Stein, Gary S.

Significance Repression of the large number of transposable elements in eukaryotic genomes is essential for genome stability. The Piwi-interacting RNA and short interfering RNA pathways are critical for repressing transposable elements in germlines, but the repression of transposable elements in somatic tissues involves other components. While the mammalian Hinfp has been shown to regulate Histone4 and cell-cycle progression, our manuscript provides evidence that a function of the Drosophila Hinfp is to maintain Histone1 expression to repress most transposable elements in somatic genomes. This Hinfp–Histone1 axis provides a venue to study maintenance of genome stability and progression of pathological outcomes.

Both genomic and non‐genomic mechanisms control the acquisition of specialized cellular phenotypes during development. When differentiated cells enter and exit mitosis, chromosomal condensation prevents gene expression. Post‐mitotic re‐activation of transcription is controlled in part by epigenetic marks on DNA and histones that control chromatin accessibility. However, mRNA molecules also appear to be transferred to progeny cells by either symmetrical or asymmetrical transmission. We have shown that the mRNA for the bone‐related transcription factor Runx2 mRNAs is elevated in the G2 phase of the cell cycle, but not translated in osteoblasts. This observation suggests that Runx2 protein is translationally rather than transcriptionally controlled after mitosis to maintain the phenotypic identity of osteoblasts. We used RNA‐seq expression profiling to assess whether there are other gene regulatory factors that are transmitted through mitosis. Our main results show that mRNAs for transcription factors are typically depleted during mitosis. For example, while Runx2 mRNA is retained in mitotic cells, expression of the closely related genes Runx1 and Runx3 is reduced. Strikingly, expression of Osterix/Sp7 and MEF2C, two transcription factors known to cooperate biologically with Runx2 in promoting the osteoblastic state, is selectively retained in mitotic cells. Our data suggest that selective somatic transmission of mRNAs for transcription factors in osteoblasts may be a general mechanism by which cells maintain their identity during cell division in skeletal development. Support or Funding Information NIH R01 AR049069 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Transcriptional control of Runx2 gene expression through two alternative promoters (P1 and P2) is critical for the execution of its function as an osteogenic cell fate determining factor. Using genomic DNaseI footprinting and in vivo DNA binding assays, we identified a helix‐loop‐helix (HLH) motif that overlaps with a Runx motif in the P1 promoter. The HLH mutation decreases P1 transcription 10‐fold in mesenchymal cells, while mutation of the Runx site enhances transcription 2 fold. Although the HLH element is not cell‐type specific, its function may depend on promoter context. Promoter deletion analysis shows that a GA‐rich region (−200 to −128) positively regulates Runx2 P1 transcription and synergizes with the HLH motif. Examination of the −200/−128 region reveals two tandemly repeated GA‐rich elements (GAY motifs) that each contain recognition motifs for purine‐rich DNA binding factors (i.e., SP1 and KLF10/TGFâ Inducible Factor [TIEG]). Forced expression of SP1 or TIEG modulates Runx2 P1 promoter activity through the GAY motifs. Taken together, our data suggest that the Runx2 gene transcription is regulated by synergistic interactions at multiple conserved composite promoter elements to control Runx2 expression and skeletal development.

AbstractTranscription networks and epigenetic mechanisms including DNA methylation, histone modifications, and noncoding RNAs control lineage commitment of multipotent mesenchymal progenitor cells. Proteins that read, write, and erase histone tail modifications curate and interpret the highly intricate histone code. Epigenetic reader proteins that recognize and bind histone marks provide a crucial link between histone modifications and their downstream biological effects. Here, we investigate the role of bromodomain‐containing (BRD) proteins, which recognize acetylated histones, during osteogenic differentiation. Using RNA‐sequencing (RNA‐seq) analysis, we screened for BRD proteins (n = 40) that are robustly expressed in MC3T3 osteoblasts. We focused functional follow‐up studies on Brd2 and Brd4 which are highly expressed in MC3T3 preosteoblasts and represent “bromodomain and extra terminal domain” (BET) proteins that are sensitive to pharmacological agents (BET inhibitors). We show that small interfering RNA depletion of Brd4 has stronger inhibitory effects on osteoblast differentiation than Brd2 loss as measured by osteoblast‐related gene expression, extracellular matrix deposition, and alkaline phosphatase activity. Similar effects on osteoblast differentiation are seen with the BET inhibitor +JQ1, and this effect is reversible upon its removal indicating that this small molecule has no lasting effects on the differentiation capacity of MC3T3 cells. Mechanistically, we find that Brd4 binds at known Runx2 binding sites in promoters of bone‐related genes. Collectively, these findings suggest that Brd4 is recruited to osteoblast‐specific genes and may cooperate with bone‐related transcription factors to promote osteoblast lineage commitment and maturation.

