Spatial positioning is a fundamental principle governing nuclear processes. Chromatin is organized as a hierarchy from nucleosomes to Mbp chromatin domains (CD) or topologically associating domains (TADs) to higher level compartments culminating in chromosome territories (CT). Microscopic and sequencing techniques have substantiated chromatin organization as a critical factor regulating gene expression.
View Article and Find Full Text PDFThere is growing evidence that chromosome territories (CT) have a probabilistic non-random arrangement within the cell nucleus of mammalian cells including radial positioning and preferred patterns of interchromosomal interactions that are cell-type specific. While it is generally assumed that the three-dimensional (3D) arrangement of genes within the CT is linked to genomic regulation, the degree of non-random organization of individual CT remains unclear. As a first step to elucidating the global 3D organization (topology) of individual CT, we performed multi-color fluorescence in situ hybridization using six probes extending across each chromosome in human WI38 lung fibroblasts.
View Article and Find Full Text PDFThe well-established human epidermal keratinocyte (HEK) differentiation model was investigated to determine possible alterations in chromosome territory (CT) association during differentiation. The seven human chromosomes (1, 4, 11, 12, 16, 17, and 18) selected for this analysis are representative of the chromosome size and gene density range of the overall human genome as well as including a majority of genes involved in epidermal development and differentiation (CT1, 12, and 17). Induction with calcium chloride (Ca(2+)) resulted in morphological changes characteristic of keratinocyte differentiation.
View Article and Find Full Text PDFRibosomal RNA (rRNA) sequences are synthesized at exceptionally high rates and, together with ribosomal proteins (r-proteins), are utilized as building blocks for the assembly of pre-ribosomal particles. Although it is widely acknowledged that tight regulation and coordination of rRNA and r-protein production are fundamentally important for the maintenance of cellular homeostasis, still little is known about the real-time kinetics of the ribosome component synthesis in individual cells. In this communication we introduce a label-free MicroRaman spectrometric approach for monitoring rRNA synthesis in live cultured cells.
View Article and Find Full Text PDFCardiac left ventricular outflow tract (LVOT) defects represent a common but heterogeneous subset of congenital heart disease for which gene identification has been difficult. We describe a 46,XY,t(1;5)(p36.11;q31.
View Article and Find Full Text PDFThe interchromosomal organization of a subset of human chromosomes (#1, 4, 11, 12, 16, 17, and 18) was examined in G1 and S phase of human WI38 lung fibroblast and MCF10A breast epithelial cells. Radial positioning of the chromosome territories (CTs) was independent of gene density, but size dependent. While no changes in radial positioning during the cell cycle were detected, there were stage-specific differences between cell types.
View Article and Find Full Text PDFDespite decades of study of chromosome territories (CT) in the interphase nucleus of mammalian cells, our understanding of the global shape and 3-D organization of the individual CT remains very limited. Past microscopic analysis of CT suggested that while many of the CT appear to be very regular ellipsoid-like shapes, there were also those with more irregular shapes. We have undertaken a comprehensive analysis to determine the degree of shape regularity of different CT.
View Article and Find Full Text PDFWe present a 3-D mapping in WI38 human diploid fibroblast cells of chromosome territories (CT) 13,14,15,21, and 22, which contain the nucleolar organizing regions (NOR) and participate in the formation of nucleoli. The nuclear radial positioning of NOR-CT correlated with the size of chromosomes with smaller CT more interior. A high frequency of pairwise associations between NOR-CT ranging from 52% (CT13-21) to 82% (CT15-21) was detected as well as a triplet arrangement of CT15-21-22 (72%).
View Article and Find Full Text PDFThe interchromosomal spatial positionings of a subset of human chromosomes was examined in the human breast cell line MCF10A (10A) and its malignant counterpart MCF10CA1a (CA1a). The nine chromosomes selected (#1, 4, 11, 12, 15, 16, 18, 21 and X) cover a wide range in size and gene density and compose ∼40% of the total human genome. Radial positioning of the chromosome territories (CT) was size dependent with certain of the CT more peripheral in CA1a.
View Article and Find Full Text PDFThe SLIP1-SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1-SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus.
View Article and Find Full Text PDFThe replication timing of nine genes commonly involved in cancer was investigated in the MCF10 cell lines for human breast cancer progression. Six of these nine genes are part of a constellation of tumor suppressor genes that play a major role in familial human breast cancer (TP53, ATM, PTEN, CHK2, BRCA1, and BRCA2). Three other genes are involved in a large number of human cancers including breast as either tumor suppressors (RB1 and RAD51) or as an oncogene (cMYC).
View Article and Find Full Text PDFPrevious reports have implicated connexin 43 (Cx43) as a tumor suppressor in early stages of tumorigenesis and in some cases as an enhancer of cell migration in later stages. To address the role of Cx43 in melanoma tumor progression, we utilized two melanoma cell lines derived from the same patient in pre-metastasis (WM793B) and following isolation from a lung metastasis in nude mice (1205Lu). Our results demonstrate a strikingly increased expression of Cx43 in both the pre-metastatic and metastatic melanoma cell lines that were actively migrating compared to non-migrating cells.
View Article and Find Full Text PDFHistone mRNAs are rapidly degraded at the end of S phase, and a 26-nucleotide stem-loop in the 3' untranslated region is a key determinant of histone mRNA stability. This sequence is the binding site for stem-loop binding protein (SLBP), which helps to recruit components of the RNA degradation machinery to the histone mRNA 3' end. SLBP is the only protein whose expression is cell cycle regulated during S phase and whose degradation is temporally correlated with histone mRNA degradation.
