Wavelet transform analysis of chromatin texture changes during heat shock.

Texture analysis can be a useful tool to investigate the organization of chromatin. Approaches based on multiscale analysis and in particular the 'à trou' wavelet analysis has already been used for microscopy (Olivo Marin). In order to analyse texture changes, the statistical properties of the wavelet coefficient images were summarized by the first four statistical orders: mean, standard deviation, skewness and kurtosis of the coefficient image histogram. The 'à trou' transform provided a representation of the wavelet coefficients and texture parameters with the same statistical robustness throughout the scale spaces. It was applied for quantifying chromatin texture and heat-induced chromatin changes in living cells. We investigated the changes by both laser scanning and spinning disk confocal microscopies and compared the texture parameters before and after increasing duration of heat shock exposure (15 min, 30 min and 1 h). Furthermore, as activation of the heat shock response also correlates with a rapid localization of HSF1 within a few nuclear structures termed nuclear stress bodies (nSBs), we compared the dynamics of nSBs formation with that of textural changes during 1 h of continuous heat shock. Next, we studied the recovery phase following a 1-h heat shock. Significant differences were observed, particularly affecting the perinucleolar region, even for the shortest heat shock time affecting mostly the skewness and standard deviation. Furthermore, progressive changes could be observed according to the duration of heat shock, mostly affecting fine details (pixel-wise changes) as revealed by the parameters, obtained from the first- and second-order wavelet coefficients. 'A trou' wavelet texture analysis provided a sensitive and efficient tool to investigate minute changes of chromatin.

Improvement of FISH mapping resolution on combed DNA molecules by iterative constrained deconvolution: a quantitative study.

Image restoration approaches, such as digital deconvolution, are becoming widely used for improving the quality of microscopic images. However, no quantification of the gain in resolution of fluorescence images is available. We show that, after iterative constrained deconvolution, fluorescent cosmid signals appear to be 25% smaller, and 1.2-kb fragment signals on combed molecules faithfully display the expected length.

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.

Genome organization in the human sperm nucleus studied by FISH and confocal microscopy.

The sperm nucleus has a unique chromatin structure where the DNA is highly condensed and associated with specific proteins, the protamines. It is a nondividing cell which is also transcriptionally inactive. After fusion with an oocyte, the sperm nucleus undergoes decondensation and, in the same time, starts replication and transcription. It has been suggested that somatic chromosomes during interphase are organized in territories which display a cell type and cell cycle specific distribution. The purpose of this work was to investigate whether chromosomes would also have a specific distribution in the sperm nucleus, which could be related to its inactive state, and have implications on the early stages of fertilization. In the present study, centromeric and telomeric sequences were detected by fluorescent techniques performed on human decondensed spermatozoa. Chromosome painting probes were used to detect the chromosome X and chromosome 13 on interphase sperm nuclei. The fluorescent signals were captured in 3D with a confocal microscope. For each of these chromatin structures, the volume, position, and distribution of the signals were analyzed in samples of 30 nuclei with the help of image analysis software. The centromeres appeared grouped in several foci that were randomly distributed within the sperm nucleus. The telomeres gave an approximately haploid number of small signals, evenly distributed throughout the nucleus. The chromosomes X and 13 occupied 4.7% and 3. 7% of the total nuclear volume, respectively. Interestingly, the X chromosome territory showed a preferential position in the anterior half of the volume of the nucleus, whereas chromosome 13 had a random position. This work shows a particular distribution of chromosome territories in the human sperm nucleus that could be related to mechanisms implicated in its specific functions. The analysis of more chromosomes and chromosomal structures, including the Y chromosome, would help to understand the structure of the human sperm chromatin, and its fundamental and clinical implications.

Quantitative assessment of the alteration of chromatin during the course of FISH procedures. Fluorescent in situ hybridization.

BACKGROUND: DNA denaturation, required for fluorescent in situ hybridization (FISH) experiments, is likely to induce chromatin alterations. Only few attempts have been made to quantify the extent of these perturbations. We propose a quality-control approach based on image analysis to monitor the effect of a procedure commonly used in FISH experiments. METHODS: Using DAPI as a probe, the same nuclei were successively imaged with a CCD camera after fixation, after permeabilization, and after thermal denaturation and hybridization with a centromeric probe. The modifications of the staining pattern were analyzed. Volumes of the FISH signals were measured using confocal imaging. RESULTS: DAPI staining combined with image analysis proved to be a sensitive tool to visualize the effects of different treatments used in FISH experiments. Permeabilization of nuclei after fixation has only limited impact on the chromatin. On the contrary, the denaturation procedure modifies the staining of DNA by DAPI, as well as the underlying chromatin structure as assessed by the increase of FISH signal volume with denaturation time. The protocol that involves a pre-fixation permeabilization step results in a more severe loss of chromatin structure. CONCLUSIONS: Our results clearly show that analysis of alterations of DAPI staining patterns is a useful monitoring tool to control and standardize hybridization procedures.

Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells.

Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.

Intron-independent association of splicing factors with active genes.

