Spatial organisation of the ß-globin locus

Alle multi-cellulaire organismen beginnen als een enkele bevruchte eicel. Gedurende de differentiatie wordt het aantal cellen vermeerderd door middel van celdeling. De cellen specialiseren zich tevens in verschillende celtypes zoals hersen-, bloed- en spiercellen. Toch bevatten al deze cellen dezelfde genetische informatie dat is opgeslagen in miljoenen base paren DNA, welke georganiseerd zijn in grote elementen die chromosomen worden genoemd. Een DNA sequentie die codeert voor een bepaalde overerfbare eigenschap (vaak een eiwit) wordt een gen genoemd. Het gehalte aan genen is gelijk in elke cel (ongeveer 25.000 genen). Het verschil tussen de diverse celtypes wordt daarom niet bepaald door de genomische opbouw van een cel maar juist hoe deze genomische opbouw gebruikt wordt, of anders gezegd; welke genen geactiveerd of juist onderdrukt worden. De activatie van genen wordt strak gereguleerd qua plaats en tijd en een gebrek aan juiste transcriptionele regulatie resulteert vaak in allerlei ziekten zoals b.v. kanker. Bij de juiste transcriptionele activatie van genen in hogere eukaryoten zijn verschillende regulerende DNA elementen betrokken; de promoter wordt vlakbij het gen gevonden terwijl andere elementen zoals enhancers zeer ver van het te activeren gen kunnen liggen. Een van de belangrijkste vragen in de moleculaire biologie is hoe deze zogeheten cis-regulerende elementen over deze aanzienlijke afstanden met de promoter van een gen kunnen communiceren. Verschillende mechanismen zijn voorgesteld voor deze communicatie: o.a. het looping model, het tracking model en het linking model. Deze modellen worden in hoofdstuk 1 in detail besproken. Een goed voorbeeld van een gespecialiseerde cel in zoogdieren is de rode bloed cel. Het meest voorkomende molecuul in rode bloedcellen is het zuurstof en kooldioxide transporterende hemoglobine dat opgebouwd is uit twee a-globine eiwitten, twee ß-globine eiwitten en vier heem groepen. Het ß-globine eiwit wordt gecodeerd door genen in het ß-globine locus. Het muizen ß-globine locus bevat vier ß-achtige genen. Twee daarvan, de embryonale ß-globine genen, komen in het embryo tot expressie terwijl de twee andere genen, de volwassen ß-globine genen actief worden in een later stadium van de ontwikkeling

Transcription factor binding at enhancers: Shaping a genomic regulatory landscape in flux

The mammalian genome is packed tightly in the nucleus of the cell. This packing is primarily facilitated by histone proteins and results in an ordered organization of the genome in chromosome territories that can be roughly divided in heterochromatic and euchromatic domains. On top of this organization several distinct gene regulatory elements on the same chromosome or other chromosomes are thought to dynamically communicate via chromatin looping. Advances in genome-wide technologies have revealed the existence of a plethora of these regulatory elements in various eukaryotic genomes. These regulatory elements are defined by particular in vitro assays as promoters, enhancers, insulators, and boundary elements. However, recent studies indicate that the in vivo distinction between these elements is often less strict. Regulatory elements are bound by a mixture of common and lineage-specific transcription factors which mediate the long-range interactions between these elements. Inappropriate modulation of the binding of these transcription factors can alter the interactions between regulatory elements, which in turn leads to aberrant gene expression with disease as an ultimate consequence. Here we discuss the bi-modal behavior of regulatory elements that act in cis (with a focus on enhancers), how their activity is modulated by transcription factor binding and the effect this has on gene regulation

Nuclear positioning rather than contraction controls ordered rearrangements of immunoglobulin loci

Progenitor-B cells recombine their immunoglobulin (Ig) loci to create unique antigen receptors. Despite a common recombination machinery, the Ig heavy and Ig light chain loci rearrange in a stepwise manner. We studied pre-pro-B cells and Rag-/- progenitor-B cells to determine whether Ig locus contraction or nuclear positioning is decisive for stepwise rearrangements. We found that both Ig loci were contracted in pro-B and pre-B cells. Igh relocated from the nuclear lamina to central domains only at the pro-B cell stage, whereas, Igê remained sequestered at the lamina, and only at the pre-B cell stage located to central nuclear domains. Finally, in vitro induced re-positioning of Ig alleles away from the nuclear periphery increased germline transcription of Ig loci in pre-pro-B cells. Thus, Ig locus contraction juxtaposes genomically distant elements to mediate efficient recombination, however, sequential positioning of Ig loci away from the nuclear periphery determines stage-specific accessibility of Ig loci

Allele-specific long-distance regulation dictates IL-32 isoform switching and mediates susceptibility to HIV-1

We integrated data obtained from HIV-1 genome-wide association studies with T cell-derived epigenome data and found that the noncoding intergenic variant rs4349147, which is statistically associated with HIV-1 acquisition, is located in a CD4+ T cell-specific deoxyribonuclease I hypersensitive region, suggesting regulatory potential for this variant. Deletion of the rs4349147 element in Jurkat cells strongly reduced expression of interleukin-32 (IL-32), approximately 10-kb upstream, and chromosome conformation capture assays identified a chromatin loop between rs4349147 and the IL-32 promoter validating its function as a long-distance enhancer. We generated single rs4349147-A or rs4349147-G allele clones and demonstrated that IL-32 enhancer activity and interaction with the IL-32 promoter are strongly allele dependent; rs4349147 -/A cells display reduced IL-32 expression and altered chromatin conformation as compared to rs4349147 G/- cells. Moreover, RNA sequencing demonstrated that rs4349147 G/- cells express a lower relative ratio of IL-32α to non-a isoforms than rs4349147 -/A cells and display increased expression of lymphocyte activation factors rendering them more prone to infection with HIV-1. In agreement, in primary CD4+ T cells, both treatment with recombinant IL-32γ (rIL-32γ) but not rIL-32α, and exogenous lentiviral overexpression of IL-32γ or IL-32β but not IL-32α resulted in a proinflammatory T cell cytokine environment concomitant with increased susceptibility to HIV infection. Our data demonstrate that rs4349147-G promotes transcription of non-IL-32α isoforms, generating a proinflammatory e

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency

A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription

Genetics of skin color variation in Europeans: genome-wide association studies with functional follow-up

In the International Visible Trait Genetics (VisiGen) Consortium, we investigated the genetics of human skin color by combining a series of genome-wide association studies (GWAS) in a total of 17,262 Europeans with functional follow-up of discovered loci. Our GWAS provide the first genome-wide significant evidence for chromosome 20q11.22 harboring the ASIP gene being explicitly associated with skin color in Europeans. In addition, genomic loci at 5p13.2 (SLC45A2), 6p25.3 (IRF4), 15q13.1 (HERC2/OCA2), and 16q24.3 (MC1R) were confirmed to be involved in skin coloration in Europeans. In follow-up gene expression and regulation studies of 22 genes in 20q11.22, we highlighted two novel genes EIF2S2 and GSS, serving as competing functional candidates in this region and providing future research lines. A genetically inferred skin color score obtained from the 9 top-associated SNPs from 9 genes in 940 worldwide samples (HGDP-CEPH) showed a clear gradual pattern in Western Eurasians similar to the distribution of physical skin color, suggesting the used 9 SNPs as suitable markers for DNA prediction of skin color in Europeans and neighboring populations, relevant in future forensic and anthropological investigations

Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development

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