Andrew J. Fritz

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

ABSTRACT 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.

C-ing the Genome: A Compendium of Chromosome Conformation Capture Methods to Study Higher-Order Chromatin Organization.

Three‐dimensional organization of the chromatin has important roles in transcription, replication, DNA repair, and pathologic events such as translocations. There are two fundamental ways to study higher‐order chromatin organization: microscopic and molecular approaches. In this review, we briefly introduce the molecular approaches, focusing on chromosome conformation capture or “3C” technology and its derivatives, which can be used to probe chromatin folding at resolutions beyond that provided by microscopy techniques. We further discuss the different types of data generated by the 3C‐based methods and how they can be used to answer distinct biological questions. J. Cell. Physiol. 230: 31–35, 2016.

SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization.

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. J. Cell. Biochem. 114: 1074–1083, 2013.

Chromosomes at Work: Organization of Chromosome Territories in the Interphase Nucleus

The organization of interphase chromosomes in chromosome territories (CTs) was first proposed more than one hundred years ago. The introduction of increasingly sophisticated microscopic and molecular techniques, now provide complementary strategies for studying CTs in greater depth than ever before. Here we provide an overview of these strategies and how they are being used to elucidate CT interactions and the role of these dynamically regulated, nuclear‐structure building blocks in directly supporting nuclear function in a physiologically responsive manner. J. Cell. Biochem. 117: 9–19, 2016.

Chromosomes at Work

The organization of interphase chromosomes in chromosome territories (CTs) was first proposed more than one hundred years ago. The introduction of increasingly sophisticated microscopic and molecular techniques, now provide complementary strategies for studying CTs in greater depth than ever before. Here we provide an overview of these strategies and how they are being used to elucidate CT interactions and the role of these dynamically regulated, nuclear‐structure building blocks in directly supporting nuclear function in a physiologically responsive manner. J. Cell. Biochem. 117: 9–19, 2016.

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.

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.

Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition

Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression.

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. J. Cell. Physiol. 230: 427–439, 2015.