The cell nucleus is organized as discrete domains, often associated with specific events involved in chromosome organization, replication, and gene expression. We have examined the spatial and functional relationship between the sites of heat shock gene transcription and the speckles enriched in splicing factors in primary human fibroblasts by combining immunofluorescence and fluorescence in situ hybridization (FISH). The hsp90alpha and hsp70 genes are inducibly regulated by exposure to stress from a low basal level to a high rate of transcription; additionally the hsp90alpha gene contains 10 introns whereas the hsp70 gene is intronless. At 37 degrees C, only 30% of hsp90alpha transcription sites are associated with speckles whereas little association is detected with the hsp70 gene, whose constitutive expression is undetectable relative to the hsp90alpha gene. Upon exposure of cells to heat shock, the heavy metal cadmium, or the amino acid analogue azetidine, transcription at the hsp90alpha and hsp70 gene loci is strongly induced, and both hsp transcription sites become associated with speckles in >90% of the cells. These results reveal a clear disconnection between the presence of intervening sequences at specific gene loci and the association with splicing factor-rich regions and suggest that subnuclear structures containing splicing factors are associated with sites of transcription.

High-resolution mapping of the X-linked lymphoproliferative syndrome region by FISH on combed DNA.

X-linked lymphoproliferative syndrome is an inherited immunodeficiency for which the responsible gene is currently unknown. Several megabase-sized deleted regions mapping to Xq25 have been identified in XLP patients, and more recently a 130-kb deletion has been reported (Lamartine et al., 1996; Lanyi et al., 1996). To establish a physical map of this deleted region and to identify the XLP gene, two cosmid contigs were established (Lamartine et al., 1996). However, the physical map of this region is still uncompleted and controversial and three points remain unsolved: (1) the centromeric-telomeric orientation of the whole region, (2) the relative orientation of the two contigs, and (3) the size of the gap between the two contigs. To provide a definitive answer to these questions, high-resolution mapping by fluorescence in situ hybridization on combed DNA and molecular approaches were combined to establish the physical map of the XLP region over 600 kb. Our results identified a gap of 150 kb between the two contigs, established the relative orientation of one contig to the other, and determine the centromeric-telomeric orientation of the whole region. Our results show that the order of the marker over this region is: cen.1D10T7-DF83-DXS982.tel.

Genomic structure and chromosomal mapping of the mouse STOP gene (Mtap6).

The microtubule associated protein STOP (Stable Tubule Only Polypeptide) is a calmodulin-regulated protein able to induce a high degree of microtubule stability. STOP is abundant in neurons which contain large subpopulations of stable microtubules. Genomic clones spanning 67 kb and encompassing the mouse STOP gene (Mtap6) have been isolated and characterized. These clones derive from a single gene mapping to the E2-F1 region of mouse chromosome 7. The gene is composed of 4 exons that exhibit conventional vertebrate splicing sequences. Transcription of the gene initiate at multiple sites in a 85 nucleotide region located 530 bases upstream the translation initiation codon. Accordingly, the 5' flanking region of the gene lacks a TATA box or an initiator element at usual position. The protein encoded by the mouse STOP gene (Mtap6) is composed of 906 amino acids and presents a 91% identities with the rat brain STOP.

Contribution of growing RNA molecules to the nuclear transcripts foci observed by FISH.

The focal distributions of RNAs observed using fluorescence in situ hybridization (FISH) in the cell nucleus may correspond either to RNAs in the course of transcription or essentially to accumulation of full-length transcripts at the sites of transcription. To determine to what extent nuclear transcript foci represent growing RNA molecules, uninterrupted hsp70 transcripts were detected by FISH in heat-shocked human fibroblasts using two probes specific for the 5' or for the 3' end of the transcripts. By comparing the size of the signals obtained with each probe, we show that transcript foci mainly represent accumulations of full-length transcripts at their site of transcription. A major contribution of RNAs in the course of transcription to the transcript foci is observed only during the first minutes of gene induction. These observations suggest the existence of a rate-limiting step in the release of newly synthesized transcripts from their site of transcription, which is independent from the step of intron excision.

Analysis of the transcriptional activity of amplified genes in tumour cells by fluorescence in situ hybridization.

In this paper we present a new application of the detection of nuclear transcripts by fluorescence in situ hybridization (FISH) for studying the transcriptional activity of amplified genes in tumour cells. As a model, we have used the A431 cell line in which several amplification sites have been identified. We focused on two amplified regions: (1) the 6p12 region, which was found amplified by using comparative genomic hybridization, and which contains an amplification of the hsp90 beta gene; (2) the 7p12-p13 region, which displays a 20- to 30-fold amplification of the gene encoding the epidermal growth factor receptor (EGFr). By using FISH to detect nuclear transcripts, we show that the extra-copies of the hsp90 beta and EGFr genes are actively transcribed within the sites of amplification. This work illustrates the potential of this method as a tool for functional in situ cytogenetic analyses.

HSF1 transcription factor concentrates in nuclear foci during heat shock: relationship with transcription sites.

