologies enables the high-throughput detection of long-range genomic interactions, via the generation of ligation products between DNA sequences, which are closely juxtaposed in vivo. These interactions involve promoter regions, enhancers and other regulatory and structural elements of chromosomes and can reveal key details of the regulation of gene expression. 3C-seq is a variant of the method for the detection of interactions between one chosen genomic element (viewpoint) and the rest of the genome. We present r3Cseq, an R/Bioconductor package designed to perform 3C-seq data analysis in a number of different experimental designs. The package reads a common aligned read input format, provides data normalization, allows the visualization of candidate interaction regions and detects statistically significant chromatin interactions, thus greatly facilitating hypothesis generation and the interpretation of experimental results. We further demonstrate its use on a series of real-world applications.
Nucleic Acids Research 05/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.
Genes & development 04/2013; 27(7):767-777. · 12.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1-8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.
[show abstract][hide abstract] ABSTRACT: The first site exhibiting hematopoietic activity in mammalian development is the yolk sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in BL-CFCs to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk sac hematopoiesis and vascular development in Ldb1(-/-) mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome-wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signalling pathways during the very early stages of hematopoietic/endothelial development and the specific signalling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways, not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells.
[show abstract][hide abstract] ABSTRACT: RUNX1 is known to be an essential transcription factor for generating hematopoietic stem cells (HSC), but much less is known about its role in the downstream process of hematopoietic differentiation. RUNX1 has been shown to be part of a large transcription factor complex, together with LDB1, GATA1, TAL1, and ETO2 (N. Meier et al., Development 133:4913-4923, 2006) in erythroid cells. We used a tagging strategy to show that RUNX1 interacts with two novel protein partners, LSD1 and MYEF2, in erythroid cells. MYEF2 is bound in undifferentiated cells and is lost upon differentiation, whereas LSD1 is bound in differentiated cells. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and microarray expression analysis were used to show that RUNX1 binds approximately 9,000 target sites in erythroid cells and is primarily active in the undifferentiated state. Functional analysis shows that a subset of the target genes is suppressed by RUNX1 via the newly identified partner MYEF2. Knockdown of Myef2 expression in developing zebrafish results in a reduced number of HSC.
Molecular and cellular biology 07/2012; 32(19):3814-22. · 6.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system.
[show abstract][hide abstract] ABSTRACT: Regulation of V(D)J recombination events at immunoglobulin (Ig) and T-cell receptor loci in lymphoid cells is complex and achieved via changes in substrate accessibility. Various studies over the last year have identified the DNA-binding zinc-finger protein CCCTC-binding factor (CTCF) as a crucial regulator of long-range chromatin interactions. CTCF often controls specific interactions by preventing inappropriate communication between neighboring regulatory elements or independent chromatin domains. Although recent gene targeting experiments demonstrated that the presence of the CTCF protein is not required for the process of V(D)J recombination per se, CTCF turned out to be essential to control order, lineage specificity and to balance the Ig V gene repertoire. Moreover, CTCF was shown to restrict activity of κ enhancer elements to the Ig κ locus. In this review, we discuss CTCF function in the regulation of V(D)J recombination on the basis of established knowledge on CTCF-mediated chromatin loop domains in various other loci, including the imprinted H19-Igf2 locus as well as the complex β-globin, MHC class II and IFN-γ loci. Moreover, we discuss that loss of CTCF-mediated restriction of enhancer activity may well contribute to oncogenic activation, when in chromosomal translocations Ig enhancer elements and oncogenes appear in a novel genomic context.
[show abstract][hide abstract] ABSTRACT: Gfi-1B is a transcriptional repressor essential for the regulation of erythropoiesis and megakaryopoiesis. Here we identify Gfi-1B p32, a Gfi-1B isoform, as essential for erythroid differentiation. Gfi-1B p32 is generated by alternative splicing and lacks the two first zinc finger domains of the protein. Selective knock down of Gfi-1B p32 compromises erythroid differentiation, whereas its ectopic expression induces erythropoiesis in the absence of erythropoietin. Gfi-1B p32 isoform binds to Gfi-1B target gene promoters and associates with the LSD1-CoREST repressor complex more efficiently than the major Gfi-1B p37 isoform. Furthermore, we show that Gfi-1B includes a KSKK motif in its SNAG domain, which recruits the repressor complex only when dimethylated on lysine 8. Mutation of lysine 8 prevents Gfi-1B p32-induced erythroid development. Our results thus highlight a key role for the alternatively spliced Gfi-1B p32 isoform in erythroid development.
[show abstract][hide abstract] ABSTRACT: The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping.
The EMBO Journal 12/2011; 31(4):986-99. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Regulation of immunoglobulin (Ig) V(D)J gene rearrangement is dependent on higher-order chromatin organization. Here, we studied the in vivo function of the DNA-binding zinc-finger protein CTCF, which regulates interactions between enhancers and promoters. By conditional deletion of the Ctcf gene in the B cell lineage, we demonstrate that loss of CTCF allowed Ig heavy chain recombination, but pre-B cell proliferation and differentiation was severely impaired. In the absence of CTCF, the Igκ light chain locus showed increased proximal and reduced distal Vκ usage. This was associated with enhanced proximal Vκ and reduced Jκ germline transcription. Chromosome conformation capture experiments demonstrated that CTCF limits interactions of the Igκ enhancers with the proximal V(κ) gene region and prevents inappropriate interactions between these strong enhancers and elements outside the Igκ locus. Thus, although Ig gene recombination can occur in the absence of CTCF, it is a critical factor determining Vκ segment choice for recombination.
[show abstract][hide abstract] ABSTRACT: Transcription factors (TFs) play a central role in the development of multicellular organisms. The sequential actions of critical TFs direct cells to adopt defined differentiation pathways leading to functional, fully differentiated tissues. Here, we describe a generic experimental pipeline that integrates biochemistry, genetics and next generation sequencing with bioinformatics to characterize TF complexes composition, function and target genes at a genome-wide scale. We show an application of this experimental pipeline which aims to unravel the molecular events taking place during hematopoietic cell differentiation.
[show abstract][hide abstract] ABSTRACT: Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell-derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.
Nature medicine 05/2010; 16(5):571-9, 1p following 579. · 27.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex-in particular, the binding of the negative regulators Eto2 and Mtgr1-are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences.
Genes & development 02/2010; 24(3):277-89. · 12.08 Impact Factor