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The increased detection and treatment of early stage breast cancer as well as ductal carcinoma in situ (DCIS) has not led to significant survival benefits. Therefore, the current standard treatment of DCIS is questionable. An informed evidence-based treatment strategy, and likely de-escalation from the current standards requires new prognostic models built from more comprehensive characterization with objective criteria. Parallel profiling of the molecular landscape and micro-environment in pure DCIS remains challenging due to histological heterogeneity and the inevitable reliance on small archived specimens. Leveraging recent methodological advances, we characterized the mutational, transcriptional, histological and microenvironmental landscape across multiple micro-dissected regions from 39 cases to generate a multi-modal breast precancer atlas. The histological architecture was associated with grade, adiposity, and intrinsic expression subtypes. Similar to previous findings, high-grade lesions had higher mutational burden, including TP53 mutations, while low-grade lesions had more frequent 16q losses and GATA3 mutations. Multi-region analysis revealed most somatic alterations, including whole genome duplication events, were clonal, but genetic divergence increased with distance between regions. In 7/12 evaluable cases, somatic mutations in putative driver genes affected a subset of regions only. This genetic heterogeneity often accompanied phenotypic heterogeneity and regions with low risk features (Normal-like, Luminal A) occurred earlier than those with high-risk features (Her2-like, Basal or necrosis) according to the phylogenetic analysis. The immune-environment was evaluated using multiplex immuno-histochemistry to measure relative stromal and epithelial densities of B lymphocyte (B-cell), T lymphocyte (T-cell) and regulatory T cells (T-reg) and identify 3 immune-states: Active, Suppressed and Excluded (lower epithelial density). All states included both DCIS and adjacent benign regions, and none associated with intrinsic subtypes. The Excluded state was enriched in high-grade DCIS and, compared to benign areas, more likely acquired in DCIS, showing transcriptional evidence of stronger immune-suppression and possible evasion. The breast pre-cancer atlas therefore reveals correlated levels of phenotypic and genotypic heterogeneity, including at sub-histological resolution. These uniquely integrated observations will help scope future studies, prioritize candidate markers for progression risk modelling and identify functional similarities in precursor lesions from other types of adenocarcinomas.

RUNX1 has recently been shown to play an important role in determination of mammary epithelial cell identity. However, mechanisms by which loss of the RUNX1 transcription factor in mammary epithelial cells leads to epithelial-to-mesenchymal transition (EMT) are not known. Here, we report that interaction between RUNX1 and its heterodimeric partner CBFβ is essential for sustaining mammary epithelial cell identity. Disruption of RUNX1-CBFβ interaction, DNA binding, and association with mitotic chromosomes alters cell morphology, global protein synthesis, and phenotype-related gene expression. During interphase, RUNX1 is organized as punctate, predominantly nuclear, foci that are dynamically redistributed during mitosis, with a subset localized to mitotic chromosomes. Genome-wide RUNX1 occupancy profiles for asynchronous, mitotically enriched, and early G1 breast epithelial cells reveal RUNX1 associates with RNA Pol II-transcribed protein coding and long non-coding RNA genes and RNA Pol I-transcribed ribosomal genes critical for mammary epithelial proliferation, growth, and phenotype maintenance. A subset of these genes remains occupied by the protein during the mitosis to G1 transition. Together, these findings establish that the RUNX1-CBFβ complex is required for maintenance of the normal mammary epithelial phenotype and its disruption leads to EMT. Importantly, our results suggest, for the first time, that RUNX1 mitotic bookmarking of a subset of epithelial-related genes may be an important epigenetic mechanism that contributes to stabilization of the mammary epithelial cell identity.

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24-/low versus CD24+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFβ1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24-/low and CD24+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFβ for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial-to-mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer-compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

Selective estrogen receptor modulators (SERMs), including the SERM/SERD bazedoxifene (BZA), are used to treat postmenopausal osteoporosis and may reduce breast cancer (BCa) risk. One of the most persistent unresolved questions regarding menopausal hormone therapy is compromised control of proliferation and phenotype because of short- or long-term administration of mixed-function estrogen receptor (ER) ligands. To gain insight into epigenetic effectors of the transcriptomes of hormone and BZA-treated BCa cells, we evaluated a panel of histone modifications. The impact of short-term hormone treatment and BZA on gene expression and genome-wide epigenetic profiles was examined in ERαneg mammary epithelial cells (MCF10A) and ERα+ luminal breast cancer cells (MCF7). We tested individual components and combinations of 17β-estradiol (E2), estrogen compounds (EC10) and BZA. RNA-seq for gene expression and ChIP-seq for active (H3K4me3, H3K4ac, H3K27ac) and repressive (H3K27me3) histone modifications were performed. Our results show that the combination of BZA with E2 or EC10 reduces estrogen-mediated patterns of histone modifications and gene expression in MCF-7ERα+ cells. In contrast, BZA has minimal effects on these parameters in MCF10A mammary epithelial cells. BZA-induced changes in histone modifications in MCF7 cells are characterized by altered H3K4ac patterns, with changes at distal enhancers of ERα-target genes and at promoters of non-ERα bound proliferation-related genes. Notably, the ERα target gene GREB1 is the most sensitive to BZA treatment. Our findings provide direct mechanistic-based evidence that BZA induces epigenetic changes in E2 and EC10 mediated control of ERα regulatory programs to target distinctive proliferation gene pathways that restrain the potential for breast cancer development.

Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas-9 (CRISPR-Cas9) technique to generate iMSCs-VPR(BMP-9+), followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of bone morphogenetic proteins (BMP)-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2. Protein expression of SMAD1/5/8 and pSMAD1/5/8 iMSCs-VPR(BMP-9+) displayed significant changes in the expression of a panel of genes involved in TGF-β/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPR(BMP-9+) exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.

