Mario Galindo

Mitotic occupancy and lineage-specific transcriptional control of rRNA genes by Runx2

Regulation of ribosomal RNA genes is a fundamental process that supports the growth of cells and is tightly coupled with cell differentiation. Although rRNA transcriptional control by RNA polymerase I (Pol I) and associated factors is well studied, the lineage-specific mechanisms governing rRNA expression remain elusive. Runt-related transcription factors Runx1, Runx2 and Runx3 establish and maintain cell identity, and convey phenotypic information through successive cell divisions for regulatory events that determine cell cycle progression or exit in progeny cells. Here we establish that mammalian Runx2 not only controls lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also acts as a repressor of RNA Pol I mediated rRNA synthesis. Within the condensed mitotic chromosomes we find that Runx2 is retained in large discrete foci at nucleolar organizing regions where rRNA genes reside. These Runx2 chromosomal foci are associated with open chromatin, co-localize with the RNA Pol I transcription factor UBF1, and undergo transition into nucleoli at sites of rRNA synthesis during interphase. Ribosomal RNA transcription and protein synthesis are enhanced by Runx2 deficiency that results from gene ablation or RNA interference, whereas induction of Runx2 specifically and directly represses rDNA promoter activity. Runx2 forms complexes containing the RNA Pol I transcription factors UBF1 and SL1, co-occupies the rRNA gene promoter with these factors in vivo, and affects local chromatin histone modifications at rDNA regulatory regions. Thus Runx2 is a critical mechanistic link between cell fate, proliferation and growth control. Our results suggest that lineage-specific control of ribosomal biogenesis may be a fundamental function of transcription factors that govern cell fate.

Mitotic retention of gene expression patterns by the cell fate-determining transcription factor Runx2

During cell division, cessation of transcription is coupled with mitotic chromosome condensation. A fundamental biological question is how gene expression patterns are retained during mitosis to ensure the phenotype of progeny cells. We suggest that cell fate-determining transcription factors provide an epigenetic mechanism for the retention of gene expression patterns during cell division. Runx proteins are lineage-specific transcription factors that are essential for hematopoietic, neuronal, gastrointestinal, and osteogenic cell fates. Here we show that Runx2 protein is stable during cell division and remains associated with chromosomes during mitosis through sequence-specific DNA binding. Using siRNA-mediated silencing, mitotic cell synchronization, and expression profiling, we identify Runx2-regulated genes that are modulated postmitotically. Novel target genes involved in cell growth and differentiation were validated by chromatin immunoprecipitation. Importantly, we find that during mitosis, when transcription is shut down, Runx2 selectively occupies target gene promoters, and Runx2 deficiency alters mitotic histone modifications. We conclude that Runx proteins have an active role in retaining phenotype during cell division to support lineage-specific control of gene expression in progeny cells.

Runx2 regulates G protein-coupled signaling pathways to control growth of osteoblast progenitors

Runt-related transcription factor 2 (Runx2) controls lineage commitment, proliferation, and anabolic functions of osteoblasts as the subnuclear effector of multiple signaling axes (e.g. transforming growth factor-β/BMP-SMAD, SRC/YES-YAP, and GROUCHO/TLE). Runx2 levels oscillate during the osteoblast cell cycle with maximal levels in G1. Here we examined what functions and target genes of Runx2 control osteoblast growth. Forced expression of wild type Runx2 suppresses growth of Runx2-/- osteoprogenitors. Point mutants defective for binding to WW domain or SMAD proteins or the nuclear matrix retain this growth regulatory ability. Hence, key signaling pathways are dispensable for growth control by Runx2. However, mutants defective for DNA binding or C-terminal gene repression/activation functions do not block proliferation. Target gene analysis by Affymetrix expression profiling shows that the C terminus of Runx2 regulates genes involved in G protein-coupled receptor signaling (e.g. Rgs2, Rgs4, Rgs5, Rgs16, Gpr23, Gpr30, Gpr54, Gpr64, and Gna13). We further examined the function of two genes linked to cAMP signaling as follows: Gpr30 that is stimulated and Rgs2 that is down-regulated by Runx2. RNA interference of Gpr30 and forced expression of Rgs2 in each case inhibit osteoblast proliferation. Notwithstanding its growth-suppressive potential, our results surprisingly indicate that Runx2 may sensitize cAMP-related G protein-coupled receptor signaling by activating Gpr30 and repressing Rgs2 gene expression in osteoblasts to increase responsiveness to mitogenic signals.

