Chromosome territories and the global regulation of the genome.

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. For example, enhancers loop back to interact with their target genes almost exclusively within TADs, distally located coregulated genes reposition into common transcription factories upon activation, and Mbp CDs exhibit dynamic motion and configurational changes in vivo. A longstanding question in the nucleus field is whether an interactive nuclear matrix provides a direct link between structure and function. The findings of nonrandom radial positioning of CT within the nucleus suggest the possibility of preferential interaction patterns among populations of CT. Sequential labeling up to 10 CT followed by application of computer imaging and geometric graph mining algorithms revealed cell-type specific interchromosomal networks (ICN) of CT that are altered during the cell cycle, differentiation, and cancer progression. It is proposed that the ICN correlate with the global level of genome regulation. These approaches also demonstrated that the large scale 3-D topology of CT is specific for each CT. The cell-type specific proximity of certain chromosomal regions in normal cells may explain the propensity of distinct translocations in cancer subtypes. Understanding how genes are dysregulated upon disruption of the normal "wiring" of the nucleus by translocations, deletions, and amplifications that are hallmarks of cancer, should enable more targeted therapeutic strategies.

Large-scale probabilistic 3D organization of human chromosome territories.

There 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. Six CT were selected ranging in size and gene density (1, 4, 12, 17, 18 and X). In-house computational geometric algorithms were applied to measure the 3D distances between every combination of probes and to elucidate data-mined structural patterns. Our findings demonstrate a high degree of non-random arrangement of individual CT that vary from chromosome to chromosome and display distinct changes during the cell cycle. Application of a classic, well-defined data mining and pattern recognition approach termed the 'k-means' generated 3D models for the best fit arrangement of each chromosome. These predicted models correlated well with the detailed distance measurements and analysis. We propose that the unique 3D topology of each CT and characteristic changes during the cell cycle provide the structural framework for the global gene expression programs of the individual chromosomes.

Reorganization of the interchromosomal network during keratinocyte differentiation.

The 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. Combined multi-fluorescence in situ hybridization (FISH) and computational image analysis on the undifferentiated (0 h) and differentiated (24 h after Ca(2+) treatment) HEK revealed that (a) increases in CT volumes correspond to overall nuclear volume increases, (b) radial positioning is gene density-dependent at 0 h but neither gene density- nor size-dependent at 24 h, (c) the average number of interchromosomal associations for each CT is gene density-dependent and similar at both time points, and (d) there are striking differences in the single and multiple pairwise interchromosomal association profiles. Probabilistic network models of the overall interchromosomal associations demonstrate major reorganization of the network during differentiation. Only ~40 % of the CT pairwise connections in the networks are common to both 0 and 24 h HEK. We propose that there is a probabilistic chromosome positional code which can be significantly altered during cell differentiation in coordination with reprogramming of gene expression.

Fluctuations and synchrony of RNA synthesis in nucleoli.

Ribosomal 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. Remarkably high and rapid fluctuations of rRNA production rates were revealed by this technique. Strikingly, the changes in the rRNA output were synchronous for ribosomal genes located in separate nucleoli of the same cell. Our findings call for the development of new concepts to elucidate the coordination of ribosomal components production. In this regard, numerical modeling further demonstrated that the production of rRNA and r-proteins can be coordinated, regardless of the fluctuations in rRNA synthesis. Overall, our quantitative data reveal a spectacular interplay of inherently stochastic rates of RNA synthesis and the coordination of gene expression.

MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus.

