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Pre-B Cell Receptor Signaling Induces Immunoglobulin κ Locus Accessibility by Functional Redistribution of Enhancer-Mediated Chromatin Interactions  [PDF]
Ralph Stadhouders,Marjolein J. W. de Bruijn,Magdalena B. Rother,Saravanan Yuvaraj,Claudia Ribeiro de Almeida,Petros Kolovos,Menno C. Van Zelm,Wilfred van Ijcken,Frank Grosveld,Eric Soler,Rudi W. Hendriks
PLOS Biology , 2014, DOI: 10.1371/journal.pbio.1001791
Abstract: During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vκ transcription. To investigate whether pre-BCR signaling modulates Vκ accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ~3.2 Mb Vκ region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3′κ enhancer with Vκ genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vκ genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vκ genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the Vκ region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vκ region, whereby the two enhancers play distinct roles.
Targeting Of Somatic Hypermutation By immunoglobulin Enhancer And Enhancer-Like Sequences  [PDF]
Jean-Marie Buerstedde ,Jukka Alinikula,Hiroshi Arakawa,Jessica J. McDonald,David G. Schatz
PLOS Biology , 2014, DOI: 10.1371/journal.pbio.1001831
Abstract: Somatic hypermutation (SH) generates point mutations within rearranged immunoglobulin (Ig) genes of activated B cells, providing genetic diversity for the affinity maturation of antibodies. SH requires the activation-induced cytidine deaminase (AID) protein and transcription of the mutation target sequence, but how the Ig gene specificity of mutations is achieved has remained elusive. We show here using a sensitive and carefully controlled assay that the Ig enhancers strongly activate SH in neighboring genes even though their stimulation of transcription is negligible. Mutations in certain E-box, NFκB, MEF2, or Ets family binding sites—known to be important for the transcriptional role of Ig enhancers—impair or abolish the activity. Full activation of SH typically requires a combination of multiple Ig enhancer and enhancer-like elements. The mechanism is evolutionarily conserved, as mammalian Ig lambda and Ig heavy chain intron enhancers efficiently stimulate hypermutation in chicken cells. Our results demonstrate a novel regulatory function for Ig enhancers, indicating that they either recruit AID or alter the accessibility of the nearby transcription units.
Evolution and Genetic Architecture of Chromatin Accessibility and Function in Yeast  [PDF]
Caitlin F. Connelly,Jon Wakefield,Joshua M. Akey
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004427
Abstract: Chromatin accessibility is an important functional genomics phenotype that influences transcription factor binding and gene expression. Genome-scale technologies allow chromatin accessibility to be mapped with high-resolution, facilitating detailed analyses into the genetic architecture and evolution of chromatin structure within and between species. We performed Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-Seq) to map chromatin accessibility in two parental haploid yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus and their diploid hybrid. We show that although broad-scale characteristics of the chromatin landscape are well conserved between these species, accessibility is significantly different for 947 regions upstream of genes that are enriched for GO terms such as intracellular transport and protein localization exhibit. We also develop new statistical methods to investigate the genetic architecture of variation in chromatin accessibility between species, and find that cis effects are more common and of greater magnitude than trans effects. Interestingly, we find that cis and trans effects at individual genes are often negatively correlated, suggesting widespread compensatory evolution to stabilize levels of chromatin accessibility. Finally, we demonstrate that the relationship between chromatin accessibility and gene expression levels is complex, and a significant proportion of differences in chromatin accessibility might be functionally benign.
