A Functional Role for 4qA/B in the Structural Rearrangement of the 4q35 Region and in the Regulation of FRG1 and ANT1 in Facioscapulohumeral Dystrophy

The number of D4Z4 repeats in the subtelomeric region of chromosome 4q is strongly reduced in patients with Facio-Scapulo-Humeral Dystrophy (FSHD). We performed chromosome conformation capture (3C) analysis to document the interactions taking place among different 4q35 markers. We found that the reduced number of D4Z4 repeats in FSHD myoblasts was associated with a global alteration of the three-dimensional structure of the 4q35 region. Indeed, differently from normal myoblasts, the 4qA/B marker interacted directly with the promoters of the FRG1 and ANT1 genes in FSHD cells. Along with the presence of a newly identified transcriptional enhancer within the 4qA allele, our demonstration of an interaction occurring between chromosomal segments located megabases away on the same chromosome 4q allows to revisit the possible mechanisms leading to FSHD

Chromatin loop domain organization within the 4q35 locus in facioscapulohumeral dystrophy patients versus normal human myoblasts

Fascioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder linked to partial deletion of integral numbers of a 3.3 kb polymorphic repeat, D4Z4, within the subtelomeric region of chromosome 4q. Although the relationship between deletions of D4Z4 and FSHD is well established, how this triggers the disease remains unclear. We have mapped the DNA loop domain containing the D4Z4 repeat cluster in human primary myoblasts and in murine–human hybrids. A nuclear matrix attachment site was found located in the vicinity of the repeat. Prominent in normal human myoblasts and nonmuscular human cells, this site is much weaker in muscle cells derived from FSHD patients, suggesting that the D4Z4 repeat array and upstream genes reside in two loops in nonmuscular cells and normal human myoblasts but in only one loop in FSHD myoblasts. We propose a model whereby the nuclear scaffold/matrix attached region regulates chromatin accessibility and expression of genes implicated in the genesis of FSHD

A nuclear matrix attachment site in the 4q35 locus has an enhancer-blocking activity in vivo: Implications for the facio-scapulo-humeral dystrophy

Facio-scapulo-humeral dystrophy (FSHD), a muscular hereditary disease with a prevalence of 1 in 20,000, is caused by a partial deletion of a subtelomeric repeat array on chromosome 4q. Earlier, we demonstrated the existence in the vicinity of the D4Z4 repeat of a nuclear matrix attachment site, FR-MAR, efficient in normal human myoblasts and nonmuscular human cells but much weaker in muscle cells from FSHD patients. We now report that the D4Z4 repeat contains an exceptionally strong transcriptional enhancer at its 5′-end. This enhancer up-regulates transcription from the promoter of the neighboring FRG1 gene. However, an enhancer blocking activity was found present in FR-MAR that in vitro could protect transcription from the enhancer activity of the D4Z4 array. In vivo, transcription from the FRG1 and FRG2 genes could be down- or up-regulated depending on whether or not FR-MAR is associated with the nuclear matrix. We propose a model for an etiological role of the delocalization of FR-MAR in the genesis of FSHD

ZNF555 protein binds to transcriptional activator site of 4qA allele and ANT1: potential implication in Facioscapulohumeral dystrophy

International audienceFacioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) β-satellite repeats (BSR), a prevalent part of the 4qA allele. Most of the recent FSHD studies have been focused on a DUX4 transcript inside D4Z4 and its tandem contraction in FSHD patients. However, the D4Z4-contraction alone is not pathological, which would also require the 4qA allele. Since little is known about BSR, we investigated the 4qA BSR functional role in the transcriptional control of the FSHD region 4q35. We have shown that an individual BSR possesses enhancer activity leading to activation of the Adenine Nucleotide Translocator 1 gene (ANT1), a major FSHD candidate gene. We have identified ZNF555, a previously uncharacterized protein, as a putative transcriptional factor highly expressed in human primary myoblasts that interacts with the BSR enhancer site and impacts the ANT1 promoter activity in FSHD myoblasts. The discovery of the functional role of the 4qA allele and ZNF555 in the transcriptional control of ANT1 advances our understanding of FSHD pathogenesis and provides potential therapeutic targets

Distinct Distribution of Ectopically Expressed Histone Variants H2A.Bbd and MacroH2A in Open and Closed Chromatin Domains

International audienceIt becomes increasingly evident that nuclesomes are far from being identical to each other. This nucleosome diversity is due partially to the existence of histone variants encoded by separate genes. Among the known histone variants the less characterized are H2A.Bbd and different forms of macroH2A. This is especially true in the case of H2A.Bbd as there are still no commercially available antibodies specific to H2A.Bbd that can be used for chromatin immunoprecipitation (ChIP)

Downregulation of TREM-like transcript-1 and collagen receptor α2 subunit, two novel RUNX1-targets, contributes to platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia

International audienc

Differences in transcription patterns between induced pluripotent stem cells produced from the same germ layer are erased upon differentiation.

