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.

Genome replication in eukaryotic cells necessitates the stringent coupling of histone biosynthesis with the onset of DNA replication at the G 1 /S phase transition. A fundamental question is the mechanism that links the restriction (R) point late in G 1 with histone gene expression at the onset of S phase. Here we demonstrate that HiNF-P, a transcriptional regulator of replication-dependent histone H4 genes, interacts directly with p220NPAT , a substrate of cyclin E/CDK2, to coactivate histone genes during S phase. HiNF-P and p220 are targeted to, and colocalize at, subnuclear foci (Cajal bodies) in a cell cycle-dependent manner. Genetic or biochemical disruption of the HiNF-P/p220 interaction compromises histone H4 gene activation at the G 1 /S phase transition and impedes cell cycle progression. Our results show that HiNF-P and p220 form a critical regulatory module that directly links histone H4 gene expression at the G 1 /S phase transition to the cyclin E/CDK2 signaling pathway at the R point.Fidelity of genome replication in eukaryotic cells is essential for cell division and necessitates the stringent coupling of histone biosynthesis with DNA replication to ensure that nascent DNA is immediately assembled into chromatin during DNA synthesis. Cell division requires staged expression of genes in response to growth factors, which induce cell growth from quiescence or maintain competency for cell cycle progression during periods of active proliferation. Stimulation of cell proliferation initially triggers a cyclin/cyclin-dependent kinase (CDK) cascade, which activates the cyclin E/CDK2 kinase complex at the restriction (R) point (17,19). The R point is the major cell cycle checkpoint that controls the commitment for DNA replication in late G 1 via CDK2-dependent release of E2F from Rb-related proteins. The R point is mechanistically linked through E2F to activate the gene regulatory program necessary for nucleotide metabolism and DNA replication (17,19). Passage beyond the R point permits growth factor-independent entry into S phase and subsequent cell cycle stages. However, cell cycle progression remains constrained by multiple checkpoints, including surveillance mechanisms that monitor DNA integrity and fidelity of chromatin assembly.We postulate that the induction of histone gene expression at the G 1 /S phase transition represents a second necessary cell cycle regulatory event. The coupling of DNA synthesis with histone protein production is maintained by coordinately inducing expression of the multiple core histone gene subtypes, including the 15 distinct histone H4 genes, at the onset of S phase (1,3,12,20,23,24). The cell cycle regulatory sequence of histone H4 genes lacks E2F binding sites (28). We have recently identified the key transcription factor of H4 genes, histone nuclear factor P (HiNF-P), which interacts with a highly conserved histone H4 subtype-specific element in the site II cell cycle regulatory domain (16). HiNF-P supports histone gene transcription at the G 1 /S phase transition indepe...

Cleidocranial dysplasia (CCD) in humans is an autosomal-dominant skeletal disease that results from mutations in the bone-specific transcription factor RUNX2 (CBFA1/AML3). However, distinct RUNX2 mutations in CCD do not correlate with the severity of the disease. Here we generated a new mouse model with a hypomorphic Runx2 mutant allele (Runx2(neo7)), in which only part of the transcript is processed to full-length (wild-type) Runx2 mRNA. Homozygous Runx2(neo7/neo7) mice express a reduced level of wild-type Runx2 mRNA (55-70%) and protein. This mouse model allowed us to establish the minimal requirement of functional Runx2 for normal bone development. Runx2(neo7/neo7) mice have grossly normal skeletons with no abnormalities observed in the growth plate, but do exhibit developmental defects in calvaria and clavicles that persist through post-natal growth. Clavicle defects are caused by disrupted endochondral bone formation during embryogenesis. These hypomorphic mice have altered calvarial bone volume, as observed by histology and microCT imaging, and decreased expression of osteoblast marker genes. The bone phenotype of the heterozygous mice, which have 79-84% of wild-type Runx2 mRNA, is normal. These results show there is a critical gene dosage requirement of functional Runx2 for the formation of intramembranous bone tissues during embryogenesis. A decrease to 70% of wild-type Runx2 levels results in the CCD syndrome, whereas levels>79% produce a normal skeleton. Our findings suggest that the range of bone phenotypes in CCD patients is attributable to quantitative reduction in the functional activity of RUNX2.