Modifications of histone cores and tails in V(D)J recombination
Kathrin Muegge
Genome Biology , 2003, DOI: 10.1186/gb-2003-4-4-211
Abstract: The organization of DNA into chromatin allows dense packaging of the genetic material and protects it against damage. Chromatin packaging also allows functional organization of the genome into active regions of 'open' euchromatin, which are accessible to regulatory factors, and silent regions of condensed heterochromatin [1,2]. The reversible switch from euchromatin to heterochromatin provides a means of controlling processes occurring on the DNA, such as replication and transcription, and has been proposed - in the so-called 'accessibility hypothesis' - to be crucial for regulating V(D)J recombination during immune-system development [3].During V(D)J recombination, gene segments that encode variable receptors within the immune system are recombined and newly assembled to allow expression of distinct immunoglobulins or T-cell receptors (TCRs) [4,5]. The V(D)J recombinase, a dimer of the site-specific recombination proteins RAG1 and RAG2, binds to recombination signal sequences that flank the V, D and J gene segments and initiates the process of cleaving the DNA sequences that are to be rearranged. Because the same recombinase is present in both T and B cells, but only B cells fully rearrange their immunoglobulin loci and only T cells their TCR loci, it has been proposed that a specific modulation of chromatin structure might open up recombination signal sequences to provide access for the recombinase. This 'chromatin accessibility' model could explain lineage and allele specificity of recombination as well as the temporal order of V(D)J rearrangement during development. A recent study from the Struhl and Oettinger laboratories [6] has provided new evidence in support of this model.The accessibility model gained support from the observation of close association between the processes of transcription and recombination of unrearranged V, D and J fragments. Deletion of cis-acting enhancer or promoter sequences frequently inhibited both processes [5,7], and it was propos
Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells  [cached]
Sumitomo Yasuhiko,Higashitsuji Hiroaki,Higashitsuji Hisako,Liu Yu
BMC Biotechnology , 2012, DOI: 10.1186/1472-6750-12-72
Abstract: Background There are a growing number of reports on the sub-physiological temperature culturing of mammalian cells for increased recombinant protein yields. However, the effect varies and the reasons for the enhancement are not fully elucidated. Expression of cold-inducible RNA-binding protein (cirp, also called cirbp or hnRNP A18) is known to be induced in response to mild, but not severe, hypothermia in mammalian cells. To clarify the molecular mechanism underlying the induction and to exploit this to improve the productivity of recombinant proteins, we tried to identify the regulatory sequence(s) in the 5′ flanking region of the mouse cirp gene. Results By transiently transfecting HEK293 cells with plasmids expressing chloramphenicol acetyltransferase as a reporter, we found that the cirp 5′ flanking region octanucleotide 5′-TCCCCGCC-3′ is a mild-cold responsive element (MCRE). When 3 copies of MCRE were placed upstream of the CMV promoter and used in transient transfection, reporter gene expression was increased 3- to 7-fold at 32°C relative to 37°C in various cell lines including HEK293, U-2 OS, NIH/3T3, BALB/3T3 and CHO-K1 cells. In stable transfectants, MCRE also enhanced the reporter gene expression at 32°C, although more copy numbers of MCRE were necessary. Sp1 transcription factor bound to MCRE in vitro. Immunohistochemistry and chromatin immunoprecipitation assays demonstrated that more Sp1, but not Sp3, was localized in the nucleus to bind to the cirp regulatory region containing MCRE at 32°C than 37°C. Overexpression of Sp1 protein increased the expression of endogenous Cirp as well as a reporter gene driven by the 5′ flanking region of the cirp gene, and down-regulation of Sp1 had the opposite effect. Mutations within the MCRE sequence in the 5′ flanking region abolished the effects of Sp1 on the reporter gene expression both at 37°C and 32°C. Conclusions Cold-induced, as well as constitutive, expression of cirp is dependent, at least partly, on MCRE and Sp1. The present novel enhancer permits conditional high-level gene expression at moderately low culture temperatures and could be utilized to increase the yield of recombinant proteins in mammalian cells.
Effective Blocking of the White Enhancer Requires Cooperation between Two Main Mechanisms Suggested for the Insulator Function  [PDF]
Olga Kyrchanova equal contributor,Oksana Maksimenko equal contributor,Viacheslav Stakhov,Tatyana Ivlieva,Alexander Parshikov,Vasily M. Studitsky,Pavel Georgiev
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003606
Abstract: Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. In this study, we examined the role of chromatin loops formed by two unrelated insulators, gypsy and Fab-7, in their enhancer-blocking activity. To test for this activity, we selected the white reporter gene that is activated by the eye-specific enhancer. The results showed that one copy of the gypsy or Fab-7 insulator failed to block the eye enhancer in most of genomic sites, whereas a chromatin loop formed by two gypsy insulators flanking either the eye enhancer or the reporter completely blocked white stimulation by the enhancer. However, strong enhancer blocking was achieved due not only to chromatin loop formation but also to the direct interaction of the gypsy insulator with the eye enhancer, which was confirmed by the 3C assay. In particular, it was observed that Mod(mdg4)-67.2, a component of the gypsy insulator, interacted with the Zeste protein, which is critical for the eye enhancer–white promoter communication. These results suggest that efficient enhancer blocking depends on the combination of two factors: chromatin loop formation by paired insulators, which generates physical constraints for enhancer–promoter communication, and the direct interaction of proteins recruited to an insulator and to the enhancer–promoter pair.