The bone remodeling process is crucial for titanium (Ti) osseointegration and involves the crosstalk between osteoclasts and osteoblasts. Considering the high osteogenic potential of Ti with nanotopography (Ti Nano) and that osteoclasts inhibit osteoblast differentiation, we hypothesized that nanotopography attenuate the osteoclast-induced disruption of osteoblast differentiation. Osteoblasts were co-cultured with osteoclasts on Ti Nano and Ti Control and non-co-cultured osteoblasts were used as control. Gene expression analysis using RNAseq showed that osteoclasts downregulated the expression of osteoblast marker genes and upregulated genes related to histone modification and chromatin organization in osteoblasts grown on both Ti surfaces. Osteoclasts also inhibited the mRNA and protein expression of osteoblast markers, and such effect was attenuated by Ti Nano. Also, osteoclasts increased the protein expression of H3K9me2, H3K27me3 and EZH2 in osteoblasts grown on both Ti surfaces. ChIP assay revealed that osteoclasts increased accumulation of H3K27me3 that represses the promoter regions of Runx2 and Alpl in osteoblasts grown on Ti Control, which was reduced by Ti Nano. In conclusion, these data show that despite osteoclast inhibition of osteoblasts grown on both Ti Control and Ti Nano, the nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by preventing the increase of H3K27me3 accumulation that represses the promoter regions of some key osteoblast marker genes. These findings highlight the epigenetic mechanisms triggered by nanotopography to protect osteoblasts from the deleterious effects of osteoclasts, which modulate the process of bone remodeling and may benefit the osseointegration of Ti implants.

Background: With the implementation of widespread breast cancer screening, the diagnosis of Ductal Carcinoma In Situ (DCIS) has increased 5 fold. Most cases are treated with combinations of surgery, radiation and endocrine therapy, reducing the risk of second events, including ipsilateral invasive cancer. Without standard markers to confidently identify the most indolent lesions, a subset of cases are likely over-treated. The mutational landscapes of DCIS and invasive ductal carcinoma (IDC) are similar and not sufficient to identify higher risk lesions with recent studies suggesting that clonal selection plays a limited role in progression. Histological analysis highlighted the role of the extracellular matrix and immune-surveillance to maintain duct integrity and limit progression. Due to the small size, limited availability and quality of research specimens, few biomarker studies investigated pure DCIS with adequate follow-up or genome-wide methods, let alone integrated more than one type of biomarker. Unlike breast cancer, there is no comprehensive, systematic, multi-modal atlas of DCIS, limiting the ability to test broad and novel hypotheses and characterize processes maintaining breast tissue homeostasis. Methods: Through sequential sectioning of pure DCIS archived specimens, a total of 70 histological regions from 40 cases were annotated and profiled using up to 3 platforms: multiplex immuno-histochemistry (mIHC), RNA-seq, and whole-exome sequencing. Stromal and epithelial spatial distribution of immune cells and states were quantified using mIHC. The epithelial compartments were microdissected and profiled using genome-wide gene expression, DNA mutations and copy number alterations. Results: Epithelial regions were classified according to expression subtypes consistent with histological markers, highlighting associations with the lesion architecture and grade. Compared to solid pattern, cribriform pattern DCIS has induced EMT processes and repressed proliferation processes, a trend reminiscent of low-recurrence-risk expression signatures measured in IDC. The DNA copy number burden increased with grade and the mutational burden was the highest in solid DCIS. Both were higher in Her2+ cases. The clustering of mutations at chromosome 17p - attributed to the APOBEC-driven Kataegis phenomenon - was observed in a subset of regions, suggesting this event can occur in pre-invasive lesions. All DCIS had somatic alterations of at least one known driver gene with some associated with grade (TP53) or expression subtype (ERBB2). Multi-region profiling available in a subset of samples revealed genetic heterogeneity of likely-driver events between proximal regions of similar histological characteristics. The density and proliferative states of selected immune cells - including T-cells, B-cells and Macrophages - highlights the diversity of the tumor immune environment with the highest densities observed in Her2+ ducts and stroma, minimal ductal infiltration in other lesions, fewer dividing B- and T-cells around the more proliferative areas and a small number of regions depleted from any adaptive immune cells. Conclusion: This first multi-modal profiling of pure DCIS reveals an unsuspected diversity of molecular and microenvironmental states and presents their association with progression risk factors. The observations support the need for stronger integration of molecular and clinicopathology features, especially at sub-histological levels, to ensure the findings can be interpreted in the correct clinical and phenotypic context. The compatibility of the approach with archived specimens supports the expansion to larger retrospective DCIS collections with outcomes. Citation Format: Olivier Harismendy, Daniela Nachmanson, Mark F. Evans, Hidetoshi Mori, Adam Officer, Christina Yau, Joseph Steward, Huazhen Yao, Thomas O'Keefe, Farnaz Hasteh, Gary S. Stein, Kristen Jepsen, Michael Campbell, Donald L. Weaver, Gillian L. Hirst, Brian L. Sprague, Laura J. Esserman, Jonathan A. Gordon, Alexander Borowsky, Janet L. Stein. The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS2-01.

Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones ("histone code"), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. J. Cell. Biochem. 118: 1262-1272, 2017. © 2016 Wiley Periodicals, Inc.

Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti-osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre-osteoblast cell line, MC3T3-E1. Ethanolic extract of CQ (CQ-E) was found to affect growth kinetics of MC3T3-E1 cells in a dosage-dependent manner. High concentrations of CQ-E (more than 10 μg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non-toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ-E. CQ-E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose-dependent effect of CQ-E with lower concentrations exhibiting anabolic and osteogenic properties. J. Cell. Physiol. 232: 540-547, 2017. © 2016 Wiley Periodicals, Inc.

RUNX1 has recently been shown to play an important role in determination of mammary epithelial cell identity. However, mechanisms by which loss of the RUNX1 transcription factor in mammary epithelial cells leads to epithelial-to-mesenchymal transition (EMT) are not known. Here, we report mitotic bookmarking of genes by RUNX1 as a potential mechanism to convey regulatory information through successive cell divisions for coordinate control of mammary cell proliferation, growth, and identity. Genome-wide RUNX1 occupancy profiles for asynchronous, mitotically enriched, and early G1 breast epithelial cells reveal RUNX1 is retained during the mitosis to G1 transition on protein coding and long non-coding RNA genes critical for mammary epithelial proliferation, growth, and phenotype maintenance. Disruption of RUNX1 DNA binding and association with mitotic chromosomes alters cell morphology, global protein synthesis, and phenotype-related gene expression. Together, these findings show for the first time that RUNX1 bookmarks a subset of epithelial-related genes during mitosis that remain occupied as cells enter the next cell cycle. Compromising RUNX1 DNA binding initiates EMT, an essential first step in the onset of breast cancer.SignificanceThis study elucidates mitotic gene bookmarking as a potential epigenetic mechanism that impacts breast epithelial cell growth and phenotype and has potential implications in breast cancer onset.