MicroRNA-34c inversely couples the biological functions of the runt-related transcription factor RUNX2 and the tumor suppressor p53 in osteosarcoma

Background: Osteosarcoma (OS) is associated with loss of tumor suppressor p53 and increased Runx2. Results: Runx2 and p53 levels are inversely correlated in OS. miR-34c, which targets Runx2, is absent in OS and elevated by p53. Conclusion: p53, miR-34c, and Runx2 form a regulatory loop that is compromised in OS. Significance: RUNX2 could be targeted by miR-34c to prevent OS growth. Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3′-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.

Lymphocyte enhancer‐binding factor 1 (Lef1) inhibits terminal differentiation of osteoblasts

Lef1 is a transcriptional regulator of the Wnt/β‐catenin signaling cascade. Wnts directly augment bone formation and osteoblast differentiation from mesenchymal stem cells by receptor‐mediated pathways involving Lrp5 and Frizzled. We previously reported that Lef1 represses Runx2‐dependent activation of the late osteoblast differentiation gene, osteocalcin. Lef1 is expressed in preosteoblasts but is undetectable in fully differentiated osteoblasts. To determine if downregulation of Lef1 is necessary for osteoblast maturation, we constitutively overexpressed Lef1 in MC3T3‐E1 preosteoblasts. Lef1‐overexpressing cells produced alkaline phosphatase (ALP) and osteocalcin later, and at lower levels than control cells. Moreover, the extracellular matrices of Lef1‐overexpressing cell cultures never mineralized. To further examine the role of Lef1 in osteoblasts, we suppressed Lef1 expression in MC3T3‐E1 cells by RNA interference. Transient expression of a Lef1 shRNA efficiently reduced murine Lef1 levels and transcriptional activity. Stable suppression of Lef1 in MC3T3 preosteoblasts did not affect proliferation or Runx2 levels; however, ALP production and matrix mineralization were accelerated by 3–4 days. Gene chip analyses identified 14 genes that are differentially regulated in Lef1‐suppressed cells. These data outline a role for Lef1 in delaying osteoblast maturation and suggest that Lef1 controls the expression of multiple genes in osteoblasts. J. Cell. Biochem. 97: 969–983, 2006.

The cancer-related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines.

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre‐osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up‐regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle‐regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre‐established levels in a given cell type triggers one or more anti‐proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. J. Cell. Physiol. 228: 714–723, 2013.

Genomic Promoter Occupancy of Runt-related Transcription Factor RUNX2 in Osteosarcoma Cells Identifies Genes Involved in Cell Adhesion and Motility

Background: The osteogenic Runt-related (RUNX) transcription factor Runx2 is frequently elevated in osseous and non-osseous tumor cells. Results: Genomic RUNX2 target genes involved in motility were identified; RUNX2 depletion reduces cell motility in osteosarcoma cells. Conclusion: RUNX2 regulates cell motility and adhesion in osteosarcoma cells. Significance: RUNX2 may also control migration of normal osteoblasts and/or tumor cells. Runt-related transcription factors (RUNX1, RUNX2, and RUNX3) are key lineage-specific regulators of progenitor cell growth and differentiation but also function pathologically as cancer genes that contribute to tumorigenesis. RUNX2 attenuates growth and stimulates maturation of osteoblasts during bone formation but is also robustly expressed in a subset of osteosarcomas, as well as in metastatic breast and prostate tumors. To assess the biological function of RUNX2 in osteosarcoma cells, we examined human genomic promoter interactions for RUNX2 using chromatin immunoprecipitation (ChIP)-microarray analysis in SAOS-2 cells. Promoter binding of both RUNX2 and RNA polymerase II was compared with gene expression profiles of cells in which RUNX2 was depleted by RNA interference. Many RUNX2-bound loci (1550 of 2339 total) exhibit promoter occupancy by RNA polymerase II and contain the RUNX consensus motif 5′-((T/A/C)G(T/A/C)GG(T/G). Gene ontology analysis indicates that RUNX2 controls components of multiple signaling pathways (e.g. WNT, TGFβ, TNFα, and interleukins), as well as genes linked to cell motility and adhesion (e.g. the focal adhesion-related genes FAK/PTK2 and TLN1). Our results reveal that siRNA depletion of RUNX2, PTK2, or TLN1 diminishes motility of U2OS osteosarcoma cells. Thus, RUNX2 binding to diverse gene loci may support the biological properties of osteosarcoma cells.