LMP1-augmented kappa intron enhancer activity contributes to upregulation expression of Ig kappa light chain via NF-kappaB and AP-1 pathways in nasopharyngeal carcinoma cells
HaiDan Liu, Hui Zheng, Zhi Duan, DuoSha Hu, Ming Li, SuFang Liu, ZiJian Li, XiYun Deng, ZhenLian Wang, Min Tang, Ying Shi, Wei Yi, Ya Cao
Molecular Cancer , 2009, DOI: 10.1186/1476-4598-8-92
Abstract: In this study, luciferase reporter plasmid containing human wild-type iEκ, and its derivative plasmids containing mutant binding sites for transcription factor NF-κB or AP-1 were constructed. Luciferase reporter assays demonstrate iEκ is active in Igκ-expressing NPC cells and LMP1 expression can upregulate the activity of iEκ in NPC cells. Mutation of the NF-κB or AP-1 site within and downstream the iEκ, inhibition of the NF-κB and AP-1 pathways by their respective chemical inhibitor Bay11-7082 and SP600125 as well as stable or transient expression of dominant-negative mutant of IκBα (DNMIκBα) or of c-Jun (TAM67) indicate that both sites are functional and LMP1-enhanced iEκ activity is partly regulated by these two sites. Gel shift assays show that LMP1 promotes NF-κB subunits p52 and p65 as well as AP-1 family members c-Jun and c-Fos binding to the κNF-κB and the κAP-1 motifs in vitro, respectively. Both chemical inhibitors and dominant negative mutants targeting for NF-κB and AP-1 pathways can attenuate the LMP1-enhanced bindings. Co-IP assays using nuclear extracts from HNE2-LMP1 cells reveal that p52 and p65, c-Jun and c-Fos proteins interact with each other at endogenous levels. ChIP assays further demonstrate p52 and p65 binding to the κB motif as well as c-Jun and c-Fos binding to the AP-1 motif of Ig kappa gene in vivo.These results suggest that human iEκ is active in Igκ-expressing NPC cells and LMP1-stimulated NF-κB and AP-1 activation results in an augmenting activation of the iEκ. LMP1 promotes the interactions of heterodimeric NF-κB (p52/p65) and heterodimeric AP-1 (c-Jun/c-Fos) transcription factors with the human iEκ enhancer region are important for the upregulation of kappa light chain in LMP1-positive nasopharyngeal carcinoma cells.While considerable evidence has shown that immunoglobulins (Igs) "unexpectly" expressed in malignant tumors of epithelial origin [1-10], much less is known about the molecular mechanisms of nonlymphoid cells expressing I
On accessibility of finitely generated groups  [PDF]
Richard Weidmann
Mathematics , 2007,
Abstract: We prove an accessibility result for finitely generated groups that combines Sela's acylindrical accessibility with Linell accessibility.
A Weakened Transcriptional Enhancer Yields Variegated Gene Expression  [PDF]
Cathy Collins, Peter Azmi, Maribel Berru, Xiaofu Zhu, Marc J. Shulman
PLOS ONE , 2006, DOI: 10.1371/journal.pone.0000033
Abstract: Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, ~1.4 kb 3′ of the promoter, but inactive ~6 kb 3′, indicating that the activity of the core enhancer was distance-dependent. A longer segment – the core enhancer plus ~1 kb of 3′ flanking material, including the 3′ matrix attachment region – was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3′ flank includes binding sites for at least two activators.
Immunoglobulin Therapy  [cached]
Y?ld?z Camc?o?lu
Cocuk Enfeksiyon Dergisi , 2009,
Abstract: The protective effect of immunoglobulin administration has been known for the last 50 years. Immune globulin (IG)is a sterile IgG product, prepared for intramuscular, subcutaeous injection and intravenous infusion. Intravenous immunoglobulin (IVIG) has several advantages over other immunoglobulins. Immunoglobulin preparations are safe and effective. Human immunoglobulin replacement therapy is the mainstay of therapy for the patients with antibody deficiencies such as X-linked agammaglobulinemia (X-LA), common variable immunodeficiencies (CVI), hyper-IgM syndrome and combined B- and T-cell defect.In this review, you will find the answers to commonly asked questions related to IVIG and recent advances in IVIG therapy.
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