Biomedical subjects

Pascal Lefevre

Publications and source records attributed to Pascal Lefevre.

10 recordsLinked to original sources

PubMed2006

Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements.

Native chromatin IP assays were used to define changes in core histone acetylation at the lysozyme locus during developmental maturation of chicken macrophages and stimulation to high-level expression by lipo-polysaccharide. In pluripotent precursors the lysozyme gene (Lys) is inactive and there is no acetylation of core histones at the gene, its promoter or at the upstream cis-control elements. In myeloblasts, where there is a very low level of Lys expression, H4 acetylation appears at the cis-control elements but not at the Lys gene or its promoter: neither H3 nor H2B become significantly acetylated in myeloblasts. In mature macrophages, Lys expression increases 5-fold: H4, H2B and H2A.Z are all acetylated at the cis-control elements but H3 remains unacetylated except at the -2.4 S silencer. Stimulation with LPS increases Lys expression a further 10-fold: this is accompanied by a rise in H3 acetylation throughout the cis-control elements; H4 and H2B acetylation remain substantial but acetylation at the Lys gene and its promoter remains low. Acetylation is thus concentrated at the cis-control elements, not at the Lys gene or its immediate promoter. H4 acetylation precedes H3 acetylation during development and H3 acetylation is most directly linked to high-level Lys expression.

Acetylation↗

PubMed2006

Analyzing histone modification using crosslinked chromatin treated with micrococcal nuclease.

Epigenetic processes involve alterations in the covalent modification of histones, which are driven by sequence-specific transcription factors recruiting the enzymatic machinery performing these reactions. Such histone modifications alter the charge or biochemical surface of the chromatin fiber and also serve as "docking" sites for transcription factor and chromatin remodelling complexes. Examining histone modification patterns is vital if we want to understand how transcription complexes establish patterns of gene expression. Histone modification can be quite localized and sometimes only involves a few nucleosomes. We therefore established a chromatin immunoprecipitation procedure that uses crosslinked chromatin allowing the isolation of small chromatin fragments while simultaneously minimizing histone movement during chromatin preparation.

Animals↗

PubMed2005

The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken.

The replacement histone H2A.Z is variously reported as being linked to gene expression and preventing the spread of heterochromatin in yeast, or concentrated at heterochromatin in mammals. To resolve this apparent dichotomy, affinity-purified antibodies against the N-terminal region of H2A.Z, in both a triacetylated and non-acetylated state, are used in native chromatin immmuno-precipitation experiments with mononucleosomes from three chicken cell types. The hyperacetylated species concentrates at the 5' end of active genes, both tissue specific and housekeeping but is absent from inactive genes, while the unacetylated form is absent from both active and inactive genes. A concentration of H2A.Z is also found at insulators under circumstances implying a link to barrier activity but not to enhancer blocking. Although acetylated H2A.Z is widespread throughout the interphase genome, at mitosis its acetylation is erased, the unmodified form remaining. Thus, although H2A.Z may operate as an epigenetic marker for active genes, its N-terminal acetylation does not.

Acetylation↗

PubMed2005

Differentiation-dependent alterations in histone methylation and chromatin architecture at the inducible chicken lysozyme gene.

It is now well established that locus-wide chromatin remodeling and dynamic alterations of histone modifications are required for the developmentally regulated activation of tissue-specific genes. However, little is known about the dynamics of these events during cell differentiation and how chromatin of an entire gene locus responds to signal transduction processes. To address this issue we investigated chromatin accessibility, linker histone distribution, and the histone methylation status at the macrophage-specific chicken lysozyme locus and the ubiquitously expressed gas41 locus in multipotent precursor cell lines and BM2 monoblast cells. The latter can be induced to go through macrophage maturation by treatment with phorbol-12-myristate acetate and can be further stimulated with bacterial lipopolysaccharide. We show that expression of the lysozyme gene in undifferentiated monoblasts is low and that a high level of gene expression requires both cell differentiation and lipopolysaccharide stimulation. However, depletion of the linker histone H1 is observed already in lysozyme non-expressing multipotent precursor cells. In undifferentiated monoblasts, the lysozyme regulatory regions are marked by the presence of monomethylated histone H3 lysine 4, which becomes increasingly converted into trimethylated H3 lysine K4 during cell differentiation. We also present evidence for extensive, differentiation-dependent alterations in nuclease accessibility at the lysozyme promoter without alterations of nucleosome and transcription factor occupancy.

Animals↗

PubMed2005

c-FMS chromatin structure and expression in normal and leukaemic myelopoiesis.

The macrophage colony-stimulating factor receptor is encoded by the c-FMS gene, and it has been suggested that altered regulation of c-FMS expression may contribute to leukaemic transformation. c-FMS is expressed in pluripotent haemopoietic precursor cells and is subsequently upregulated during monocytic differentiation, but downregulated during granulopoiesis. We have examined transcription factor occupancy and aspects of chromatin structure of the critical c-FMS regulatory element located within the second intron (FIRE - fms intonic regulatory element) during normal and leukaemic myelopoiesis. Granulocytic differentiation from normal and leukaemic precursors is accompanied by loss of transcription factors at FIRE and downregulated c-FMS expression. The presence of AML1-ETO in leukaemic cells does not prevent this disassembly. In nonleukaemic cells, granulocytic differentiation is accompanied by reversal to a chromatin fine structure characteristic of c-FMS-nonexpressing cells. In addition, we show that low-level expression of the gene in leukaemic blast cells and granulocytes does not associate with increased CpG methylation across the c-FMS locus.

Base Sequence↗

PubMed2004

Epigenetic silencing of the c-fms locus during B-lymphopoiesis occurs in discrete steps and is reversible.