Histone genes in trypanosomatids.

Histone genes in Trypanosomatids are of considerable interest because these flagellates do not condense their chromatin during mitosis. In contrast to higher eukaryotes, histone genes in Trypanosomatids are found on separate chromosomes, and their transcripts are polyadenylated. Sequence similarity of Trypanosomatid core histones with those of higher eukaryotes is found predominantly in the globular region; the N-terminal is highly divergent. Finally, in general, Trypanosomatid histones H1 are of low molecular weight, bearing closest homology to the C-terminal region of the higher eukaryote histones H1. These features constitute interesting targets for a rational approach to the study of these protozoa, as discussed here by Norbel Galanti and colleagues.

Runx2, p53, and pRB status as diagnostic parameters for deregulation of osteoblast growth and differentiation in a new pre-chemotherapeutic osteosarcoma cell line (OS1).

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS‐2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre‐chemotherapeutic tumor in the femur of a 6‐year‐old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase‐PCR, Western blotting and flow cytometry. OS1 have aberrant G‐banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS1 had ossification profiles similar to human fetal osteoblasts rather than SAOS‐2 which ossifies ab initio (P < 0.05). Absence of p53 correlates with increased Runx2 expression, while the slow proliferation of OS1 cells is perhaps attenuated by pRB retention. OS1 express mesenchymal stem cell markers (CD44, CD105) and differ in relative expression of CD29, CD63, and CD71 to SAOS‐2. (P < 0.05). Cell cycle synchronization with nocodazole did not affect Runx2 and CDK1 levels but decreased cyclin‐E and increased cyclin‐A (P < 0.05). Xenotransplantion of OS1 in SCID mice yields spontaneous tumors that were larger and grew faster than SAOS‐2 transplants. Hence, OS1 is a new osteosarcoma cell culture model derived from a pre‐chemotherapeutic ethnic Chinese patient, for mechanistic studies and development of therapeutic strategies to counteract metastasis and deregulation of mesenchymal development. J. Cell. Physiol. 221: 778–788, 2009.

The Osteogenic Transcription Factor Runx2 Controls Genes Involved in Sterol/Steroid Metabolism, Including Cyp11a1 in Osteoblasts

Steroid hormones including (1,25)-dihydroxyvitamin D3, estrogens, and glucocorticoids control bone development and homeostasis. We show here that the osteogenic transcription factor Runx2 controls genes involved in sterol/steroid metabolism, including Cyp11a1, Cyp39a1, Cyp51, Lss, and Dhcr7 in murine osteoprogenitor cells. Cyp11a1 (P450scc) encodes an approximately 55-kDa mitochondrial enzyme that catalyzes side-chain cleavage of cholesterol and is rate limiting for steroid hormone biosynthesis. Runx2 is coexpressed with Cyp11a1 in osteoblasts as well as nonosseous cell types (e.g. testis and breast cancer cells), suggesting a broad biological role for Runx2 in sterol/steroid metabolism. Notably, osteoblasts and breast cancer cells express an approximately 32-kDa truncated isoform of Cyp11a1 that is nonmitochondrial and localized in both the cytoplasm and the nucleus. Chromatin immunoprecipitation analyses and gel shift assays show that Runx2 binds to the Cyp11a1 gene promoter in osteoblasts, indicating that Cyp11a1 is a direct target of Runx2. Specific Cyp11a1 knockdown with short hairpin RNA increases cell proliferation, indicating that Cyp11a1 normally suppresses osteoblast proliferation. We conclude that Runx2 regulates enzymes involved in sterol/steroid-related metabolic pathways and that activation of Cyp11a1 by Runx2 may contribute to attenuation of osteoblast growth.