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.

Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia (AML). Multiple aspects of platelet function are impaired in these patients, associated with altered expression of genes regulated by RUNX1 We aimed to identify RUNX1-targets involved in platelet function by combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes (MK). Down-regulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrin subunit alpha (α)-2 (ITGA2) of collagen receptor α2-beta (ß)-1, which are involved in platelet aggregation and adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks was demonstrated for both genes using chromatin immunoprecipitation. Cloning of these regions upstream of the respective promoters in lentivirus allowing mCherry reporter expression showed that RUNX1 positively regulates TREML1 and ITGA2, and this regulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced in patient MK and platelets. A blocking anti-TLT-1 antibody was able to block aggregation of normal but not patient platelets, whereas recombinant soluble TLT-1 potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiency in the platelet function defect. Low levels of α2 integrin subunit were demonstrated in patient platelets and MK, coupled with reduced platelet and MK adhesion to collagen, both under static and flow conditions. In conclusion, we show that gene expression profiling of RUNX1 knock-down or mutated MK provides a suitable approach to identify novel RUNX1 targets, among which downregulation of TREML1 and ITGA2 clearly contribute to the platelet phenotype of familial platelet disorder with predisposition to AML.

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

Facioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) ß-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.

Molecular turnover, the H3.3 dilemma and organismal aging (hypothesis).

The H3.3 histone variant has been a subject of increasing interest in the field of chromatin studies due to its two distinguishing features. First, its incorporation into chromatin is replication independent unlike the replication-coupled deposition of its canonical counterparts H3.1/2. Second, H3.3 has been consistently associated with an active state of chromatin. In accordance, this histone variant should be expected to be causally involved in the regulation of gene expression, or more generally, its incorporation should have downstream consequences for the structure and function of chromatin. This, however, leads to an apparent paradox: In cells that slowly replicate in the organism, H3.3 will accumulate with time, opening the way to aberrant effects on heterochromatin. Here, we review the indications that H3.3 is expected both to be incorporated in the heterochromatin of slowly replicating cells and to retain its functional downstream effects. Implications for organismal aging are discussed.

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.

Distinct distribution of ectopically expressed histone variants H2A.Bbd and MacroH2A in open and closed chromatin domains.

BACKGROUND: It 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). METHODS: We have generated HeLa S3 cell lines stably expressing epitope-tagged versions of macroH2A1.1, H2A.Bbd or canonical H2A and analyzed genomic distribution of the tagged histones using ChIP-on-chip technique. RESULTS: The presence of histone H2A variants macroH2A1.1 and H2A.Bbd has been analyzed in the chromatin of several segments of human chromosomes 11, 16 and X that have been chosen for their different gene densities and chromatin status. Chromatin immunoprecipitation (ChIP) followed by hybridization with custom NimbleGene genomic microarrays demonstrated that in open chromatin domains containing tissue-specific along with housekeeping genes, the H2A.Bbd variant was preferentially associated with the body of a subset of transcribed genes. The macroH2A1.1 variant was virtually absent from some genes and underrepresented in others. In contrast, in closed chromatin domains which contain only tissue-specific genes inactive in HeLa S3 cells, both macroH2A1.1 and H2A.Bbd histone variants were present and often colocalized. CONCLUSIONS: Genomic distribution of macro H2A and H2A.Bbd does not follow any simple rule and is drastically different in open and closed genomic domains.

Loop domain organization of the p53 locus in normal and breast cancer cells correlates with the transcriptional status of the TP53 and the neighboring genes.

P53 is a tumor suppressor protein critical for genome integrity. Although its control at the protein level is well known, the transcriptional regulation of the TP53 gene is still unclear. We have analyzed the organization of the TP53 gene domain using DNA arrays in several breast cancer and control cell lines. We have found that in the control breast epithelial cell line, HB2, the TP53 gene is positioned within a relatively small DNA domain, encompassing 50 kb, delimited by two nuclear matrix attachment sites. Interestingly, this domain structure was found to be radically different in the studied breast cancer cell lines, MCF7, T47D, MDA-MB-231, and BT474, in which the domain size was increased and TP53 transcription was decreased. We propose a model in which the organization of the TP53 gene domain correlates with the transcriptional status of TP53 and neighboring genes.

Interaction in vivo between the two matrix attachment regions flanking a single chromatin loop.