Nuclear organization is functionally linked to genetic and epigenetic regulation of gene expression for biological control and is modified in cancer. Nuclear organization supports cell growth and phenotypic properties of normal and cancer cells by facilitating physiologically responsive interactions of chromosomes, genes and regulatory complexes at dynamic three-dimensional microenvironments. We will review nuclear structure/function relationships that include: 1. Epigenetic bookmarking of genes by phenotypic transcription factors to control fidelity and plasticity of gene expression as cells enter and exit mitosis; 2. Contributions of chromatin remodeling to breast cancer nuclear morphology, metabolism and effectiveness of chemotherapy; 3. Relationships between fidelity of nuclear organization and metastasis of breast cancer to bone; 4. Dynamic modifications of higher-order inter- and intra-chromosomal interactions in breast cancer cells; 5. Coordinate control of cell growth and phenotype by tissue-specific transcription factors; 6. Oncofetal epigenetic control by bivalent histone modifications that are functionally related to sustaining the stem cell phenotype; and 7. Noncoding RNA-mediated regulation in the onset and progression of breast cancer. The discovery of components to nuclear organization that are functionally related to cancer death and compromise gene expression have the potential for translation to innovative cancer diagnosis and targeted therapy.

Multiple mechanisms of epigenetic control that include DNA methylation, histone modification, noncoding RNAs, and mitotic gene bookmarking play pivotal roles in stringent gene regulation during lineage commitment and maintenance. Experimental evidence indicates that bivalent chromatin domains, i.e., genome regions that are marked by both H3K4me3 (activating) and H3K27me3 (repressive) histone modifications, are a key property of pluripotent stem cells. Bivalency of developmental genes during the G1 phase of the pluripotent stem cell cycle contributes to cell fate decisions. Recently, some cancer types have been shown to exhibit partial recapitulation of bivalent chromatin modifications that are lost along with pluripotency, suggesting a mechanism by which cancer cells reacquire properties that are characteristic of undifferentiated, multipotent cells. This bivalent epigenetic control of oncofetal gene expression in cancer cells may offer novel insights into the onset and progression of cancer and may provide specific and selective options for diagnosis as well as for therapeutic intervention.

Long suspected, recently recognized, and increasingly studied, non protein-coding RNAs (ncRNAs) are emerging as key drivers of biological control and pathology. Since their discovery in 1993, microRNAs (miRNAs) have been the subject of intense research focus and investigations have revealed striking findings, establishing that these molecules can exert a substantial level of biological control in numerous tissues. More recently, long ncRNAs (lncRNAs), the lesser-studied siblings of miRNA, have been suggested to have a similar robust role in developmental and adult tissue regulation. Mesenchymal stem cells (MSCs) are an important source of multipotent cells for normal and therapeutic tissue repair. Much is known about the critical role of miRNAs in biogenesis and differentiation of MSCs however; recent studies have suggested lncRNAs may play an equally important role in the regulation of these cells. Here we highlight the role of lncRNAs in the regulation of mesenchymal stem cell lineages including adipocytes, chondrocytes, myoblasts, and osteoblasts. In addition, the potential for these noncoding RNAs to be used as biomarkers for disease or therapeutic targets is also discussed.

Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for differentiation of precursor cells into mature osteoblasts. Although organization and control of the bone-specific Runx2-P1 promoter have been studied extensively, long-range regulation has not been explored. In this study, we investigated higher-order organization of the Runx2-P1 promoter during osteoblast differentiation. Mining the ENCODE database revealed interactions between Runx2-P1 and Supt3h promoters in several non-mesenchymal human cell lines. Supt3h is a ubiquitously expressed gene located within the first intron of Runx2. These two genes show shared synteny across species from humans to sponges. Chromosome conformation capture analysis in the murine pre-osteoblastic MC3T3-E1 cell line revealed increased contact frequency between Runx2-P1 and Supt3h promoters during differentiation. This increase was accompanied by enhanced DNaseI hypersensitivity along with RUNX2 and CTCF binding at the Supt3h promoter. Furthermore, interplasmid-3C and luciferase reporter assays showed that the Supt3h promoter can modulate Runx2-P1 activity via direct association. Taken together, our data demonstrate physical proximity between Runx2-P1 and Supt3h promoters, consistent with their syntenic nature. Importantly, we identify the Supt3h promoter as a potential regulator of the bone-specific Runx2-P1 promoter.

Human mutations and in vitro studies indicate that DLX3 has a crucial function in bone development, however, the in vivo role of DLX3 in endochondral ossification has not been established. Here, we identify DLX3 as a central attenuator of adult bone mass in the appendicular skeleton. Dynamic bone formation, histologic and micro-computed tomography analyses demonstrate that in vivo DLX3 conditional loss of function in mesenchymal cells (Prx1-Cre) and osteoblasts (OCN-Cre) results in increased bone mass accrual observed as early as 2 weeks that remains elevated throughout the lifespan owing to increased osteoblast activity and increased expression of bone matrix genes. Dlx3OCN-conditional knockout mice have more trabeculae that extend deeper in the medullary cavity and thicker cortical bone with an increased mineral apposition rate, decreased bone mineral density and increased cortical porosity. Trabecular TRAP staining and site-specific Q-PCR demonstrated that osteoclastic resorption remained normal on trabecular bone, whereas cortical bone exhibited altered osteoclast patterning on the periosteal surface associated with high Opg/Rankl ratios. Using RNA sequencing and chromatin immunoprecipitation-Seq analyses, we demonstrate that DLX3 regulates transcription factors crucial for bone formation such as Dlx5, Dlx6, Runx2 and Sp7 as well as genes important to mineral deposition (Ibsp, Enpp1, Mepe) and bone turnover (Opg). Furthermore, with the removal of DLX3, we observe increased occupancy of DLX5, as well as increased and earlier occupancy of RUNX2 on the bone-specific osteocalcin promoter. Together, these findings provide novel insight into mechanisms by which DLX3 attenuates bone mass accrual to support bone homeostasis by osteogenic gene pathway regulation.

