Differences of size and shape of active and inactive X-chromosome domains in human amniotic fluid cell nuclei

It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46, XX and 46, XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITC-conjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi- and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISS-hybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area AXi for the inactive X-domain was smaller than the area AXa for the active domain (mean ratio AXa/AXi = 1.9 ± 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 m). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 ± 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 ± 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 ± 2.6 S.D.)%. These findings indicate that differences in the 3D expansion of active and inactive X-chromosome domains are less pronounced than previously thought. A current model suggests that chromosome domains consist of a compact core surrounded by loosely coiled outer chromatin fiber loops. The latter fraction may be considerably larger in Xa- as compared to Xi-domains. We suggest that the interactive outlining procedure used in the 2D analyses included the loosely structured domain periphery more accurately, while the threshold algorithm applied to light optical sections delineated the more compact core of the domains, leading to smaller and more similar volume estimates of Xa and Xi. Present limitations of nuclear and chromosome domain volume measurements using confocal laser scanning microscopy are discussed

Towards many colors in FISH on 3D-preserved interphase nuclei

The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + widefield microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested. Copyright (c) 2006 S. Karger AG, Basel

Tackling tumour cell heterogeneity at the super-resolution level in human colorectal cancer tissue

Tumour cell heterogeneity, and its early individual diagnosis, is one of the most fundamental problems in cancer diagnosis and therapy. Single molecule localisation microscopy (SMLM) resolves subcellular features but has been limited to cultured cell lines only. Since nuclear chromatin architecture and microRNAs are critical in metastasis, we introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma (CRC) patient tissue sections. Chromatin density profiles proved to differ for cells in normal and carcinoma colorectal tissues. In tumour sections, nuclear size and chromatin compaction percentages were significantly different in carcinoma versus normal epithelial and other cells of colorectal tissue. SMLM analysis in nuclei from normal colorectal tissue revealed abrupt changes in chromatin density profiles at the nanoscale, features not detected by conventional widefield microscopy. SMLM for microRNAs relevant for metastasis was achieved in colorectal cancer tissue at the nuclear level. Super-resolution microscopy with quantitative image evaluation algorithms provide powerful tools to analyse chromatin nanostructure and microRNAs of individual cells from normal and tumour tissue at the nanoscale. Our new perspectives improve the differential diagnosis of normal and (metastatically relevant) tumour cells at the single-cell level within the heterogeneity of primary tumours of patients

Nejire/dCBP-mediated control of H3 acetylation and transcriptional regulation by testis-specific Plus3 domain proteins during Drosophila spermatogenesis

Spermatogenesis describes the development from germ line stem cells to highly specialized sperm. Drosophila melanogaster spermatogenesis is a good model system for chromatin remodelling processes as many of these processes are similar in mammals and in flies. Histone modifications are a prerequisite for the exchange of histones by protamines during these chromatin remodelling processes but also transcription processes in earlier germ cell stages can be compared. In this thesis, the histone acetyltransferase Nejire/dCBP has been characterised as being responsible for the modification of histone H3 at lysine 18 and lysine 27 during post-meiotic germ cell development as well as in the spermatocyte stage. An RNAi-mediated knock-down revealed that the function of Nejire/dCBP is essential for fertility of male flies. Efficient mRNA synthesis of postmeiotic chromatin components depends on Nejire/dCBP whereas incorporation of protamines into the chromatin does not seem to depend on Nejire/dCBP function. Drosophila spermatogenesis is featured by a special regulation of transcription and translation. While most transcripts are synthesised in spermatocytes, a large portion has to be translationally repressed until required in later stages of germ cell development. Transcriptional regulation is supported by testis-specific variants of the general transcription machinery. This includes among others the tTAFs, the tMAC complex and bromodomain proteins. Bromodomain proteins are able to recognise and bind acetylated lysine residues on N-terminal histone chains. The bromodomain proteins tBRD-1 and tBRD-2 are expressed in spermatocytes and can interact with tTAFs, this might facilitate recruitment of the TFIID complex to certain chromatin areas. Further testis-specific variants of ubiquitously expressed proteins are the Plus3 domain proteins. Here, the expression and function of the testis-enriched proteins tPlus3a and tPlus3b have been examined. Both proteins share the conserved Plus3 domain of Rtf1. RNAseq analysis using RNA from mutant testes revealed that tPlus3a and tPlus3b likely contribute to the regulation of transcription in spermatocytes. Furthermore, genes which also depend on tBRD-1 function were identified. We hypothesise that tPlus3a and tPlus3b regulate a group of genes overlapping with tBRD-1-depending genes but not with genes depending on tTAFs. tPlus3a and tPlus3b might therefore contribute to diversification of transcriptional regulation in spermatocytes

Evolution of histone 2A for chromatin compaction in eukaryotes.

During eukaryotic evolution, genome size has increased disproportionately to nuclear volume, necessitating greater degrees of chromatin compaction in higher eukaryotes, which have evolved several mechanisms for genome compaction. However, it is unknown whether histones themselves have evolved to regulate chromatin compaction. Analysis of histone sequences from 160 eukaryotes revealed that the H2A N-terminus has systematically acquired arginines as genomes expanded. Insertion of arginines into their evolutionarily conserved position in H2A of a small-genome organism increased linear compaction by as much as 40%, while their absence markedly diminished compaction in cells with large genomes. This effect was recapitulated in vitro with nucleosomal arrays using unmodified histones, indicating that the H2A N-terminus directly modulates the chromatin fiber likely through intra- and inter-nucleosomal arginine-DNA contacts to enable tighter nucleosomal packing. Our findings reveal a novel evolutionary mechanism for regulation of chromatin compaction and may explain the frequent mutations of the H2A N-terminus in cancer

Differences in the localization and morphology of chromosomes in the human nucleus

Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized