The connection between BRG1, CTCF and topoisomerases at TAD boundaries

The eukaryotic genome is partitioned into topologically associating domains (TADs). Despite recent advances characterizing TADs and TAD boundaries, the organization of these structures is an important dimension of genome architecture and function that is not well understood. Recently, we demonstrated that knockdown of BRG1, an ATPase driving the chromatin remodeling activity of mammalian SWI/SNF enzymes, globally alters long-range genomic interactions and results in a reduction of TAD boundary strength. We provided evidence suggesting that this effect may be due to BRG1 affecting nucleosome occupancy around CTCF sites present at TAD boundaries. In this review, we elaborate on our findings and speculate that BRG1 may contribute to the regulation of the structural and functional properties of chromatin at TAD boundaries by affecting the function or the recruitment of CTCF and DNA topoisomerase complexes

Nuclear structure-gene expression interrelationships: implications for aberrant gene expression in cancer

There is long-standing recognition that transformed and tumor cells exhibit striking alterations in nuclear morphology as well as in the representation and intranuclear distribution of nucleic acids and regulatory factors. Parameters of nuclear structure support cell growth and phenotypic properties of cells by facilitating the organization of genes, replication and transcription sites, chromatin remodeling complexes, transcripts, and regulatory factors in structurally and functionally definable subnuclear domains within the three-dimensional context of nuclear architecture. The emerging evidence for functional interrelationships of nuclear structure and gene expression is consistent with linkage of tumor-related modifications in nuclear organization to compromised gene regulation during the onset and progression of cancer

The Varus Knee Reveals Differential Expression Patterns of miRNAs in Spared vs. Non-spared Compartments