Multipotent mesenchymal stromal cells (MSCs) are critical for regeneration of multiple tissues. Epigenetic mechanisms are fundamental regulators of lineage specification and cell fate, and as such, we addressed the question of which epigenetic modifications characterize the transition of nascent MSCs to a tissue specific MSC-derived phenotype. By profiling the temporal changes of seven histone marks correlated to gene expression during proliferation, early commitment, matrix deposition, and mineralization stages, we identified distinct epigenetic mechanisms that regulate transcriptional programs necessary for tissue-specific phenotype development. Patterns of stage-specific enrichment of histone modifications revealed distinct modes of repression and activation of gene expression that would not be detected using single endpoint analysis. We discovered that at commitment, H3K27me3 is removed from genes that are upregulated and is not acquired on downregulated genes. Additionally, we found that the absence of H3K4me3 modification at promoters defined a subset of osteoblast-specific upregulated genes, indicating that acquisition of acetyl modifications drive activation of these genes. Significantly, loss or gain of H3K36me3 was the primary predictor of dynamic changes in temporal gene expression. Using unsupervised pattern discovery analysis the signature of osteogenic-related histone modifications identified novel functional cis regulatory modules associated with enhancer regions that control tissue-specific genes. Our work provides a cornerstone to understand the epigenetic regulation of transcriptional programs that are important for MSC lineage commitment and lineage, as well as insights to facilitate MSC-based therapeutic interventions.

Alterations in the epigenetic landscape are fundamental drivers of aberrant gene expression that contribute to cancer progression and pathology. Understanding specific modes of epigenetic regulation can be used to identify novel biomarkers or targets for therapeutic intervention to clinically treat solid tumors and leukemias. The bivalent marking of gene promoters by H3K4me3 and H3K27me3 is a primary mechanism to poise genes for expression in pluripotent embryonic stem cells (ESC). In this study we interrogated three well-established mammary cell lines to model epigenetic programming observed among breast cancer subtypes. Evidence is provided for a distinct bivalent signature, activating and repressive histone marks co-residing at the same gene promoter, in the MCF7 (ESR/PGR+) luminal breast cancer cell line. We identified a subset of genes, enriched for developmental pathways that regulate cellular phenotype and signaling, and partially recapitulate the bivalent character observed in ESC. We validated the biological relevance of this "oncofetal epigenetic" signature using data from ESR/PGR+ tumor samples from breast cancer patients. This signature of oncofetal epigenetic control is an informative biomarker and may provide novel therapeutic targets, selective for both recurring and treatment-resistant cancers. J. Cell. Physiol. 231: 2474-2481, 2016. © 2016 Wiley Periodicals, Inc.

Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.

Skeletal metastasis is a serious complication of many primary cancers. A common feature of tumor cells that metastasize to the bone marrow microenvironment is that they initiate a cascade of events, recruiting and presumably/potentially altering the phenotype of bone marrow mesenchymal stromal cells (MSC) to produce an environment that allows for tumor growth and in some cases, drug-resistant dormancy of latent cancer cells. Consequently the MSC population can contribute to metastatic disease through several distinct mechanisms by differentiating into cancer-associated fibroblasts (CAFs). Understanding the expression and epigenetic changes that occur as normal MSCs become associated with metastatic tumors would reveal possible therapeutic targets for treating skeletal metastasis.

Abdominal-class homeodomain-containing (Hox) factors form multimeric complexes with TALE-class homeodomain proteins (Pbx, Meis) to regulate tissue morphogenesis and skeletal development. Here we have established that Pbx1 negatively regulates Hoxa10-mediated gene transcription in mesenchymal cells and identified components of a Pbx1 complex associated with genes in osteoblasts. Expression of Pbx1 impaired osteogenic commitment of C3H10T1/2 multipotent cells and differentiation of MC3T3-E1 preosteoblasts. Conversely, targeted depletion of Pbx1 by short hairpin RNA (shRNA) increased expression of osteoblastrelated genes. Studies using wild-type and mutated osteocalcin and Bsp promoters revealed that Pbx1 acts through a Pbx-binding site that is required to attenuate gene activation by Hoxa10. Chromatin-associated Pbx1 and Hoxa10 were present at osteoblast-related gene promoters preceding gene expression, but only Hoxa10 was associated with these promoters during transcription. Our results show that Pbx1 is associated with histone deacetylases normally linked with chromatin inactivation. Loss of Pbx1 from osteoblast promoters in differentiated osteoblasts was associated with increased histone acetylation and CBP/p300 recruitment, as well as decreased H3K9 methylation. We propose that Pbx1 plays a central role in attenuating the ability of Hoxa10 to activate osteoblast-related genes in order to establish temporal regulation of gene expression during osteogenesis. Embryonic patterning of the skeleton is a complex process responsible for regulating the positional identity, shape, and size of skeletal primordial elements by an intricate orchestration of signaling molecules and transcriptional regulators. The

We have developed a novel Ribonucleoprotein Immunoprecipitation (RNP-IP) method to isolate miR-RISC complexes, associated microRNAs and target mRNAs. Our method characterizes mRNAs present in immunoprecipitates containing miR-RISC complexes that were obtained using GW182 and AGO2 antibodies. MicroRNA bound transcripts were reverse transcribed and amplified using seed sequence and 3'UTR derived primers. This flexible IP-based assay is a straightforward method to identify miRs participating in gene regulation and their cognate mRNAs in real time.

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways.