Lineage progression in osteoblasts and chondrocytes is stringently controlled by the cell-fate-determining transcription factor Runx2. In this study, we directly addressed whether microRNAs (miRNAs) can control the osteogenic activity of Runx2 and affect osteoblast maturation. A panel of 11 Runx2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, miR-218, and miR-338) is expressed in a lineage-related pattern in mesenchymal cell types. During both osteogenic and chondrogenic differentiation, these miRNAs, in general, are inversely expressed relative to Runx2. Based on 3′UTR luciferase reporter, immunoblot, and mRNA stability assays, each miRNA directly attenuates Runx2 protein accumulation. Runx2-targeting miRNAs differentially inhibit Runx2 protein expression in osteoblasts and chondrocytes and display different efficacies. Thus, cellular context contributes to miRNA-mediated regulation of Runx2. All Runx2-targeting miRNAs (except miR-218) significantly impede osteoblast differentiation, and their effects can be reversed by the corresponding anti-miRNAs. These findings demonstrate that osteoblastogenesis is limited by an elaborate network of functionally tested miRNAs that directly target the osteogenic master regulator Runx2.osteogenesis | chondrogenesis | post-transcriptional regulation C ell-fate determination and subsequent lineage progression of phenotype-committed cells are mediated by master regulatory transcription factors that integrate multiple cell-signaling inputs and generate epigenetic changes in chromatin to modulate gene expression. Transcription factors are components of positive and negative feedback loops that initiate or maintain the acquisition of distinct biological states. Epigenomic mechanisms, including attenuation of mRNA and protein expression by small noncoding microRNAs (miRNAs) (1), permit effective control of gene expression beyond genomic interactions between transcription factors and their cognate elements in gene promoters. The biological potency of miRNAs, which are generated by the RNA processing enzyme Dicer, is based on their ability to control mRNA accumulation and/or protein synthesis through specific interactions with the 3′UTRs of target genes (1). Gene regulatory networks involving transcription factors and miRNAs may mutually reinforce cell fates and support phenotypic maturation of lineage-committed cells.Osteogenic differentiation provides an effective cell model in which to define both epigenetic and epigenomic mechanisms required for cell-fate determination and phenotypic differentiation. Differentiation of multipotent mesenchymal stem cells into the osteoblast lineage and maturation of osteoprogenitors are controlled by multiple extracellular ligands [e.g., BMPs, WNTs, and FGFs] (2-4) that direct the activities of key transcription factors, including Runx2, Osterix, and different classes of homeodomain proteins (5-8). Runx2 is a critical regulator of the osteogenic lineage, and its epigenetic functions modulate ...

Background: MicroRNAs control cell growth and differentiation in part by inhibiting transcription factor expression. Results: Specific microRNAs in mesenchymal cells block osteoblastic and chondrocytic but not adipocytic cell fate. Conclusion: Select microRNA-transcription factor networks control mesenchymal cell fate. Significance: MicroRNAs can be used to manipulate stem cells for musculoskeletal tissue regeneration.

Arsenic is a well-known carcinogen that possibly promotes tumors and the development of various types of cancer in individuals chronically exposed to arsenic in their work or living environment. Many studies have demonstrated the activation of mitogen-activated protein kinase (MAPK) in several cell types by using lethal concentrations of arsenic in the range of 50-500 micro M. Since the exposure of humans to arsenic is normally at a much lower level in the workplace or in daily life, it is more relevant to study the effects of arsenic at this lower exposure level. In the present study we aimed at redefining the role of signal transduction pathways in arsenic-induced malignant transformation as well as apoptosis using our established in vitro rat lung epithelial cell model system. Our results indicate a molecular mechanism by which MAPK pathways might differentially contribute to cell growth regulation and cell death in response to different dosages of arsenite. A low level (2 micro M) of arsenite stimulated extracellular signal-regulated kinase (ERK) signaling pathway and enhanced cell proliferation, and this arsenite-induced ERK activity was blocked by MEK inhibitor, PD98059. In contrast, a high level (40 micro M) of arsenite stimulated the c-Jun N-terminal kinase (JNK) signaling pathway and induced cell apoptosis, and this arsenite-induced JNK activity was blocked by JNK inhibitor II, SP600125. The implications of these findings are that a high concentration of arsenic exposure causes apoptosis, whereas a low concentration of arsenic exposure is carcinogenic and may result in aberrant cell accumulation.

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. In all vertebrates examined to date, the bone related P1 promoter contains a purine-rich region (؊303 to ؊128 bp in the rat) that separates two regulatory domains. The length of this region differs dramatically between species even within the same order. Using deletion analysis, we show that part of this purine-rich region (؊200 to ؊128) containing a duplicated element (Y-repeat) positively regulates Runx2 P1 transcription. Electrophoretic mobility assays and chromatin immunoprecipitations reveal that Y-repeat binds at least two different classes of transcription factors related to GC box binding proteins (e.g. SP1 and SP7/Osterix) and ETS-like factors (e.g. ETS1 and ELK1). Forced expression of SP1 increases Runx2 P1 promoter activity through the Y-repeats, and small interfering RNA depletion of SP1 decreases Runx2 expression. Similarly, exogenous expression of wild type ELK1, but not a defective mutant that cannot be phosphorylated, enhances Runx2 gene expression. SP1 is most abundant in proliferating cells, and ELK1 is most abundant in postconfluent cells; during MC3T3-E1 osteoblast differentiation, both proteins are transiently co-expressed when Runx2 expression is enhanced. Taken together, our data suggest that basal Runx2 gene transcription is regulated by dynamic interactions between SP1 and ETS-like factors during progression of osteogenesis.

Several recent clinical trials have successfully incorporated a costimulatory domain derived from either CD28 or 4-1BB with the original CD3ζ T cell activating domain to form second-generation chimeric antigen receptors (CARs) that can increase the responsiveness and survival of CAR-engineered T (CAR-T) cells. However, a rigorous assessment of the individual benefits of these costimulatory components relative to the in vivo performance of infused T cells in patients is still lacking. Therefore, we have designed a study that allows us to investigate and compare the impact of different costimulatory signal domains on CAR-T cells in vivo. Patients with B cell leukemia were infused with a mixture of two types of CD19-specific CAR-T cells, individually bearing CD28 (28ζ) and 4-1BB (BBζ) costimulatory signaling domains. We found that such a clinical procedure was feasible and safe. Complete remission (CR) was observed in five of seven enrolled patients, with two patients exhibiting durable CR lasting more than 15 months. The in vivo expansion pattern of 28ζ and BBζ CAR-T cells varied significantly among individual patients. These results confirm a feasible method of comparing different CAR designs within individual patients, potentially offering objective insights that may facilitate the development of optimal CAR-T cell-based immunotherapies.