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.

Abstract Osteosarcomas are bone tumors that frequently metastasize to the lung. Aberrant expression of the transcription factor, runt‐related transcription factor 2 (RUNX2), is a key pathological feature in osteosarcoma and associated with loss of p53 and miR‐34 expression. Elevated RUNX2 may transcriptionally activate genes mediating tumor progression and metastasis, including the RUNX2 target gene osteopontin ( OPN/SPP1 ). This gene encodes a secreted matricellular protein produced by osteoblasts to regulate bone matrix remodeling and tissue calcification. Here we investigated whether and how the RUNX2/OPN axis regulates lung metastasis of osteosarcoma. Importantly, RUNX2 depletion attenuates lung metastasis of osteosarcoma cells in vivo. Using next‐generation RNA‐sequencing, protein‐based assays, as well as the loss‐ and gain‐of‐function approaches in selected osteosarcoma cell lines, we show that osteopontin messenger RNA levels closely correlate with RUNX2 expression and that RUNX2 controls the levels of secreted osteopontin. Elevated osteopontin levels promote heterotypic cell–cell adhesion of osteosarcoma cells to human pulmonary microvascular endothelial cells, but not in the presence of neutralizing antibodies. Collectively, these findings indicate that the RUNX2/OPN axis regulates the ability of osteosarcoma cells to attach to pulmonary endothelial cells as a key step in metastasis of osteosarcoma cells to the lung.

AbstractThe RUNX1 transcription factor has recently been shown to be obligatory for normal development. RUNX1 controls the expression of genes essential for proper development in many cell lineages and tissues including blood, bone, cartilage, hair follicles, and mammary glands. Compromised RUNX1 regulation is associated with many cancers. In this review, we highlight evidence for RUNX1 control in both invertebrate and mammalian development and recent novel findings of perturbed RUNX1 control in breast cancer that has implications for other solid tumors. As RUNX1 is essential for definitive hematopoiesis, RUNX1 mutations in hematopoietic lineage cells have been implicated in the etiology of several leukemias. Studies of solid tumors have revealed a context‐dependent function for RUNX1 either as an oncogene or a tumor suppressor. These RUNX1 functions have been reported for breast, prostate, lung, and skin cancers that are related to cancer subtypes and different stages of tumor development. Growing evidence suggests that RUNX1 suppresses aggressiveness in most breast cancer subtypes particularly in the early stage of tumorigenesis. Several studies have identified RUNX1 suppression of the breast cancer epithelial‐to‐mesenchymal transition. Most recently, RUNX1 repression of cancer stem cells and tumorsphere formation was reported for breast cancer. It is anticipated that these new discoveries of the context‐dependent diversity of RUNX1 functions will lead to innovative therapeutic strategies for the intervention of cancer and other abnormalities of normal tissues.

Breast cancer is the most common cancer in women, and accounts for ~30% of new cancer cases and 15% of cancer‐related deaths. Tumor relapse and metastasis are primary factors contributing to breast cancer‐related deaths. Therefore, the challenge for breast cancer treatment is to sustain remission. A driving force behind tumor relapse is breast cancer heterogeneity (both intertumor, between different patients, and intratumor, within the same tumor). Understanding breast cancer heterogeneity is necessary to develop preventive interventions and targeted therapies. A recently emerging concept is that intratumor heterogeneity is driven by cancer stem cells (CSCs) that are capable of giving rise to a multitude of different cells within a tumor. Studies have highlighted linkage of CSC formation with epithelial‐to‐mesenchymal transition (EMT). In this review, we summarize the current understanding of breast cancer heterogeneity, links between EMT and CSCs, regulation of EMT by Runx transcription factors, and potential therapeutic strategies targeting these processes.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher‐order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA‐seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal‐like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub‐clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub‐clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in‐between them, suggesting that the histone sub‐clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub‐nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher‐order chromatin organization of the major HLB and its regulation during breast cancer progression.

ABSTRACTOsteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over‐expression of the runt‐related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/β‐catenin signaling is over‐activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/β‐catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/β‐catenin pathway controls the tumor‐related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of β‐catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, β‐catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of β‐catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3β inhibitor SB216763. Furthermore, small interfering RNA (siRNA)‐mediated depletion of β‐catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/β‐catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662–3674, 2017. © 2017 Wiley Periodicals, Inc.