Introduction MicroRNAs (miRNAs) function by repressing cellular protein levels to provide a sophisticated level of gene regulation that coordinates a broad spectrum of biological processes. MiRNA inhibition of mRNA translation has emerged as an important regulator of chondrogenic and osteogenic development, osteoblast, osteoclast and chondrocyte cell growth and differentiation, and tissue homeostasis in the adult skeleton. MiRNAs control many layers of regulation in adult tissues connected to both normal biological and pathologic cellular activities. The study of miRNAs in skeletal disorders is in its infancy. Osteoarthritis (OA) is a disease that progresses from degeneration of the articular cartilage to remodeling of the underlying subchondral bone over many years. While miRNAs have been identified with the inflammatory pathogenesis of rheumatoid arthritis (RA), only a few studies have been performed on OA tissue (1,2) . Here we performed a systematic analysis of the articular cartilage from varus OA knee replacements, comparing multiple tissue samples from the lateral (spared) and medial (diseased) compartments. Before proceeding to a miRNA profiling, each sample was analyzed for expression of a small set of miRNAs that have been reported in association with RA, OA and cartilage formation. These preliminary findings have identified a spectrum of changes in surface cartilage between control and diseased tissue. Methods Human tissues: 6 individual articular cartilage samples were harvested from a total of 5 osteoarthritic varus human knees. Cartilage samples were exempt from IRB review as they are discarded materials. Samples were removed with a biopsy punch and were approximately 6 x 2mm (diameter x thickness). Cartilage specimens were harvested from the more normal-appearing lateral (‘spared’) compartments and from the more OA-affected, medial compartments of the knees. This sampling technique allows direct comparison of more significantly OA-affected cartilage samples with those of lower OA grade from the same set of individuals. Knee ages ranged from 53-74 years old and averaged 65 years old. RNA and miRNA Isolation: Each osteochondral specimen was placed in RNA Later (Sigma) immediately following surgical removal, in order to preserve the integrity of the total RNA. Specimens were transported to the lab where individual samples were removed carefully with RNase-treated tools and were transferred intofresh RNA Later solution and incubated overnight at 4C to allow penetration and maximal inhibition of RNase activity. Samples were then removed from RNA Later, blotted briefly and frozen in liquid N2, and then pulverized using a Bessman tissue pulverizer (Fisher). The pulverized samples were immediately placed into Trizol (InVitrogen) and homogenized using a polytron device. Total RNA was isolated to include small RNAs of \u3e17 nucleotides, according to the manufacturer’s protocol (InVitrogen). Purified RNA was obtained using precipitated total RNAs filtered through glass columns according to the manufacturer’s protocol (Zymo Research). RNAs were reverse-transcribed into DNA using 900ng of each purified RNA sample using the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems). TaqMan qPCR analysis for small RNAs was performed using the following human primer-probe sets from Applied Biosystems: hsa-miRs-: 9, 22, 27a, 29a and 34a. Human U6 was used to normalize all qPCR data and data was plotted as normalized relative values. Normalized relative values were averaged for each of the complement of medial vs. lateral samples. Results MiRs were found to be either up- or down-regulated in a manner that suggests a mechanism of de-repression of pro-inflammatory cytokine signaling and repression of pro-inflammatory events in medial vs. lateral varus knee OA cartilage samples, respectively. MiRs 9, 27a, and 29a were found to up-regulated in lateral varus knee cartilage samples vs. medial varus knee cartilage samples (Fig.1. A,C,E,F). Conversely, miRs 22 and 34a were found to upregulated in medial vs. lateral cartilage samples (Fig1, B, D). Discussion The functional characterization of global gene expression patterns through miRNAs in OA is lacking. Particularly, the roles of miRs in OA disease development, as biomarkers, and in disease outcomes are at question. A few large-scale microarray approaches have previously identified expression signatures of potential OA-involved miRNAs (2). By comparing cartilage samples that derive from more advanced (medial) vs. less advanced (lateral) OA stages in varus human knees, we seek to combine miRNA expression analysis with clinicopathologic features. MiRs -9, -22 and -34a are known to be involved in regulating pro-inflammatory events in OA. Higher levels of miRs, -9, -27a & -140 in less-affected lateral compartment cartilage are consistent with previous reports of reduced TNFa, MMP-13 & ADAMTS-5 expression events, respectively (Fig.1, F, E & A) (3,4,5). MiRs -22 and -34a have been shown to be associated with promoting tissue catabolism by their presence and are here shown to be increased in more affected medial compartment cartilage (Fig.1, B, D) (4). In addition, miR-34a deficiency has been previously shown to inhibit chondrocyte apoptosis, consistent with the lower expression level found in lateral cartilage (Fig.1, D) (6). MiR-29a was found in a previous microarray analysis to be the highest-fold down-regulated miRNA in OA vs. normal cartilage, consistent with our finding of under-expression in medial cartilage samples (Fig.1, C) (1). The goal of these studies is to begin to understand how miRNAs can both contribute to and protect against OA. Here we show that the comparison of cartilage-derived miRNAs in medial and lateral compartment pairs from the same knee may facilitate validation of candidate OA miRNAs. Significance The aims of this project are to provide an internally-controlled platform of study for the miRNAs of OA using the natural disease differences inherent in spared vs. non-spared cartilage compartments from a varus OA knee. Such efforts may provide an alternative methodology when compared to the significant barrier of obtaining age-matched, non-OA control knee cartilage. References 1.) Iliopoulus D. PLoS One. 2008;3(11):e3740. Epub 2008 Nov 17. 2.) Goldring MB. Curr Opin Rheumatol. 2011 Jul 22. [Epub]. 3.) Yu C. J Int Med Res. 2011;39(1):1-9. 4.) Alcaraz MJ. Biochem Pharmacol. 2010 Jul 1;80(1):13-21. 5.) Miyaki S. Genes Dev. 2010 Jun 1;24(11):1173-85. 6.) Abouheif MM. Rheumatology. 2010 Nov;49(11):2054-60

Expression of cell cycle regulatory factors in differentiating osteoblasts: postproliferative up-regulation of cyclins B and E