The Runx1 transcription factor, known for its essential role in normal hematopoiesis, was reported in limited studies to be mutated or associated with human breast tumor tissues. Runx1 increases concomitantly with disease progression in the MMTV-PyMT transgenic mouse model of breast cancer. Compelling questions relate to mechanisms that regulate Runx1 expression in breast cancer. Here, we tested the hypothesis that dysregulation of Runx1-targeting microRNAs (miRNAs) allows for pathologic increase of Runx1 during breast cancer progression. Microarray profiling of the MMTV-PyMT model revealed significant downregulation of numerous miRNAs predicted to target Runx1. One of these, miR-378, was inversely correlated with Runx1 expression during breast cancer progression in mice and in human breast cancer cell lines MCF7 and triple-negative MDA-MB-231 that represent early- and late-stage diseases, respectively. MiR-378 is nearly absent in MDA-MB-231 cells. Luciferase reporter assays revealed that miR-378 binds the Runx1 3' untranslated region (3'UTR) and inhibits Runx1 expression. Functionally, we demonstrated that ectopic expression of miR-378 in MDA-MB-231 cells inhibited Runx1 and suppressed migration and invasion, while inhibition of miR-378 in MCF7 cells increased Runx1 levels and cell migration. Depletion of Runx1 in late-stage breast cancer cells resulted in increased expression of both the miR-378 host gene PPARGC1B and pre-miR-378, suggesting a feedback loop. Taken together, our study identifies a novel and clinically relevant mechanism for regulation of Runx1 in breast cancer that is mediated by a PPARGC1B-miR-378-Runx1 regulatory pathway. Our results highlight the translational potential of miRNA replacement therapy for inhibiting Runx1 in breast cancer.

Osteosarcoma is a highly metastatic form of bone cancer in adolescents and young adults that is resistant to existing treatments. Development of an effective therapy has been hindered by very limited understanding of the mechanisms of osteosarcomagenesis. Here, we used genetically engineered mice to investigate the effects of deleting the tumor suppressor Wwox selectively in either osteoblast progenitors or mature osteoblasts. Mice with conditional deletion of Wwox in preosteoblasts (WwoxΔosx1) displayed a severe inhibition of osteogenesis accompanied by p53 upregulation, effects that were not observed in mice lacking Wwox in mature osteoblasts. Deletion of p53 in WwoxΔosx1 mice rescued the osteogenic defect. In addition, the Wwox;p53Δosx1 double knockout mice developed poorly differentiated osteosarcomas that resemble human osteosarcoma in histology, location, metastatic behavior, and gene expression. Strikingly, the development of osteosarcomas in these mice was greatly accelerated compared with mice lacking p53 only. In contrast, combined WWOX and p53 inactivation in mature osteoblasts did not accelerate osteosarcomagenesis compared with p53 inactivation alone. These findings provide evidence that a WWOX–p53 network regulates normal bone formation and that disruption of this network in osteoprogenitors results in accelerated osteosarcoma. The Wwox;p53Δosx1 double knockout establishes a new osteosarcoma model with significant advancement over existing models. Cancer Res; 76(20); 6107–17. ©2016 AACR.

Bone sialoprotein (BSP) is an acidic, noncollagenous glycoprotein abundantly expressed in mineralized tissues. Although BSP is frequently used as a marker of osteoblast differentiation, the role of the protein in osteoblast function is unclear. BSP belongs to the SIBLING (Small Integrin-binding LIgand N-linked Glycoprotein) family of RGD-containing matrix proteins, several members of which have been shown to affect cell differentiation. The normal levels of BSP expression in osteoblasts were specifically altered by CMV-mediated adenoviral overexpression in primary osteoblasts or inhibition by an RNA interference-based strategy in the MC3T3E1 cell line. Alternatively, osteoblast cultures were supplemented with recombinant BSP protein. Quantitative real-time PCR was used to monitor the mRNA levels of the osteoblast-related transcription factors Osterix and Runx2 as well as the osteoblast-specific gene osteocalcin. As markers of osteoblast differentiation, alkaline phosphatase enzyme activity, Runx2-luciferase reporter activity and calcein incorporation into mineralized cultures were also measured. The overexpression of BSP increased osteoblast-related gene expression as well as calcium incorporation and nodule formation by osteoblast cultures. Similarly, supplementation of osteoblast cultures with recombinant BSP increased several markers of osteoblast differentiation. Conversely, suppression of BSP expression by small-hairpin RNA-encoding plasmids inhibited expression of osteoblast markers and nodule formation. Overexpression of several functional-domain mutants of BSP demonstrated that increases in osteoblast-related gene expression and matrix mineralization observed in BSP overexpression models are mediated by the integrin-binding RGD motif found near the C-terminus of the protein. These results demonstrate that BSP may serve as a matrix-associated signal directly promoting osteoblast differentiation resulting in the increased production of a mineralized matrix.

The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that result in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was over-represented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways.

Background Many leukemias result from chromosomal rearrangements. The t(8;21) chromosomal translocation produces AML1-ETO, an oncogenic fusion protein that compromises the function of AML1, a transcription factor critical for myeloid cell differentiation. Because of the pressing need for new therapies in the treatment of acute myleoid leukemia, we investigated the genome-wide occupancy of AML1-ETO in leukemic cells to discover novel regulatory mechanisms involving AML-ETO bound genes. Results We report the co-localization of AML1-ETO with the N-CoR co-repressor to be primarily on genomic regions distal to transcriptional start sites (TSSs). These regions exhibit over-representation of the motif for PU.1, a key hematopoietic regulator and member of the ETS family of transcription factors. A significant discovery of our study is that genes co-occupied by AML1-ETO and N-CoR (e.g., TYROBP and LAPTM5) are associated with the leukemic phenotype, as determined by analyses of gene ontology and by the observation that these genes are predominantly up-regulated upon AML1-ETO depletion. In contrast, the AML1-ETO/p300 gene network is less responsive to AML1-ETO depletion and less associated with the differentiation block characteristic of leukemic cells. Furthermore, a substantial fraction of AML1-ETO/p300 co-localization occurs near TSSs in promoter regions associated with transcriptionally active loci. Conclusions Our findings establish a novel and dominant t(8;21) AML leukemia signature characterized by occupancy of AML1-ETO/N-CoR at promoter-distal genomic regions enriched in motifs for myeloid differentiation factors, thus providing mechanistic insight into the leukemic phenotype. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1445-0) contains supplementary material, which is available to authorized users.

