Michel Robert-Nicoud

Higher concentrations of histone macroH2A in the Barr body are correlated with higher nucleosome density

Histone macroH2A, which is a subtype of histone H2A, possesses a histone H2A-like portion fused to a relatively long non-histone portion. MacroH2A has been shown to associate preferentially with the inactive X chromosome [1]. To investigate the specificity of this association, the nuclear distribution of macroH2A was compared with that of regular core histones. In normal human female fibroblasts, all anti-histone antibodies that were tested (including anti-macroH2A antibody) preferentially labeled the inactive X chromosome. Moreover, when expressed as green fluorescent protein (GFP) fusions, both histone H2A and macroH2A were concentrated in the Barr body. These data clearly show the presence of a higher density of nucleosomes in the inactive X chromosome. Accordingly, the specificity of the macroH2A association with the inactive X chromosome should be reconsidered. While investigating the role of macroH2A, we found that the proximity of the non-histone region of macroH2A to a promoter could lead to a specific repression of transcription, suggesting that the incorporation of macroH2A into chromatin might help to establish the stable pattern of gene expression in differentiated cells.

Comparison of Chromosomal Imbalances in Neuroendocrine and Non-Small-Cell Lung Carcinomas

Lung carcinomas are represented by non-small-cell lung carcinomas (NSCLC) and neuroendocrine carcinomas (NE) which differ in their clinical presentation and prognosis. We used comparative genomic hybridization (CGH) to characterize and compare the chromosomal pattern of 11 NSCLC and 11 high-grade NE lung carcinomas. Overall, the total number of aberrations was higher in NSCLC than in high-grade NE lung tumors (p < 0.05) and gains predominated over losses in NSCLC (p < 0.0003). Gains common to both lung tumor phenotypes were detected in 1p, 1q, 3q, 5p, 6p, 8q, 12, 17q, 19p, 19q, 20p, 20q, and X, whereas common losses were found in 2q, 3p, 4p, 4q, 5q, 8p, 9p, 10p, 11p, 11q, 13q, and 17p. Major gains on 18q and losses on 2p and 16q were exclusively detected in high-grade NE lung tumors. On the other hand, major gains on 2p and 15q and losses on 21q were found only in NSCLC. Furthermore, gains within 22q11-q12 and 7p12-p15 were associated with NSCLC (p < 0.05). The differences in the pattern and distribution of genetic changes observed in NSCLC as opposed to high-grade NE lung carcinomas suggest the existence of distinct tumorigenic pathways between these two major classes of lung tumors.

Optimization of Nuclear Transcript Detection by FISH and Combination with Fluorescence Immunocytochemical Detection of Transcription Factors

Detection of specific nuclear transcripts by fluorescence in situ hybridization (FISH) has constituted a major breakthrough in the study of the organization of transcription in the cell nucleus. Using the model of heat shock genes, we present an optimized procedure for nuclear transcripts that provides high efficiency for RNA detection and good preservation of cell morphology and nuclear texture. Using this procedure, we designed an original high-efficiency methodology combining FISH and fluorescence immunocytochemistry (FICC), which is used here for the simultaneous detection of heat-shock protein (hsp) nuclear transcripts and the specific heat-shock transcription factor 1 (HSF1). We show that the nuclear accumulation sites of HSF1 in heat-shocked cells do not correspond to the sites of transcription of the hsp70 gene.

Nonrandom Distribution of Metaphase AgNOR Staining Patterns on Human Acrocentric Chromosomes

The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes.

Image restoration applied to x-ray microscopy: application to images with low signal-to-noise ratio

X-ray microscopy makes it possible to obtain images at a higher spatial resolution (about 20 nm) as compared to optical microscopy. Moreover, x-ray microscopy permits direct acquisition from the specimen in 2D or 3D mode, without any preparation step (staining, fixation, ...), which is not possible in electron microscopy. Here we present deblurring methods to restore images after the acquisition process. An additive Poisson noise is generated by the use of x rays and also contributes to image degradation. Our purpose is to analyze such noise and to restore images. Due to the optical properties of the Fresnel zone plate, we presently associate it to an optical circular lens as is done for the optical microscope. Here we use the Richardson Lucy algorithm to deconvolute. A first step is to observe results of restoration obtained on an image grating (a star pattern) with inner zones of dimensions near the resolution. The next step involves the suppression of noise effects arising from the deconvolution process. The characteristics of the noise after deconvolution are evaluated by Fourier analysis. These effects are eliminated by a filtering process in the Fourier spectrum. This filtering is applied on images with different signal to noise ratio (obtained after different time exposures), in order to compare results obtained on noisy images with long time exposure images.

Assignment1 of the STOP gene (MAP6) to human chromosome bands 6p12→p11 by fluorescence in situ hybridization

STOP proteins are microtubule-associated proteins acting on the stability and dynamics of microtubules. These proteins are expressed at high level in neuronal cells where they stabilize neurite microtubules. When STOP proteins are inhibited by antibody micro-injection into neuronal cells, microtubule cold and drug stability are abolished. Moreover, cells micro-injected during differentiation show impaired neurite outgrowth (Guillaud et al., 1998). Therefore STOP proteins are the unique effectors of microtubule stabilization in neurons. Recently, we have described the structure of the mouse STOP gene (Denarier et al. 1998). This gene has been assigned to the E2–F1 region of chromosome 7. Here we have determined the chromosome assignment of the human STOP gene (MAP6) by fluorescent in situ hybridization.