View Article and Find Full Text PDFSelective induction of apoptosis in melanoma cells is optimal for therapeutic development. To achieve this goal, a non-thermal helium plasma torch was modified for use on cultured cells in a temperature-controlled environment. Melanoma cells were targeted with this torch (1) in parallel cultures with keratinocytes, (2) in co-culture with keratinocytes and (3) in a soft agar matrix.
View Article and Find Full Text PDFGenomic DNA in mammalian cells is organized into ~1 Mbp chromatin domains (ChrD) which represent the basic structural units for DNA compaction, replication, and transcription. Remarkably, ChrD are highly dynamic and undergo both translational movement and configurational changes. In this study, we introduce an automated motion tracking analysis to measure, both in 2D and 3D, the linear displacement of early, mid and late S-phase replicated ChrD over short time periods (<1 sec).
View Article and Find Full Text PDFMed Image Comput Comput Assist Interv
November 2010
In this paper, we study the problem of finding organization patterns of chromosomes inside the cell nucleus from microscopic nucleus images. Emerging evidence from cell biology research suggests that global chromosome organization has a vital role in fundamental cell processes related to gene expression and regulation. To understand how chromosome territories are neighboring (or associated) to each other, in this paper we present a novel technique for computing a common association pattern, represented as a Maximum Association Graph (MAG), from the nucleus images of a population of cells.
View Article and Find Full Text PDFUndifferentiated human epidermal keratinocytes are self-renewing stem cells that can be induced to undergo a program of differentiation by varying the calcium chloride concentration in the culture media. We utilize this model of cell differentiation and a 3D chromosome painting technique to document significant changes in the radial arrangement, morphology, and interchromosomal associations between the gene poor chromosome 18 and the gene rich chromosome 19 territories at discrete stages during keratinocyte differentiation. We suggest that changes observed in chromosomal territorial organization provides an architectural basis for genomic function during cell differentiation and provide further support for a chromosome territory code that contributes to gene expression at the global level.
View Article and Find Full Text PDFWe used a combination of spectral karyotyping, array comparative genomic hybridization, and cDNA microarrays to gain insights into the structural and functional changes of the genome in the MCF10 human breast cancer progression model cell lines. Spectral karyotyping data showed several chromosomal aberrations and array comparative genomic hybridization analysis identified numerous genomic gains and losses that might be involved in the progression toward cancer. Analysis of the expression levels of genes located within these genomic regions revealed a lack of correlation between chromosomal gains and losses and corresponding up-regulation or down-regulation for the majority of the approximately 1,000 genes analyzed in this study.
View Article and Find Full Text PDFMatrin 3 (matr3), an abundant protein of the internal nuclear matrix, has been linked to a variety of functional events. As a step toward defining its multifunctional nature, we have studied the association of matr3 with chromosome territories and identified potential interacting proteins. A similar staining pattern of matr3 was observed in fixed WI38 fibroblast cells and in live HeLa cells using a matr3-GFP construct.
View Article and Find Full Text PDFThere is growing evidence that chromosome territories have a probabilistic non-random arrangement within the cell nucleus of mammalian cells. Other than their radial positioning, however, our knowledge of the degree and specificity of chromosome territory associations is predominantly limited to studies of pair-wise associations. In this study we have investigated the association profiles of eight human chromosome pairs (numbers 1, 2, 3, 4, 6, 7, 8, 9) in the cell nuclei of G(0)-arrested WI38 diploid lung fibroblasts.
View Article and Find Full Text PDFNumerous studies indicate that the genome of higher eukaryotes is organized into distinct chromosome territories and that the 3-D arrangement of these territories may be closely connected to genomic function and the global regulation of gene expression. Despite this progress, the degree of non-random arrangement remains unclear and no overall model has been proposed for chromosome territory associations. To address this issue, a re-FISH approach was combined with computational analysis to analysis the pair-wise associations for six pairs of human chromosomes (chr #1, 4, 11, 12, 16, 18) in the G(0) state of normal human WI38 lung fibroblast and MCF10A epithelial breast cells.
View Article and Find Full Text PDFDiscrete chromatin domains (ChrD), containing an average of approximately 1 Mbp DNA, represent the basic structural units for the regulation of DNA organization and replication in situ. In this study, a bio-computational approach is employed to simultaneously measure the translational motion of large populations of ChrD in the cell nucleus of living cells. Both movement and configurational changes are strikingly higher in early S-phase replicating ChrD compared to those that replicate in mid and late S-phase.
View Article and Find Full Text PDFThe organization of the amplified type I interferon (IFN) gene cluster and surrounding chromosomal regions was studied in the interphase cell nucleus of the human osteosarcoma cell line MG63. Rather than being arranged in a linear ladder-like array as in mitotic chromosomes, a cluster of approximately 15 foci was detected that was preferentially associated along the periphery of both the cell nucleus and a chromosome territory containing components of chromosomes 4, 8, and 9. Interspersed within the IFN gene foci were corresponding foci derived from amplified centromere 4 and 9 sequences.
View Article and Find Full Text PDFThe organization of the type I interferon (IFN) gene cluster (9p21.3) was studied in a human osteosarcoma cell line (MG63). Array comparative genomic hybridization (aCGH) showed an amplification of approximately 6-fold which ended at both ends of the gene cluster with a deletion that extended throughout the 9p21.
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