The murine c-fms (Csf1r) gene encodes the macrophage colony-stimulating factor receptor, which is essential for macrophage development. It is expressed at a low level in haematopoietic stem cells and is switched off in all non-macrophage cell types. To examine the role of chromatin structure in this process we studied epigenetic silencing of c-fms during B-lymphopoiesis. c-fms chromatin in stem cells and multipotent progenitors is in the active conformation and bound by transcription factors. A similar result was obtained with specified common myeloid and lymphoid progenitor cells. In developing B cells, c-fms chromatin is silenced in distinct steps, whereby first the binding of transcription factors and RNA expression is lost, followed by a loss of nuclease accessibility. Interestingly, regions of de novo DNA methylation in B cells overlap with an intronic antisense transcription unit that is differently regulated during lymphopoiesis. However, even at mature B cell stages, c-fms chromatin is still in a poised conformation and c-fms expression can be re-activated by conditional deletion of the transcription factor Pax5.

Animals↗

PubMed2003

Dynamic reorganization of chromatin structure and selective DNA demethylation prior to stable enhancer complex formation during differentiation of primary hematopoietic cells in vitro.

In order to gain insights in the true molecular mechanisms involved in cell fate decisions, it is important to study the molecular details of gene activation where such decisions occur, which is at the level of the chromatin structure of individual genes. In the study presented here we addressed this issue and examined the dynamic development of an active chromatin structure at the chicken lysozyme locus during the differentiation of primary myeloid cells from transgenic mouse bone marrow. Using in vivo footprinting we found that stable enhancer complex assembly and high-level gene expression are late events in cell differentiation. However, even before the onset of gene expression and stable transcription factor binding, specific chromatin alterations are observed. This includes changes in DNA topology and the selective demethylation of CpG dinucleotides located in the cores of critical transcription factor binding sites, but not in flanking DNA. These results firmly support the idea that epigenetic programs guiding blood cell differentiation are engraved into the chromatin of lineage-specific genes and that such chromatin changes are implemented before cell lineage specification.

Animals↗

PubMed2003

Differential transcription factor occupancy but evolutionarily conserved chromatin features at the human and mouse M-CSF (CSF-1) receptor loci.

The c-FMS gene encodes the macrophage colony-stimulating factor receptor (M-CSFR or CSF1-R), which is a tyrosine kinase growth factor receptor essential for macrophage development. We have previously characterized the chromatin features of the mouse gene; however, very little is known about chromatin structure and function of the human c-FMS locus. Here we present a side-by-side comparison of the chromatin structure, histone modification, transcription factor occupancy and cofactor recruitment of the human and the mouse c-FMS loci. We show that, similar to the mouse gene, the human c-FMS gene possesses a promoter and an intronic enhancer element (c-fms intronic regulatory element or FIRE). Both elements are evolutionarily conserved and specifically active in macrophages. However, we demonstrate by in vivo footprinting that both murine and human c-FMS cis-regulatory elements are recognised by an overlapping, but non-identical, set of transcription factors. Despite these differences, chromatin immunoprecipitation experiments show highly similar patterns of histone H3 modification and a similar distribution of chromatin modifying and remodelling activities at individual cis-regulatory elements and across the c-FMS locus. Our experiments support the hypothesis that the same regulatory principles operate at both genes via conserved cores of transcription factor binding sites.

3T3 Cells↗

PubMed2003

Epigenetic consequences of AML1-ETO action at the human c-FMS locus.

Although many leukaemia-associated nuclear oncogenes are well characterized, little is known about the molecular details of how they alter gene expression. Here we examined transcription factor complexes and chromatin structure of the human c-FMS gene in normal and leukaemic cells. We demonstrate by in vivo footprinting and chromatin immunoprecipitation assays that this gene is bound by the transcription factor AML1 (RUNX1). In t(8;21) leukaemic cells expressing the aberrant fusion protein AML1-ETO, we demonstrate that this protein is part of a transcription factor complex binding to extended sequences of the c-FMS intronic regulatory region rather than the promoter. The AML1-ETO complex does not disrupt binding of other transcription factors, indicating that c-FMS is not irreversibly epigenetically silenced. However, AML1-ETO binding correlates with changes in the histone modification pattern and increased association of histone deacetylases. Our experiments provide for the first time a direct insight into the chromatin structure of an AML1-ETO-bound target gene.

Acute Disease↗

PubMed2003

Developmentally regulated recruitment of transcription factors and chromatin modification activities to chicken lysozyme cis-regulatory elements in vivo.

Expression of the chicken lysozyme gene is upregulated during macrophage differentiation and reaches its highest level in bacterial lipopolysaccharide (LPS)-stimulated macrophages. This is accompanied by complex alterations in chromatin structure. We have previously shown that chromatin fine-structure alterations precede the onset of gene expression in macrophage precursor cells and mark the lysozyme chromatin domain for expression later in development. To further examine this phenomenon and to investigate the basis for the differentiation-dependent alterations of lysozyme chromatin, we studied the recruitment of transcription factors to the lysozyme locus in vivo at different stages of myeloid differentiation. Factor recruitment occurred in several steps. First, early-acting transcription factors such as NF1 and Fli-1 bound to a subset of enhancer elements and recruited CREB-binding protein. LPS stimulation led to an additional recruitment of C/EBPbeta and a significant change in enhancer and promoter structure. Transcription factor recruitment was accompanied by specific changes in histone modification within the lysozyme chromatin domain. Interestingly, we present evidence for a transient interaction of transcription factors with lysozyme chromatin in lysozyme-nonexpressing macrophage precursors, which was accompanied by a partial demethylation of CpG sites. This indicates that a partially accessible chromatin structure of lineage-specific genes is a hallmark of hematopoietic progenitor cells.

Animals↗