There is growing recognition that epigenetic regulation contributes significantly to the fidelity of biological control. Compromised epigenetic pathways have now been shown to provide indications of transformation and tumor progression; they suggest potential targets for cancer prevention and intervention [1]. A formidable challenge is to deconvolute the complexity and plasticity of histone modifications associated with tumorigenesis. Although challenging, identification of epigenetic signals that are functionally associated with oncogenesis will provide a better understanding of the mechanisms underlying their impact. Post translational modifications of histone3 lysine4 (H3K4) may constitute epigenetically mediated regulatory signatures that can be informative for the molecular characterization of breast cancer pathology. Both methylation and acetylation of H3K4 are associated with gene activation. Tri-methylation of histone3 lysine4 has been extensively studied and is a well-documented marker for active or poised gene transcription. The enzyme complexes that regulate H3K4me3 have been implicated in tumorigenesis, pointing to the relevance of this epigenetic modification in cancer. Histone acetylation as well as histone acetyltransferases and deacetylating enzymes are recognized as important in tumor progression. However, the acetylation of H3K4 in particular has been less extensively investigated to date. Messier, et al., addressed the coordinated contributions of H3K4me3 and H3K4ac to breast cancer tumorigenesis in three well-established human mammary cell lines that represent a normal-like subtype (MCF10A; fibrocystic disease) and two cancer subtypes, luminal (MCF7; ER+/PR+) and basal-like metastatic (MDA-MB-231; ER−/PR−/HER2−) [2]. These cell lines recapitulate the phenotypic and proliferative transitions that accompany tumor progression states. Utilizing ChIP-seq analysis, a genome-wide map of H3K4me3 and H3K4ac patterns was developed, establishing dynamic alterations in the overall histone H3K4 epigenetic landscape in the three cell lines. An observed global increase in H3K4ac in MCF7 and MDA-MB-231 cell lines that is accompanied by a global increase in H3K4me3 in the MDA-MB-231 metastatic cell line is consistent with increased representation of both histone marks during oncogenic progression. Modifications in H3K4 tri-methylation and acetylation were found to occur on specific chromosomes, including the X-chromosome. This striking change in H3K4 tri-methylation and acetylation may account for the loss of X-chromosome inactivation and epigenomic instability of the inactive X-chromosome seen in breast cancer [3]. In contrast to methylation and acetylation of H3K9 and H3K27, which activate or repress transcription respectively, methylation and acetylation of H3K4 are both associated with gene activation. Observed increases in H3K4 acetylation marks at specific gene promoters in both the MCF7 and MDA-MB-231 cell lines, compared to normal-like MCF10A cells, suggests that H3K4 acetylation may be an early step in cancer initiation or progression. The global increase in H3K4me3 at gene promoters in MDA-MB-231 cells suggests alterations in the epigenetic landscape that may be functionally related to late stage, metastatic breast cancer. From a mechanistic perspective, the presence of H3K4ac and H3K4me3 at specific gene promoters provides a blueprint for regulatory pathways that are functionally linked to epigenetic control of breast cancer onset and progression. The dynamic acetylation of H3K4 is associated with estrogen-receptor responsiveness in the MCF7 cells and the initial loss of mammary tissue–specific gene expression in MCF7 cells that represent early stage breast cancer. Enhancing the pivotal role of H3K4ac in initial stages of breast cancer tumorigenesis, this epigenetic mark appears to poise genes for expression that leverages continued acquisition and persistence of metastatic properties. In contrast, H3K4me3, with persistence of H3K4ac reflects the epithelial to mesenchymal transition in MDA-MB-231 cells that represent late stage breast cancer. Important mechanistic linkages between histone H3K4ac and H3K4me3 modifications and breast cancer tumorigenesis remain to be established. While H3K4ac is emerging as an informative predictor of pathways that are deregulated during the onset and progression of breast cancer, the full potential for exploitation of this epigenetic modification as a biomarker and therapeutic target remains to be explored. Future studies must further define the enzymes that support acetylation and deacetylation of H3K4. Furthermore, the dynamic balance between H3K4 acetylation and methylation must be elucidated within the context of the full spectrum of histone modifications that contribute to combinatorial control of the epigenetic landscape. This will provide a drastically improved understanding of how epigenetic modifications and chromatin organization regulate mammary epithelial phenotype and gene expression related to transformation and tumor progression—the complexity of chromatin organization that supports breast cancer initiation and progression is increasingly evident. Consequently, the clinical value of epigenetic biomarkers and related therapeutic targets can be expanded by detailed characterization of the regulatory signals responsible for the deposition of specific “histone marks”. Adding to the biological challenge inherent in these analyses, the plasticity observed in post translational histone modifications must also be considered. It will be important to establish the relationships between histone modifications and other parameters of epigenetic control and chromatin organization in breast cancer that include perturbations in inter- and intra-chromosomal interactions [4] and oncofetal recapitulation of bivalent epigenetic regulatory mechanisms [5-8]. Although challenging, the complexity of epigenetic control in breast cancer provides the potential for increased capabilities in breast cancer risk assessment, early diagnosis and therapy with enhanced specificity and minimal off-target consequences. Figure 1 Dynamics of H3K4ac epigenetic modifications in cell lines representative of breast/breast cancer subtypes

Bone sialoprotein (BSP) is a highly modified, anionic phosphoprotein that is expressed almost exclusively in mineralizing connective tissues and has been shown to be a potent nucleator of hydroxyapatite (HA). Two polyglutamic acid (poly[E]) regions, predicted to be in an alpha-helical conformation and located in the amino-terminal half of the molecule, are believed to be responsible for this activity. Using a prokaryotic expression system, full-length rat BSP was expressed and tested for HA nucleating activity in a steady-state agarose gel system. The unmodified protein is less potent than native bone BSP, indicating a role for the post-translational modifications in HA nucleation. Site-directed mutagenesis of the poly[E] regions in full-length BSP was performed, replacing the poly[E] with either polyaspartic acid (poly[D]) or polyalanine (poly[A]) to examine role of charge and conformation, respectively, in HA nucleation. Replacement of single domains with either poly[A] or poly[D] did not alter nucleating activity nor did replacement of both domains with poly[D]. Replacement of both domains with poly[A], however, significantly decreased nucleating activity. In addition, two recombinant peptides, each encompassing one of the two poly[E] domains, were expressed and tested for nucleating activity. Whereas the peptide encompassing the second poly[E] domain was capable of nucleating HA, the first domain peptide showed no activity. The conformation of the wild-type and mutated proteins and peptides were studied by circular dichroism and small angle x-ray scattering, and no secondary structure was evident. These results demonstrate that a sequence of at least eight contiguous glutamic acid residues is required for the nucleation of HA by BSP and that this nucleating "site" is not alpha-helical in conformation.

