Michael J. Zeitz

Long noncoding RNA-mediated intrachromosomal interactions promote imprinting at the Kcnq1 locus

A long noncoding RNA directly builds an intrachromosomal interaction complex to establish allele-specific transcriptional gene silencing over a large chromosomal domain.

Matrin 3: Chromosomal distribution and protein interactions

Matrin 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. Matr3 was detected throughout autosomal and the active X chromosome territories. Conversely, matr3 was strikingly excluded from the inactive X chromosome as well as within both the perinuclear and perinucleolar heterochromatin. Yeast two hybrid analysis identified matr3 interactions with 33 unique nuclear localized proteins and also revealed its propensity for self association. A majority of these proteins are involved in RNA metabolism and chromatin remodeling while others function in protein translation, DNA replication/repair and apoptosis. Further analysis of a selection of these proteins and scaffold attachment factor A (SAFA) by co‐localization and co‐immunoprecipitation experiments using HeLa cells confirmed their interactions with matr3. J. Cell. Biochem. 108: 125–133, 2009.

A probabilistic model for the arrangement of a subset of human chromosome territories in WI38 Human fibroblasts

There 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 G0‐arrested WI38 diploid lung fibroblasts. Associations between heterologous chromosome combinations ranged from 52% to 78% while the homologous chromosome pairs had much lower levels of association (3–25%). A geometric computational method termed the Generalized Median Graph enabled identification of the most probable arrangement of these eight chromosome pairs. Approximately 41% of the predicted associations are present in any given nucleus. The association levels of several chromosome pairs were very similar in a series of lung fibroblast cell lines but strikingly different in skin and colon derived fibroblast cells. We conclude that a large subset of human chromosomes has a preferred probabilistic arrangement in WI38 cells and that the resulting chromosomal associations show tissue origin specificity. J. Cell. Physiol. 221: 120–129, 2009.

Genomic Interaction Profiles in Breast Cancer Reveal Altered Chromatin Architecture

Gene transcription can be regulated by remote enhancer regions through chromosome looping either in cis or in trans. Cancer cells are characterized by wholesale changes in long-range gene interactions, but the role that these long-range interactions play in cancer progression and metastasis is not well understood. In this study, we used IGFBP3, a gene involved in breast cancer pathogenesis, as bait in a 4C-seq experiment comparing normal breast cells (HMEC) with two breast cancer cell lines (MCF7, an ER positive cell line, and MDA-MB-231, a triple negative cell line). The IGFBP3 long-range interaction profile was substantially altered in breast cancer. Many interactions seen in normal breast cells are lost and novel interactions appear in cancer lines. We found that in HMEC, the breast carcinoma amplified sequence gene family (BCAS) 1–4 were among the top 10 most significantly enriched regions of interaction with IGFBP3. 3D-FISH analysis indicated that the translocation-prone BCAS genes, which are located on chromosomes 1, 17, and 20, are in close physical proximity with IGFBP3 and each other in normal breast cells. We also found that epidermal growth factor receptor (EGFR), a gene implicated in tumorigenesis, interacts significantly with IGFBP3 and that this interaction may play a role in their regulation. Breakpoint analysis suggests that when an IGFBP3 interacting region undergoes a translocation an additional interaction detectable by 4C is gained. Overall, our data from multiple lines of evidence suggest an important role for long-range chromosomal interactions in the pathogenesis of cancer.

Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C

BackgroundSeveral recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.ResultsWe describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.ConclusionTM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.

Implications of COMT long-range interactions on the phenotypic variability of 22q11.2 deletion syndrome

22q11.2 deletion syndrome (22q11DS) results from a hemizygous microdeletion on chromosome 22 and is characterized by extensive phenotypic variability. Penetrance of signs, including congenital heart, craniofacial, and neurobehavioral abnormalities, varies widely and is not well correlated with genotype. The three-dimensional structure of the genome may help explain some of this variability. The physical interaction profile of a given gene locus with other genetic elements, such as enhancers and co-regulated genes, contributes to its regulation. Thus, it is possible that regulatory interactions with elements outside the deletion region are disrupted in the disease state and modulate the resulting spectrum of symptoms. COMT, a gene within the commonly deleted ~3 Mb region has been implicated as a contributor to the neurological features frequently found in 22q11DS patients. We used this locus as bait in a 4C-seq experiment to investigate genome-wide interaction profiles in B lymphocyte and fibroblast cell lines derived from both 22q11DS and unaffected individuals. All normal B lymphocyte lines displayed local, conserved chromatin looping interactions with regions that are lost in atypical and distal deletions, which may mediate similarities between typical, atypical, and distal 22q11 deletion phenotypes. There are also distinct clusterings of cis interactions based on disease state. We identified regions of differential trans interactions present in normal, and lost in deletion-carrying, B lymphocyte cell lines. This data suggests that hemizygous chromosomal deletions such as 22q11DS can have widespread effects on chromatin organization, and may contribute to the inherent phenotypic variability.

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.

Ladder-like amplification of the type I interferon gene cluster in the human osteosarcoma cell line MG63

The 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 ∼6-fold which ended at both ends of the gene cluster with a deletion that extended throughout the 9p21.3 band. Spectral karyotyping (SKY) combined with fluorescence in-situ hybridization (FISH) identified an arrangement of the gene cluster in a ladder-like array of 5–7 ‘bands’ spanning a single chromosome termed the ‘IFN chromosome’. Chromosome painting revealed that the IFN chromosome is derived from components of chromosomes 4, 8 and 9. Labelling with centromeric probes demonstrated a ladder-like amplification of centromeric 4 and 9 sequences that co-localized with each other and a similar banding pattern of chromosome 4, as well as alternating with the IFN gene clusters. In contrast, centromere 8 was not detected on the IFN chromosome. One of the amplified centromeric 9 bands was identified as the functional centromere based on its location at the chromosome constriction and immunolocalization of the CENP-C protein. A model is presented for the generation of the IFN chromosome that involves breakage–fusion–bridge events.