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Analysis of β-globin Chromatin Micro-Environment Using a Novel 3C Variant, 4Cv  [PDF]
Ryan C. Pink,Christopher H. Eskiw,Daniel P. Caley,David R. F. Carter
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0013045
Abstract: Higher order chromatin folding is critical to a number of developmental processes, including the regulation of gene expression. Recently developed biochemical techniques such as RNA TRAP and chromosome conformation capture (3C) have provided us with the tools to probe chromosomal structures. These techniques have been applied to the β-globin locus, revealing a complex pattern of interactions with regions along the chromosome that the gene resides on. However, biochemical and microscopy data on the nature of β-globin interactions with other chromosomes is contradictory. Therefore we developed a novel 4C variant, Complete-genome 3C by vectorette amplification (4Cv), which allows an unbiased and quantitative method to examine chromosomal structure. We have used 4Cv to study the microenvironment of the β-globin locus in mice and show that a significant proportion of the interactions of β-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, β-globin is more likely to interact with other chromosomes, compared to the brain where the gene is silent and is more likely to interact with other regions along the same chromosome. Our data suggest that transcriptional activation of the β-globin locus leads to a change in nuclear position relative to the chromosome territory.
Spatial organization of the chicken beta-globin gene domain in erythroid cells of embryonic and adult lineages
Sergey V Ulianov, Alexey A Gavrilov, Sergey V Razin
Epigenetics & Chromatin , 2012, DOI: 10.1186/1756-8935-5-16
Abstract: Using chromosome conformation capture (3C), we have compared the spatial configuration of the β-globin gene domain in chicken red blood cells (RBCs) expressing embryonic (3-day-old RBCs) and adult (9-day-old RBCs) β-globin genes. In contrast to observations made in the mouse model, we found that in the chicken, the early embryonic β-globin gene, Ε, did not interact with the locus control region in RBCs of embryonic lineage (3-day RBCs), where this gene is actively transcribed. In contrast to the mouse model, a strong interaction of the promoter of another embryonic β-globin gene, ρ, with the promoter of the adult β-globin gene, βA, was observed in RBCs from both 3-day and 9-day chicken embryos. Finally, we have demonstrated that insulators flanking the chicken β-globin gene domain from the upstream and from the downstream interact with each other, which places the area characterized by lineage-specific sensitivity to DNase I in a separate chromatin loop.Taken together, our results strongly support the ACH model but show that within a domain of tissue-specific genes, the active status of a promoter does not necessarily correlate with the recruitment of this promoter to the ACH.The domain of chicken beta-globin genes is located on chromosome 1 and has a length of approximately 33 kb. It includes a cluster of four beta-globin genes: ρ (HBG1), βH (HBE1), βA (HBG2) and Ε (HBE) and several distant regulatory regions, which are marked with sites of hypersensitivity to DNase I (HS) and are necessary for the regulation of transcription, replication and chromatin status of the domain [1,2]. The locus control region of the domain (LCR) is located upstream of the embryonic β-globin gene ρ and is composed of three blocks co-localizing with the erythroid cell-specific HSs 1 to 3 [3,4]. A constitutive HS4 located upstream of the LCR marks the position of the well-studied CTCF-dependent (CTCF ‐ D?D?D?TC-binding protein factor) insulator [5-9]. The CTCF-dependent enhancer-blocking e
Role of CTCF Protein in Regulating FMR1 Locus Transcription  [PDF]
Stella Lanni,Martina Goracci,Loredana Borrelli,Giorgia Mancano,Pietro Chiurazzi,Umberto Moscato,Fabrizio Ferrè,Manuela Helmer-Citterich,Elisabetta Tabolacci,Giovanni Neri
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003601
Abstract: Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the FMR1 gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (FMR1-AS1), starting from both promoter and intron 2 of the FMR1 gene, was demonstrated in transcriptionally active alleles, but not in silent FXS alleles. Moreover, a DNA methylation boundary, which is lost in FXS, was recently identified upstream of the FMR1 gene. Several nuclear proteins bind to this region, like the insulator protein CTCF. Here we demonstrate for the first time that rare unmethylated full mutation (UFM) alleles present the same boundary described in wild type (WT) alleles and that CTCF binds to this region, as well as to the FMR1 gene promoter, exon 1 and intron 2 binding sites. Contrariwise, DNA methylation prevents CTCF binding to FXS alleles. Drug-induced CpGs demethylation does not restore this binding. CTCF knock-down experiments clearly established that CTCF does not act as insulator at the active FMR1 locus, despite the presence of a CGG expansion. CTCF depletion induces heterochromatinic histone configuration of the FMR1 locus and results in reduction of FMR1 transcription, which however is not accompanied by spreading of DNA methylation towards the FMR1 promoter. CTCF depletion is also associated with FMR1-AS1 mRNA reduction. Antisense RNA, like sense transcript, is upregulated in UFM and absent in FXS cells and its splicing is correlated to that of the FMR1-mRNA. We conclude that CTCF has a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis.