Post-translational modifications of chromatin such as DNA methylation and different types of histone acetylation, methylation and phosphorylation are well-appreciated epigenetic mechanisms that confer information to progeny cells during lineage commitment. These distinct epigenetic modifications have defined roles in bone, development, tissue regeneration, cell commitment and differentiation, as well as disease etiologies. In this review, we discuss the role of these chromatin modifications and the enzymes regulating these marks (methyltransferases, demethylases, acetyltransferases, and deacetylases) in progenitor cells, osteoblasts and bone-related cells. In addition, the clinical relevance of deregulated histone modifications and enzymes as well as current and potential therapeutic interventions targeting chromatin modifiers are addressed.

Runx2 is a developmentally regulated gene in vertebrates and is essential for bone formation and skeletal homeostasis. The induction of runx2-P1 isoform transcripts is a hallmark of early osteoblastogenesis. Although previous in vitro studies have defined a minimal Runx2-P1 promoter sequence with well-characterized functional elements, several lines of evidence suggest that transcription of the Runx2-P1 isoform relies on elements that extend beyond the previously defined P1 promoter boundaries. In this study, we examined Runx2-P1 transcriptional regulation in a cellular in vivo context during early osteoblastogenesis of MC3T3-E1 cultures and BMSCs induced towards the bone lineage by multi-layered analysis of the Runx2-P1 gene promoter using the following methodologies: 1) sequence homology among several mammalian species, 2) DNaseI hypersensitivity coupled with massively parallel sequencing (DNase-seq), and 3) chromatin immunoprecipitation of activating histone modifications coupled with massively parallel sequencing (ChIP-seq). These epigenetic features have allowed the demarcation of boundaries that redefine the minimal Runx2-P1 promoter to include a 336-bp sequence that mediates responsiveness to osteoblast differentiation. We also find that an additional level of control is contributed by a regulatory region in the 5′-UTR of Runx2-P1.

Epigenetic regulation utilizes different mechanisms to convey heritable traits to progeny cells that are independent of DNA sequence, including DNA silencing, post-translational modifications of histone proteins and the post-transcriptional modulation of RNA transcript levels by non-coding RNAs. Although long non-coding RNAs have recently emerged as important regulators of gene imprinting, but their functions during osteogenesis are as yet unexplored. In contrast, microRNAs (miRNAs) are well characterized for their control of osteogenic and osteoclastic pathways; thus, further defining how gene regulatory networks essential for skeleton functions are coordinated and finely tuned through the activities of miRNAs. Roles of miRNAs are constantly expanding as new studies uncover associations with skeletal disorders. The distinct functions of epigenetic regulators and evidence for integrating their activities to control normal bone gene expression and bone disease will be presented. In addition, potential for using “signature microRNAs” to identify, manage and therapeutically treat osteosarcoma will be discussed in this review.

MicroRNAs (miRNAs) negatively and post-transcriptionally regulate expression of multiple target genes to support anabolic pathways for bone formation. Here, we show that miR-218 is induced during osteoblast differentiation and has potent osteogenic properties. miR-218 promotes commitment and differentiation of bone marrow stromal cells by activating a positive Wnt signaling loop. In a feed forward mechanism, miR-218 stimulates the Wnt pathway by down-regulating three Wnt signaling inhibitors during the process of osteogenesis: Sclerostin (SOST), Dickkopf2 (DKK2), and secreted frizzled-related protein2 (SFRP2). In turn, miR-218 expression is up-regulated in response to stimulated Wnt signaling and functionally drives Wnt-related transcription and osteoblast differentiation, thereby creating a positive feedback loop. Furthermore, in metastatic breast cancer cells but not in normal mammary epithelial cells, miR-218 enhances Wnt activity and abnormal expression of osteoblastic genes (osteomimicry) that contribute to homing and growth of cells metastatic to bone. Thus, miR-218/Wnt signaling circuit amplifies both the osteoblast phenotype and osteomimicry-related tumor activity.

Multiple microRNAs (miRNAs) that target the osteogenic Runt-related transcription factor 2 (RUNX2) define an interrelated network of miRNAs that control osteoblastogenesis. We addressed whether these miRNAs have functional targets beyond RUNX2 that coregulate skeletal development. Here, we find that seven RUNX2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-205, and miR-217) also regulate the chondrogenic GATA transcription factor tricho-rhino-phalangeal syndrome I (TRPS1). Although the efficacy of each miRNA to target RUNX2 or TRPS1 differs in osteoblasts and chondrocytes, each effectively blocks maturation of precommitted osteoblasts and chondrocytes. Furthermore, these miRNAs can redirect mesenchymal stem cells into adipogenic cell fate with concomitant up-regulation of key lineage-specific transcription factors. Thus, a program of multiple miRNAs controls mesenchymal lineage progression by selectively blocking differentiation of osteoblasts and chondrocytes to control skeletal development.