The representation of cyclins and cyclin-dependent kinases (cdks) was analyzed during progressive development of the bone cell phenotype in cultures of normal diploid rat calvarial osteoblasts. Three developmental stages were examined: (a) proliferation; (b) monolayer confluency; and (c) mineralization of the bone extracellular matrix. We demonstrate that the presence of cyclins and cdks is not restricted to the proliferation period. Consistent with their role in cell cycle progression, cdc2 and cdk2 decrease postproliferatively. However, cdk4 and cyclins A, B, and D1 persist in confluent cells. Cyclin E is significantly up-regulated during the extracellular matrix mineralization developmental period. Examination of the cytoplasmic levels of these cell cycle regulatory proteins indicates a marked increase in cyclin B in the late differentiation stage. The elevation of nuclear cyclin E and cytoplasmic cyclin B is not observed in osteoblasts maintained under culture conditions that do not support differentiation. Furthermore, treatment with transforming growth factor beta for 48 h during the proliferation period renders the cells incompetent for differentiation and abrogates the postproliferative up-regulation of cyclins B and E. Density-induced growth inhibition of ROS 17/2.8 osteosarcoma cells is not accompanied by up-regulation of nuclear cyclin E and cytoplasmic cyclin B when compared to the proliferation period. This observation is consistent with abrogation of both growth control and differentiation regulatory mechanisms in tumor cells. These results suggest that cell cycle regulatory proteins function not only during proliferation but may also play a role in normal diploid osteoblast differentiation

runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2alphaA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development

Runx2 mediates epigenetic silencing of the bone morphogenetic protein-3B (BMP-3B/GDF10) in lung cancer cells

BACKGROUND: The Runt-related transcription factor Runx2 is essential for bone development but is also implicated in progression of several cancers of breast, prostate and bone, where it activates cancer-related genes and promotes invasive properties. The transforming growth factor beta (TGF-beta) family member bone morphogenetic protein-3B (BMP-3B/GDF10) is regarded as a tumor growth inhibitor and a gene silenced in lung cancers; however the regulatory mechanisms leading to its silencing have not been identified. RESULTS: Here we show that Runx2 is highly expressed in lung cancer cells and downregulates BMP-3B. This inverse relationship between Runx2 and BMP-3B expression is further supported by increased expression of BMP-3B in mesenchymal cells from Runx2 deficient mice. The ectopic expression of Runx2, but not DNA binding mutant Runx2, in normal lung fibroblast cells and lung cancer cells resulted in suppression of BMP-3B levels. The chromatin immunoprecipitation studies identified that the mechanism of Runx2-mediated suppression of BMP-3B is due to the recruitment of Runx2 and histone H3K9-specific methyltransferase Suv39h1 to BMP-3B proximal promoter and a concomitant increase in histone methylation (H3K9) status. The knockdown of Runx2 in H1299 cells resulted in decreased histone H3K9 methylation on BMP-3B promoter and increased BMP-3B expression levels. Furthermore, co-immunoprecipitation studies showed a direct interaction of Runx2 and Suv39h1 proteins. Phenotypically, Runx2 overexpression in H1299 cells increased wound healing response to TGFbeta treatment. CONCLUSIONS: Our studies identified BMP-3B as a new Runx2 target gene and revealed a novel function of Runx2 in silencing of BMP-3B in lung cancers. Our results suggest that Runx2 is a potential therapeutic target to block tumor suppressor gene silencing in lung cancer cells

Antagonizing miR-218-5p attenuates Wnt signaling and reduces metastatic bone disease of triple negative breast cancer cells