Cardiac 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;q31.2)dn translocation carrier with pervasive developmental delay who also exhibited LVOT defects, including bicuspid aortic valve (BAV), coarctation of the aorta (CoA) and patent ductus arteriosus (PDA). The 1p breakpoint disrupts the 5' UTR of AHDC1, which encodes AT-hook DNA-binding motif containing-1 protein, and AHDC1-truncating mutations have recently been described in a syndrome that includes developmental delay, but not congenital heart disease [Xia, F., Bainbridge, M.N., Tan, T.Y., Wangler, M.F., Scheuerle, A.E., Zackai, E.H., Harr, M.H., Sutton, V.R., Nalam, R.L., Zhu, W. et al. (2014) De Novo truncating mutations in AHDC1 in individuals with syndromic expressive language delay, hypotonia, and sleep apnea. Am. J. Hum. Genet., 94, 784-789]. On the other hand, the 5q translocation breakpoint disrupts the 3' UTR of MATR3, which encodes the nuclear matrix protein Matrin 3, and mouse Matr3 is strongly expressed in neural crest, developing heart and great vessels, whereas Ahdc1 is not. To further establish MATR3 3' UTR disruption as the cause of the proband's LVOT defects, we prepared a mouse Matr3(Gt-ex13) gene trap allele that disrupted the 3' portion of the gene. Matr3(Gt-ex13) homozygotes are early embryo lethal, but Matr3(Gt-ex13) heterozygotes exhibit incompletely penetrant BAV, CoA and PDA phenotypes similar to those in the human proband, as well as ventricular septal defect (VSD) and double-outlet right ventricle (DORV). Both the human MATR3 translocation breakpoint and the mouse Matr3(Gt-ex13) gene trap insertion disturb the polyadenylation of MATR3 transcripts and alter Matrin 3 protein expression, quantitatively or qualitatively. Thus, subtle perturbations in Matrin 3 expression appear to cause similar LVOT defects in human and mouse.

Non-Random Patterns in the Distribution of NOR-Bearing Chromosome Territories in Human Fibroblasts: A Network Model of Interactions.

We 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%). The associations of homologous CT were significantly lower (24-36%). Moreover, singular contacts between CT13-14 or CT13-22 were found in the majority of cells, while CT13-15 or CT13-21 predominantly exhibited multiple interactions. In cells with multiple nucleoli, one of the nucleoli (termed "dominant") always associated with a higher number of CT. Moreover, certain CT pairs more frequently contributed to the same nucleolus than to others. This nonrandom pattern suggests that a large number of the NOR-chromosomes are poised in close proximity during the postmitotic nucleolar recovery and through their NORs may contribute to the formation of the same nucleolus. A global data mining program termed the chromatic median determined the most probable interchromosomal arrangement of the entire NOR-CT population. This interactive network model was significantly above randomized simulation and was composed of 13 connections among the NOR-CT. We conclude that the NOR-CT form a global interactive network in the cell nucleus that may be a fundamental feature for the regulation of nucleolar and other genomic functions.

Cell type specific alterations in interchromosomal networks across the cell cycle.

The 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. Each CT was in close proximity (interaction) with a similar number of other CT except the gene rich CT17 which had significantly more interactions. Furthermore, CT17 was a member of the highest pairwise CT combinations with multiple interactions. Major differences were detected in the pairwise interaction profiles of MCF10A versus WI38 including cell cycle alterations from G1 to S. These alterations in interaction profiles were subdivided into five types: overall increase, overall decrease, switching from 1 to ≥2 interactions, vice versa, or no change. A global data mining program termed the chromatic median determined the most probable overall association network for the entire subset of CT. This probabilistic interchromosomal network was nearly completely different between the two cell lines. It was also strikingly altered across the cell cycle in MCF10A, but only slightly in WI38. We conclude that CT undergo multiple and preferred interactions with other CT in the nucleus and form preferred -albeit probabilistic- interchromosomal networks. This network of interactions is altered across the cell cycle and between cell types. It is intriguing to consider the relationship of these alterations to the corresponding changes in the gene expression program across the cell cycle and in different cell types.

Gene density and chromosome territory shape.

Despite 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. To be representative of the whole human genome, 12 different CT (~41 % of the genome) were selected that ranged from the largest (CT 1) to the smallest (CT 21) in size and from the highest (CT 19) to lowest (CT Y) in gene density. Using both visual inspection and algorithms that measure the degree of shape ellipticity and regularity, we demonstrate a strong inverse correlation between the degree of regular CT shape and gene density for those CT that are most gene-rich (19, 17, 11) and gene-poor (18, 13, Y). CT more intermediate in gene density showed a strong negative correlation with shape regularity, but not with ellipticity. An even more striking correlation between gene density and CT shape was determined for the nucleolar-associated NOR-CT. Correspondingly, striking differences in shape between the X active and inactive CT implied that aside from gene density, the overall global level of gene transcription on individual CT is also an important determinant of chromosome territory shape.