Little is known about differences between induced pluripotent stem cells produced from tissues originating from the same germ layer. We have generated human myoblast-derived iPS cells by retroviral transduction of human primary myoblasts with the OCT3/4, SOX2, KLF4 and MYC coding sequences and compared them to iPS produced from human primary fibroblasts. When cultivated in vitro, these iPS cells proved similar to human embryonic stem cells in terms of morphology, expression of embryonic stemness markers and gene promoter methylation patterns. Embryonic bodies were derived that expressed endodermal, mesodermal as well as ectodermal markers. A comparative analysis of transcription patterns revealed significant differences in the gene expression pattern between myoblast- and fibroblast-derived iPS cells. However, these differences were reduced in the mesenchymal stem cells derived from the two iPS cell types were compared

Downregulation of TREM-like transcript (TLT)-1 and collagen receptor α2 subunit, two novel RUNX1-targets, contributes to platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia

Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familialplatelet disorder with predisposition to acute myelogenous leukemia. Multiple aspects ofplatelet function are impaired in these patients, associated with altered expression of genesregulated by RUNX1. We aimed to identify RUNX1-targets involved in platelet functionby combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes.Downregulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrinsubunit α2 (ITGA2) of collagen receptor α2β1, which are involved in platelet aggregationand adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks wasdemonstrated for both genes using chromatin immunoprecipitation. Cloning of theseregions upstream of the respective promoters in lentivirus allowing mCherry reporterexpression showed that RUNX1 positively regulates TREML1 and ITGA2 and thisregulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced inpatient megakaryocytes and platelets. A blocking anti-TLT-1 antibody was able to blockaggregation of normal but not patient platelets, whereas recombinant soluble TLT-1potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiencyin the platelet function defect. Low levels of α2 integrin subunit were demonstrated inpatient platelets and megakaryocytes, coupled with reduced platelet and megakaryocyteadhesion to collagen, both under static and flow conditions. In conclusion, we show thatgene expression profiling of RUNX1 knock-down or mutated megakaryocytes provides asuitable approach to identify novel RUNX1-targets, among which downregulation ofTREML1 and ITGA2 clearly contribute to the platelet phenotype of familial plateletdisorder with predisposition to acute myelogenous leukemia.Fil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Sliwa, Dominika. Institut Gustave Roussy; FranciaFil: Bluteau, Dominique. Ecole Pratique des Hautes Etudes; Francia. Institut Gustave Roussy; FranciaFil: Balayn, Nathalie. Institut Gustave Roussy; FranciaFil: Marin Oyarzún, Cecilia Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Raimbault, Anna. Institut Gustave Roussy; FranciaFil: Bordas, Marie. Institut Gustave Roussy; FranciaFil: Droin, Nathalie. Institut Gustave Roussy; FranciaFil: Pirozhkova, Iryna. Institut Gustave Roussy; FranciaFil: Washington, Valance. Universidad de Puerto Rico; Puerto RicoFil: Goette, Nora Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Marta, Rosana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Favier, Rémi. Institut Gustave Roussy; FranciaFil: Raslova, Hana. Institut Gustave Roussy; FranciaFil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Subnuclear localisation of macroH2A and H2A.Bbd in HeLa S3cells.

<p>HeLa S3 cells stably transfected with either the pOZ -macroH2A1.1 (<b>a</b>) or pOZ- H2A.Bbd (<b>b</b>) plasmid and stained with antibodies against FLAG (green) and H3k27Me3 or nucleophosmin (red). <b>a</b>: macroH2A1.1 is preferentially localised to a single region in interphase HeLa cells and coinsides with H3k27Me3 staining (red). <b>b</b>: H2A.Bbd has a punctate nuclear staining with an exclusion zone (marked by a white arrow) and a strong perinucleolar staining (nucleophosmin, red). The figure shows representative confocal sections. Scale bar: 5 µm.</p