5′HS5 of the Human β-globin Locus Control Region Is Dispensable for the Formation of the β-globin Active Chromatin Hub  [PDF]
Ping Kei Chan, Albert Wai, Sjaak Philipsen, Kian-Cheng Tan-Un
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002134
Abstract: Hypersensitive site 5 (5′HS5) of the β-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 5′HS5 in the three dimensional organization of the β-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that when 5′HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 5′HS5 does not have an appreciable effect on the three dimensional organization of the β-globin locus. This rules out models in which 5′HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 5′HS5 by using chromatin immunoprecipitation (ChIP) assays. We detect low levels of CTCF binding to 5′HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 5′HS5 in definitive erythroid cells. Thus, binding of CTCF to 5′HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality.
Eos Negatively Regulates Human γ-globin Gene Transcription during Erythroid Differentiation  [PDF]
Hai-Chuan Yu, Hua-Lu Zhao, Zhi-Kui Wu, Jun-Wu Zhang
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0022907
Abstract: Background Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4), a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. Methodology/Principal Findings Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs). DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3) of the β-globin locus control region (LCR), the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C) assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. Conclusions/Significance Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation.
CTCF Mediates the Cell-Type Specific Spatial Organization of the Kcnq5 Locus and the Local Gene Regulation  [PDF]
Licheng Ren, Yang Wang, Minglei Shi, Xiaoning Wang, Zhong Yang, Zhihu Zhao
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031416
Abstract: Chromatin loops play important roles in the dynamic spatial organization of genes in the nucleus. Growing evidence has revealed that the multivalent functional zinc finger protein CCCTC-binding factor (CTCF) is a master regulator of genome spatial organization, and mediates the ubiquitous chromatin loops within the genome. Using circular chromosome conformation capture (4C) methodology, we discovered that CTCF may be a master organizer in mediating the spatial organization of the kcnq5 gene locus. We characterized the cell-type specific spatial organization of the kcnq5 gene locus mediated by CTCF in detail using chromosome conformation capture (3C) and 3C-derived techniques. Cohesion also participated in mediating the organization of this locus. RNAi-mediated knockdown of CTCF sharply diminished the interaction frequencies between the chromatin loops of the kcnq5 gene locus and down-regulated local gene expression. Functional analysis showed that the interacting chromatin loops of the kcnq5 gene locus can repress the gene expression in a luciferase reporter assay. These interacting chromatin fragments were a series of repressing elements whose contacts were mediated by CTCF. Therefore, these findings suggested that the dynamical spatial organization of the kcnq5 locus regulates local gene expression.
Cohesin Is Required for Higher-Order Chromatin Conformation at the Imprinted IGF2-H19 Locus  [PDF]
Raffaella Nativio equal contributor,Kerstin S. Wendt equal contributor,Yoko Ito,Joanna E. Huddleston,Santiago Uribe-Lewis,Kathryn Woodfine,Christel Krueger,Wolf Reik,Jan-Michael Peters ? ,Adele Murrell ?
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000739
Abstract: Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi–mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF–mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin's function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion.