Wide-scale alterations in interchromosomal organization in breast cancer cells: defining a network of interacting chromosomes.

The 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. Each CT was in close proximity (interaction) with a similar number of other CT except the inactive CTXi. It had lower levels of interchromosomal partners in 10A which increased strikingly in CA1a. Major alterations from 10A to CA1a were detected in the pairwise interaction profiles which were subdivided into five types of altered interaction profiles: overall increase, overall decrease, switching from 1 to ≥2, vice versa or no change. A global data mining program termed the chromatic median calculated the most probable overall association network for the entire subset of CT. This interchromosomal network was drastically altered in CA1a with only 1 of 20 shared connections. We conclude that CT undergo multiple and preferred interactions with other CT in the cell nucleus and form preferred-albeit probabilistic-interchromosomal networks. This network of interactions is highly altered in malignant human breast cells. It is intriguing to consider the relationship of these alterations to the corresponding changes in the gene expression program of these malignant cancer cells.

Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1.

The 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. The bacterially expressed unphosphorylated SLIP1-SLBP complex forms a 2:2 high-affinity (K(D) < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has a weak affinity (K(D) ~3 µM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem-loop motif followed by association with SLIP1 is required to form an "active" ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1-SLBP heterodimer. Using alanine scanning mutagenesis, we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single-point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced the abundance of histone mRNA in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP-SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo, protecting it from proteolytic degradation as an inactive heterotetramer, or alternatively, formation of the SLIP1-SLBP heterotetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation.

Chromatin dynamics in living cells: identification of oscillatory motion.

Genomic 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). We conclude that previously identified large-scale transitions in the spatial position and configuration of chromatin, originate from asymmetric oscillations of the ChrD detectable in fractions of a second. The rapid oscillatory motion correlates with the replication timing of the ChrD with early S replicated ChrD showing the highest levels of motion and late S-phase chromatin the lowest. Virtually identical levels of oscillatory motion were detected when ChrD were measured during active DNA replication or during inhibition of transcription with DRB or α-amanitin. While this motion is energy independent, the oscillations of early S and mid S, but not late S replicated chromatin, are reduced by cell permeabilization. This suggests involvement of soluble factors in the regulation of chromatin dynamics. The DNA intercalating agent actinomycin D also significantly inhibits early S-labeled chromatin oscillation. We propose that rapid asymmetric oscillations of <1 sec are the basis for translational movements and configurational changes in ChrD previously detected over time spans of minutes-hours, and are the result of both the stochastic collisions of macromolecules and specific molecular interactions.

A dominant negative Cx43 mutant differentially affects tumorigenic and invasive properties in human metastatic melanoma cells.

Previous 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. To further investigate the role of Cx43 in these melanoma cells, we overexpressed wild type (wt) Cx43 as well as a mutant dominant negative Cx43 mutant that causes closed channels (T154A). The metastatic 1205Lu cells expressing Cx43-T154A showed a twofold decrease in colony formation on soft agar while the nonmetastatic WM793B cells showed no significant change. In invasion assays through a collagen matrix, the same Cx43-T154A 1205Lu cells demonstrated a three- to fourfold increase in the invasion index compared to either wt Cx43 or vector control cells. The increase in invasiveness was eliminated by migration towards media with charcoal-stripped serum, suggesting that migration may be directed towards a lipophilic compound(s). Our findings demonstrate that a dominant negative Cx43 mutant deficient in channel formation exhibits a dual pattern of regulation in metastatic melanoma cells with a decrease in anchorage-independent growth and an increase in invasive potential.

Alterations in replication timing of cancer-related genes in malignant human breast cancer cells.

The 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). Five of these nine genes (TP53, RAD51, ATM, PTEN, and cMYC) show significant differences (P < 0.05) in replication timing between MCF10A normal human breast cells and the corresponding malignant MCF10CA1a cells. These differences are specific to the malignant state of the MCF10CA1a cells since there were no significant differences in the replication timing of these genes between normal MCF10A cells and the non-malignant cancer MCF10AT1 cells. Microarray analysis further demonstrated that three of these five genes (TP53, RAD51, and cMYC) showed significant changes in gene expression (≥2-fold) between normal and malignant cells. Our findings demonstrate an alteration in the replication timing of a small subset of cancer-related genes in malignant breast cancer cells. These alterations partially correlate with the major transcriptional changes characteristic of the malignant state in these cells.