In this paper, we show that upon heat shock, HSF1 concentrates in the nucleus of diploid human fibroblasts in two large foci. The relative distribution of HSF1 nuclear foci and active heat shock protein (hsp) genes was investigated by combining fluorescence in situ hybridization (FISH) for the detection of hsp nuclear transcripts and immunofluorescence for the detection of HSF1. We show that the HSF1 foci are distinct from the sites of hsp70 and hsp90 genes transcription. This is the second report of ploidy-dependent foci of transcription factors that are independent of their specific transcription sites. However, the correlation between the number of HSF1 foci and the ploidy of the cells strongly supports the existence of a specific chromosomal target for HSF1 foci.

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.

A mouse chromosome-specific YAC probe collection for in situ hybridization.

To facilitate the identification of mouse metaphase chromosomes by fluorescence in situ hybridization (FISH), a complete collection of mouse chromosome-specific markers has been established. Yeast artificial chromosome libraries were screened by polymerase chain reaction using primers for known loci. DNAs from positive clones were then tested by FISH. One probe per chromosome was selected on the basis of high specificity (nonchimerism) and strong fluorescence.

Transgenic mice carrying an Xist-containing YAC.

The initiation of X-chromosome inactivation in female mammals is controlled by a key locus, the X-inactivation centre (Xic). The Xist gene, which maps to the candidate region for Xic and is expressed exclusively from the inactive X chromosome, is thought to be an essential component of the Xic. To test whether sequences spanning several hundred kilobases and including Xist from the Xic region are capable of initiating inactivation, we have created a series of transgenic mice using a 460 kb yeast artificial chromosome (YAC). Analysis in these mice of the expression of Xist, of a LacZ reporter gene and of two genes in the region that are normally silent on the inactive X chromosome, suggests that essential sequences for Xist expression and X-inactivation may be absent in these transgenic animals.

Metaphase and interphase mapping by FISH: improvement of chromosome banding and signal resolution in interphase nuclei by means of iterative deconvolution.

FISH images obtained with conventional epifluorescence microscopes are always blurred by glare and out of focus light emissions. In order to restore high contrast images, a procedure based on a modelling of the optical system in the microscope was developed and used for the processing of images acquired with a cooled CCD camera mounted on a fluorescence microscope. This procedure was tested on images of both mouse and human chromosomes stained with DAP1 and on images of interphase nuclei hybridized with pairs of cosmid probes. This method improves the definition and the sharpness of the DAPI G-banding and thus facilitates and speeds up the identification of chromosomes. When performed on images of interphase cell nuclei, this procedure allows the discrimination of fluorescent signals which appear partially overlapping on raw images. This significant improvement of spatial resolution is of particular interest for ordering sets of probes on DNA fibers.

Cell cycle-dependent distribution of telomeres, centromeres, and chromosome-specific subsatellite domains in the interphase nucleus of mouse lymphocytes.

Fluorescence in situ hybridization and 3-D image analysis were combined to study the distribution of specific chromosome subdomains through the course of the cell cycle in cultured mouse lymphocytes. DNA probes specific for major satellite DNA, minor satellite DNA, telomeric DNA and to chromosome X-, 8-, and 14-specific subsatellite DNA sequences were used. We demonstrate that a redistribution of the chromatin occurs during the cell cycle in the interphase nucleus, and that the profile of the rearrangement is highly dependent on the nature of the domain considered (centromeres, telomeres, or subsatellite regions). However, the relative arrangement of chromosome homologs to each other does not appear to be spatially defined or regulated.

Cell cycle regulation of the chicken hsp90 alpha expression.

The heat-shock protein of 90 kDa (hsp90), constitutively expressed in most cells, is up-regulated by thermal stress and by developmental and mitogenic stimuli. When the serum-starved chicken hepatoma cell line DU249 is stimulated by serum, insulin, or growth factors acting via tyrosine kinase receptors, a transient induced expression of the hsp90 alpha gene takes place at both the messenger RNA and the protein synthesis level. This response is protein synthesis dependent and DNA synthesis independent. The maximum level of hsp90 alpha mRNA accumulation always precedes the maximum level of thymidine incorporation, suggesting a possible link between cell cycle and hsp90 alpha regulation (Jérôme, V., J. Léger, J. Devin, E.E. Baulieu, and M. G. Catelli. Growth Factors 4:317-327, 1991). Herein, we examine the subcellular distribution of hsp90 and the cell cycle-dependent regulation of the hsp90 alpha mRNA level. We show that, in contrast to hsp70, the 35S metabolically-labeled hsp90, which accumulates in the cytosoluble fraction 6 to 8 h after serum treatment, is not preferentially translocated to the nuclear compartment, although a small fraction is always present in the nucleus. We also demonstrated that serum- or insulin-induced accumulation of hsp90 alpha mRNA results from an activation of gene transcription and that hsp90 alpha promotor activity, which is low in quiescent DU249 cells, is induced approximately fivefold after serum stimulation. Finally, in cell culture synchronized by nocodazole or aphidicholin, hsp90 alpha mRNA accumulation seems an event specific to G1/S transition.

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.