In interphase nuclei as in metaphase chromosomes, the genome is organized into topologically closed loop domains. Here, we have mapped the ends of the loop domain that contains the Ifng (interferon-gamma) gene in primary and cultured murine T-lymphocytes. To determine whether the ends of the loop are located in close proximity to each other in the nuclear space, the 3C (chromosome conformation capture) technique, which detects protein-mediated DNA-DNA interactions, was utilized. A strong interaction was demonstrated between the two ends of the loop, which were close enough to become cross-linked in vivo in the presence of paraformaldehyde. Chromatin immunoprecipitation combined with the 3C technique demonstrated that topoisomerase IIalpha and MeCP2, but not topoisomerase IIbeta, heterochromatin-associated protein HP1 or CTCF, were involved in this interaction. The present findings have important implications in terms of mechanisms of illegitimate recombination that can result in chromosomal translocations and deletions.

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.

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.

Recruitment of RNA polymerase II in the Ifng gene promoter correlates with the nuclear matrix association in activated T helper cells.

Recruitment of the RNA polymerase II transcription complex to the promoter of the Ifng gene has been studied by chromatin immunoprecipitation (ChIP) in activated functionally different CD4+ T helper (Th) cell subsets. In parallel, analysis of association of the nuclear scaffold/matrix with the Ifng gene promoter has been carried out. The RNA polymerase II (RNA pol II) interacted with the Ifng gene promoter in analyzed activated neutral Th cells, IFN-gamma producing Th1 cells and IFN-gamma silent Th2 cells. However, the interaction of the Ifng gene promoter with the nuclear matrix occurred differentially in a lineage-specific manner. The pattern of the nuclear matrix interaction correlated directly with the gene expression. Strong association of the promoter with the nuclear matrix was observed only in the Th1 cell subset where the Ifng gene was actively transcribed. We propose that it is the interaction of the Ifng gene promoter with the nuclear matrix that may set off transcription in activated Th cells by promoter-associated RNA pol II.

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.

High-affinity glutamate transporter GLAST/EAAT1 regulates cell surface expression of glutamine/neutral amino acid transporter ASCT2 in human fetal astrocytes.

Neutral amino acid transporter ASCT2, together with high-affinity glutamate transporters, belongs to the SLC1 gene family of Na(+)-dependent solute carriers and is one of the major transporters of glutamine in cultured astrocytes. Besides glutamine and other high-affinity substrates--alanine, serine, cysteine or threonine, ASCT2 can also translocate protonated glutamate. The present study elucidated substrate-dependent trafficking of ASCT2 in differentiated primary cultures of human fetal astrocytes. The differentiation induced by 8-bromo-cAMP caused dramatic up-regulation of two co-localized and functionally linked astroglial proteins--glutamate transporter GLAST, that is the only high-affinity router of glutamate into cultured astrocytes, and glutamine synthetase (GS), a cytosolic enzyme that converts at least a part of the arriving glutamate into glutamine. In order to distinguish individual intracellular effects of these two substrates on ASCT2, in some cultures glutamine synthetase was effectively knocked down using siRNA silencing technique. In control conditions, regardless of GS levels, almost the entire ASCT2 immunoreactivity was restricted to the cytosol. Both glutamine and alanine, though to different extents, induced partial redistribution of ASCT2 from the cytosolic compartment to the plasma membrane. However, in cultures with high GS expression, micromolar concentrations of glutamate exhibited more pronounced effect on ASCT2 trafficking than the preferred substrates of this carrier. In contrast, glutamate had no effect on ASCT2 distribution in cultures devoid of GS. D-Aspartate, a metabolically inert substrate effectively transported by GLAST, had no effect in any cell culture utilized. It seems that intracellular glutamine produced by GS from glutamate that, in turn, is supplied by GLAST, is a more potent inducer of ASCT2 trafficking to the cell surface than the ASCT2-mediated translocation of extracellular substrates. At lower pH values (6.2-6.7), the cell surface pool of ASCT2 was significantly larger than at physiological pH. In addition, high concentrations of glutamate, independently from GLAST or glutamate receptor activation, induced further arrival of ASCT2 to the plasma membrane. The pH-dependent functional activation of ASCT2 and the ASCT2-mediated glutamate uptake may play important roles during ischemic acidosis or synaptic activity-induced local acidification.