Transcriptional and Proteolytic Regulation of the Toxin-Antitoxin Locus vapBC10 (ssr2962/slr1767) on the Chromosome of Synechocystis sp. PCC 6803  [PDF]
Degang Ning, Shuibing Liu, Weidong Xu, Qiang Zhuang, Chongwei Wen, Xiaoxia Tang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0080716
Abstract: VapBC toxin-antitoxin (TA) systems are defined by the association of a PIN-domain toxin with a DNA-binding antitoxin, and are thought to play important physiological roles in bacteria and archaea. Recently, the PIN-associated gene pair PIN-COG2442 was proposed to encode VapBC-family TA system and found to be abundant in cyanobacteria. However, the features of these predicted TA loci remain under investigation. We here report characterization of the PIN-COG2442 locus vapBC10 (ssr2962/slr1767) on the chromosome of Synechocystis sp. PCC 6803. RT-PCR analysis revealed that the vapBC10 genes were co-transcribed under normal growth conditions. Ectopic expression of the PIN-domain protein VapC10 caused growth arrest of Escherichia coli that does not possess vapBC TA locus. Coincidentally, this growth-inhibition effect could be neutralized by either simultaneous or subsequent production of the COG2442-domain protein VapB10 through formation of the TA complex VapBC10 in vivo. In contrast to the transcription repression activity of the well-studied antitoxins, VapB10 positively auto-regulated the transcription of its own operon via specific binding to the promoter region. Furthermore, in vivo experiments in E. coli demonstrated that the Synechocystis protease ClpXP2s, rather than Lons, could cleave VapB10 and proteolytically activate the VapC10 toxicity. Our results show that the PIN-COG2442 locus vapBC10 encodes a functional VapBC TA system with an alternative mechanism for the transcriptional auto-regulation of its own operon.
Upstream Distal Regulatory Elements Contact the Lmo2 Promoter in Mouse Erythroid Cells  [PDF]
Anandi Bhattacharya, Chih-Yu Chen, Sara Ho, Jennifer A. Mitchell
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0052880
Abstract: The Lim domain only 2 (Lmo2) gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Several distal regulatory elements have been identified upstream of the Lmo2 gene in the human and mouse genomes that are capable of enhancing reporter gene expression in erythroid cells and may be responsible for the high level transcription of Lmo2 in the erythroid lineage. In this study we investigate how these elements regulate transcription of Lmo2 and whether or not they function cooperatively in the endogenous context. Chromosome conformation capture (3C) experiments show that chromatin-chromatin interactions exist between upstream regulatory elements and the Lmo2 promoter in erythroid cells but that these interactions are absent from kidney where Lmo2 is transcribed at twelve fold lower levels. Specifically, long range chromatin-chromatin interactions occur between the Lmo2 proximal promoter and two broad regions, 3–31 and 66–105 kb upstream of Lmo2, which we term the proximal and distal control regions for Lmo2 (pCR and dCR respectively). Each of these regions is bound by several transcription factors suggesting that multiple regulatory elements cooperate in regulating high level transcription of Lmo2 in erythroid cells. Binding of CTCF and cohesin which support chromatin loops at other loci were also found within the dCR and at the Lmo2 proximal promoter. Intergenic transcription occurs throughout the dCR in erythroid cells but not in kidney suggesting a role for these intergenic transcripts in regulating Lmo2, similar to the broad domain of intergenic transcription observed at the human β-globin locus control region. Our data supports a model in which the dCR functions through a chromatin looping mechanism to contact and enhance Lmo2 transcription specifically in erythroid cells. Furthermore, these chromatin loops are supported by the cohesin complex recruited to both CTCF and transcription factor bound regions.
Uncovering Enhancer Functions Using the α-Globin Locus  [PDF]
Douglas Vernimmen
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004668
Abstract: Over the last three decades, studies of the α- and β-globin genes clusters have led to elucidation of the general principles of mammalian gene regulation, such as RNA stability, termination of transcription, and, more importantly, the identification of remote regulatory elements. More recently, detailed studies of α-globin regulation, using both mouse and human loci, allowed the dissection of the sequential order in which transcription factors are recruited to the locus during lineage specification. These studies demonstrated the importance of the remote regulatory elements in the recruitment of RNA polymerase II (PolII) together with their role in the generation of intrachromosomal loops within the locus and the removal of polycomb complexes during differentiation. The multiple roles attributed to remote regulatory elements that have emerged from these studies will be discussed.
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