The prolyl isomerase Pin1 targets stem-loop binding protein (SLBP) to dissociate the SLBP-histone mRNA complex linking histone mRNA decay with SLBP ubiquitination.

Histone 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. Here we report that chemical inhibition of the prolyl isomerase Pin1 or downregulation of Pin1 by small interfering RNA (siRNA) increases the mRNA stability of all five core histone mRNAs and the stability of SLBP. Pin1 regulates SLBP polyubiquitination via the Ser20/Ser23 phosphodegron in the N terminus. siRNA knockdown of Pin1 results in accumulation of SLBP in the nucleus. We show that Pin1 can act along with protein phosphatase 2A (PP2A) in vitro to dephosphorylate a phosphothreonine in a conserved TPNK sequence in the SLBP RNA binding domain, thereby dissociating SLBP from the histone mRNA hairpin. Our data suggest that Pin1 and PP2A act to coordinate the degradation of SLBP by the ubiquitin proteasome system and the exosome-mediated degradation of the histone mRNA by regulating complex dissociation.

Preferential induction of apoptotic cell death in melanoma cells as compared with normal keratinocytes using a non-thermal plasma torch.

Selective 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. Melanoma cells displayed high sensitivity to reactive oxygen species generated by the torch and showed a 6-fold increase in cell death compared with keratinocytes. The extent of cell death was compared between melanoma cells and normal human keratinocytes in both short-term (5 min) co-culture experiments and longer assessments of apoptotic cell death (18-24 h). Following a 10 sec plasma exposure there was a 4.9-fold increase in the cell death of melanoma vs. keratinocytes as measured after 24 h at the target site of the plasma beam. When the treatment time was increased to 30 sec, a 98% cell death was reported for melanoma cells, which was 6-fold greater than the extent of cell death in keratinocytes. Our observations further indicate that this preferential cell death is largely due to apoptosis.. In addition, we report that this non-thermal plasma torch kills melanoma cells growing in soft agar, suggesting that the plasma torch is capable of inducing melanoma cell death in 3D settings. We demonstrate that the presence of gap junctions may increase the area of cell death, likely due to the "bystander effect" of passing apoptotic signals between cells. Our findings provide a basis for further development of this non-invasive plasma torch as a potential treatment for melanoma.

Computing maximum association graph in microscopic nucleus images.

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. Our approach is based on an interesting integer linear programming formulation of the problem and utilizes inherent observations of the problem to yield optimal solutions. A two-stage technique is also introduced for producing near optimal approximations for large data sets.

Chromosomal rearrangements during human epidermal keratinocyte differentiation.

Undifferentiated 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.

Cytogenetic and cDNA microarray expression analysis of MCF10 human breast cancer progression cell lines.

We 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. We conclude that other mechanisms of gene regulation that are not directly related to chromosomal gains and losses play a major role in breast cancer progression.

Cell type specific chromosome territory organization in the interphase nucleus of normal and cancer cells.

Numerous 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. Similar levels of associations were found in WI38 and MCF10A for several of the chromosomes whereas others showed striking differences. A novel computational geometric approach, the generalized median graph (GMG), revealed a preferred probabilistic arrangement distinct for each cell line. Statistical analysis demonstrated that approximately 50% of the associations depicted in the GMG models are present in each individual nucleus. A nearly twofold increase of chromosome 4/16 associations in a malignant breast cancer cell line (MCFCA1a) compared to the related normal epithelial cell line (MCF10A) further demonstrates cancer related changes in chromosome arrangements. Our findings of highly preferred chromosome association profiles that are cell type specific and undergo alterations in cancer cells, lead us to propose a probabilistic chromosome code whereby the 3-D association profile of chromosomes contributes to the functional landscape of the cell nucleus, the global regulation of gene expression and the epigenetic state of chromatin.