Wnt signaling is implicated in bone formation and activated in breast cancer cells promoting primary and metastatic tumor growth. A compelling question is whether osteogenic miRNAs that increase Wnt activity for bone formation are aberrantly expressed in breast tumor cells to support metastatic bone disease. Here we report that miR-218-5p is highly expressed in bone metastases from breast cancer patients, but is not detected in normal mammary epithelial cells. Furthermore, inhibition of miR-218-5p impaired the growth of bone metastatic MDA-MB-231 cells in the bone microenvironment in vivo. These findings indicate a positive role for miR-218-5p in bone metastasis. Bioinformatic and biochemical analyses revealed a positive correlation between aberrant miR-218-5p expression and activation of Wnt signaling in breast cancer cells. Mechanistically, miR-218-5p targets the Wnt inhibitors Sclerostin (SOST) and sFRP-2, which highly enhances Wnt signaling. In contrast, delivery of antimiR-218-5p decreased Wnt activity and the expression of metastasis-related genes, including bone sialoprotein (BSP/IBSP), osteopontin (OPN/SPP1) and CXCR-4, implicating a Wnt/miR-218-5p regulatory network in bone metastatic breast cancer. Furthermore, miR-218-5p also mediates the Wnt-dependent up-regulation of PTHrP, a key cytokine promoting cancer-induced osteolysis. Antagonizing miR-218-5p reduced the expression of PTHrP and Rankl, inhibited osteoclast differentiation in vitro and in vivo, and prevented the development of osteolytic lesions in a preclinical metastasis model. We conclude that pathological elevation of miR-218-5p in breast cancer cells activates Wnt signaling to enhance metastatic properties of breast cancer cells and cancer-induced osteolytic disease, suggesting that miR-218-5p could be an attractive therapeutic target for preventing disease progression

Cells Capable of Bone Production Engraft from Whole Bone Marrow Transplants in Nonablated Mice

Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the individual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface

A specific targeting signal directs Runx2/Cbfa1 to subnuclear domains and contributes to transactivation of the osteocalcin gene

Key components of DNA replication and the basal transcriptional machinery as well as several tissue-specific transcription factors are compartmentalized in specialized nuclear domains. In the present study, we show that determinants of subnuclear targeting of the bone-related Runx2/Cbfa1 protein reside in the C-terminus. With a panel of C-terminal mutations, we further demonstrate that targeting of Runx2 to discrete subnuclear foci is mediated by a 38 amino acid sequence (aa 397-434). This nuclear matrix-targeting signal (NMTS) directs the heterologous Gal4 protein to nuclear-matrix-associated Runx2 foci and enhances transactivation of a luciferase gene controlled by Gal4 binding sites. Importantly, we show that targeting of Runx2 to the NM-associated foci contributes to transactivation of the osteoblast-specific osteocalcin gene in osseous cells. Taken together, these findings identify a critical component of the mechanisms mediating Runx2 targeting to subnuclear foci and provide functional linkage between subnuclear organization of Runx2 and bone-specific transcriptional control

Nuclear matrix proteins distinguish normal diploid osteoblasts from osteosarcoma cells

Interrelationships between nuclear architecture and gene expression were examined by comparing the representation of nuclear matrix proteins in ROS 17/2.8 rat and MG-63 human osteosarcoma cells with those in normal diploid osteoblasts. The tumor-derived cells coexpress genes which are expressed in a sequential and mutually exclusive manner during the progressive stages of osteoblast differentiation. In osteosarcoma cells two-dimensional electrophoretic analysis indicates a composite representation of nuclear matrix proteins characteristic of both the proliferative and postproliferative periods of osteoblast phenotype development. In addition, nuclear matrix proteins unique to the tumor cells and the absence of nuclear matrix proteins found only in normal diploid osteoblasts are observed. Tumor-specific nuclear matrix proteins include those expressed in a proliferation-dependent and independent manner. There is a parallel relationship between nuclear matrix proteins and the expression of cell growth and tissue-specific genes during osteoblast differentiation and in osteosarcoma cells where the developmental sequence of gene expression has been abrogated. Nuclear matrix proteins therefore provide markers reflecting defined periods of bone cell differentiation and phenotypic characteristics of an osteosarcoma