Induced osteogenesis includes a program of microRNAs (miRs) to repress the translation of genes that act as inhibitors of bone formation. How expression of bone-related miRs is regulated remains a compelling question. Here we report that Runx2, a transcription factor essential for osteoblastogenesis, negatively regulates expression of the miR cluster 23a∼27a∼24-2. Overexpression, reporter, and chromatin immunoprecipitation assays established the presence of a functional Runx binding element that represses expression of these miRs. Consistent with this finding, exogenous expression of each of the miRs suppressed osteoblast differentiation, whereas antagomirs increased bone marker expression. The biological significance of Runx2 repression of this miR cluster is that each miR directly targets the 3′ UTR of SATB2, which is known to synergize with Runx2 to facilitate bone formation. The findings suggest Runx2-negative regulation of multiple miRs by a feed-forward mechanism to cause derepression of SATB2 to promote differentiation. We find also that miR-23a represses Runx2 in the terminally differentiated osteocyte, representing a feedback mechanism to attenuate osteoblast maturation. We provide direct evidence for an interdependent relationship among transcriptional inhibition of the miR cluster by Runx2, translational repression of Runx2 and of SATB2 by the cluster miRs during progression of osteoblast differentiation. Furthermore, miR cluster gain of function (i.e., inhibition of osteogenesis) is rescued by the exogenous expression of SATB2. Taken together, we have established a regulatory network with a central role for the miR cluster 23a∼27a∼24-2 in both progression and maintenance of the osteocyte phenotype.

Gene and genome duplications create novel genetic material on which evolution can work and have therefore been recognized as a major source of innovation for many eukaryotic lineages. Following duplication, the most likely fate is gene loss; however, a considerable fraction of duplicated genes survive. Not all genes have the same probability of survival, but it is not fully understood what evolutionary forces determine the pattern of gene retention. Here, we use genome sequence data as well as large-scale phosphoproteomics data from the baker’s yeast Saccharomyces cerevisiae , which underwent a whole-genome duplication ∼100 mya, and show that the number of phosphorylation sites on the proteins they encode is a major determinant of gene retention. Protein phosphorylation motifs are short amino acid sequences that are usually embedded within unstructured and rapidly evolving protein regions. Reciprocal loss of those ancestral sites and the gain of new ones are major drivers in the retention of the two surviving duplicates and in their acquisition of distinct functions. This way, small changes in the sequences of unstructured regions in proteins can contribute to the rapid rewiring and adaptation of regulatory networks.

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP-induced migration and tumor survival were examined in breast and prostate cancer cells (MDA-MB-231, Hs578T and PC3). Additionally, the effects of exogenous TGF-beta1 and EGF, cytokines associated with tumor metastasis and present in high-levels in the bone microenvironment, were examined in BSP-expressing cancer cells. Expression of BSP but not an integrin-binding mutant (BSP-KAE) in tumor cell lines resulted in increased levels of alpha(v)-containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP-1-family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP-2, MMP-9, and MMP-14. The BSP-mediated activation of the FAK-associated pathway resulted in increased cancer cell invasion in a Matrigel-coated Boyden-chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF-beta1 to the BSP-expressing cell lines significantly increased ERK phosphorylation, AP-1 activation, MMP-2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF-beta1 and EGF.

The osteoinductive BMP2 signal facilitates commitment to the osteoblast phenotype by inducing several classes of early response genes. Among these are bone-related HOX factors, homeodomain, RUNX and OSTERIX proteins. Here we demonstrate molecular events among BMP2-induced transcription factors that constitute a network of molecular switches on promoters of bone-related genes to coordinate their temporal expression during cellular differentiation. Our studies provide evidence for (1) selective association of HOXA10, MSX2, DLX3 and DLX5 homeodomain transcription factors on Runx2 and OC genes at stages of osteoblast maturation as well as (2) participation of these factors with RUNX2 in chromatin remodeling of bone-specific genes for repression, activation and attenuation of transcription. These findings reveal the requirement for multiple levels of control for the appropriate timing of osteoblast-related gene expression.

The t(8;21) chromosomal translocation produces AML1-ETO, an oncogenic fusion protein that compromises the function of AML1, a transcription factor critical for the myeloid cell differentiation. The differentiation arrest and self-renewal phenotypes of cells expressing AML1-ETO have been chiefly studied at the single gene level. In order to understand how the genome-wide occupancy of AML1-ETO and associated co-regulators contributes to the leukemic properties of Kasumi-1 cells, a model system for human t(8;21) leukemia, chromatin immunoprecipitation-sequencing (ChIP-seq) experiments were employed. Genome-wide DNA binding profiles for AML1 and AML1-ETO in Kasumi-1 cells showed that both proteins were enriched at introns and distal upstream regulatory regions, the latter implying that distal elements are likely important for target gene regulation. Importantly, A substantial fraction of AML1/AML1-ETO-p300 co-localization occurs near transcriptional start sites (TSSs) in promoter regions associated with transcriptionally active loci, as indicated by an observed enrichment of the H3K4me3 histone modification. A significant discovery of the current study is the co-localization of AML1/AML1-ETO with N-CoR on regions that are primarily distal to TSSs. Analyses of both gene expression data and gene ontology suggest that the AML1/AML1-ETO co-localization with N-CoR, more so than with p300, is associated with the leukemic phenotype of Kasumi-1 cells. De novo motif discovery reveals that PU.1 motifs are over-represented in regions co-occupied by AML1-ETO and N-CoR compared to other AML1-ETO bound regions. Our findings establish a novel and predominant AML leukemia signature characterized by occupancy of the AML1-ETO/N-CoR complex at genomic regions enriched in motifs for myeloid differentiation factors. We propose that this property is critical for maintenance of the leukemic phenotype. Citation Format: Daniel J. Trombly, Troy W. Whitfield, Srivatsan Padmanabhan, Jonathan A. Gordon, Jane B. Lian, Andre J. van Wijnen, Sayyed K. Zaidi, Janet L. Stein, Gary S. Stein. Co-occupancy of AML1-ETO and N-CoR defines a dominant phenotypic signature in leukemic cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B28.

Background: Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. Results: By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation - proliferation, matrix deposition and mineralization - we identify Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing over the course of these stages, we identify approximately 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibit distinct patterns during osteogenesis, and are associated with proximal promoters and also non-promoter regions: upstream, introns, exons, transcription termination site regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identify novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of the extracellular matrix. We demonstrate by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. Conclusions: Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis. Background The development of bone tissue, new bone formation in the adult, mineral homeostasis and maintenance of bone mass are mediated by cells of the osteoblast lineage. These bone forming cells progress through a highly regulated differentiation program, with each subpopulation of cells acquiring stage-specific phenotypes that are characterized by distinct profiles of expressed genes [1]. Osteoblast com