Ronglin Xie

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.

HiNF-P directly links the cyclin E/CDK2/p220NPAT pathway to histone H4 gene regulation at the G1/S phase cell cycle transition

ABSTRACT Genome replication in eukaryotic cells necessitates the stringent coupling of histone biosynthesis with the onset of DNA replication at the G1/S phase transition. A fundamental question is the mechanism that links the restriction (R) point late in G1 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 G1/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 G1/S phase transition to the cyclin E/CDK2 signaling pathway at the R point.

A Runx2 threshold for the cleidocranial dysplasia phenotype

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.

Control of Mesenchymal Lineage Progression by MicroRNAs Targeting Skeletal Gene Regulators Trps1 and Runx2

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. 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.

Co-stimulation of the Bone-related Runx2 P1 Promoter in Mesenchymal Cells by SP1 and ETS Transcription Factors at Polymorphic Purine-rich DNA Sequences (Y-repeats)

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.

The Cell Cycle Control Element of Histone H4 Gene Transcription Is Maximally Responsive to Interferon Regulatory Factor Pairs IRF-1/IRF-3 and IRF-1/IRF-7

Interferon regulatory factors (IRFs) are transcriptional mediators of interferon-responsive signaling pathways that are involved in antiviral defense, immune response, and cell growth regulation. To investigate the role of IRF proteins in the regulation of histone H4 gene transcription, we compared the transcriptional contributions of IRF-1, IRF-2, IRF-3, and IRF-7 using transient transfection assays with H4 promoter/luciferase (Luc) reporter genes. These IRF proteins up-regulate reporter gene expression but IRF-1, IRF-3, and IRF-7 are more potent activators of the H4 promoter than IRF-2. Forced expression of different IRF combinations reveals that IRF-2 reduces IRF-1 or IRF-3 dependent activation, but does not affect IRF-7 function. Thus, IRF-2 may have a dual function in histone H4 gene transcription by acting as a weak activator at low dosage and a competitive inhibitor of other strongly activating IRFs at high levels. IRF-1/IRF-3 and IRF-1/IRF-7 pairs each mediate the highest levels of site II-dependent promoter activity and can up-regulate transcription by 120–150-fold. We also find that interferon γ up-regulates IRF-1 and site II-dependent promoter activity. This up-regulation is not observed when the IRF site is mutated or if cells are preloaded with IRF-1. Our results indicate that IRF-1, IRF-2, IRF-3, and IRF-7 can all regulate histone H4 gene expression. The pairwise utilization of distinct IRF factors provides a flexible transcriptional mechanism for integration of diverse growth-related signaling pathways.

Definitive hematopoiesis requires Runx1 C-terminal-mediated subnuclear targeting and transactivation

Runx1 is a key hematopoietic transcription factor required for definitive hematopoiesis and is a frequent target of leukemia-related chromosomal translocations. The resulting fusion proteins, while retaining DNA binding activity, display loss of subnuclear targeting and associated transactivation functions encoded by the C-terminus of the protein. To define the precise contribution of the Runx1 C-terminus in development and leukemia, we created a knock-in mouse with a C-terminal truncation by introducing a single nucleic acid substitution in the native Runx1 locus. This mutation (Runx1(Q307X)) models genetic lesions observed in patients with leukemia and myeloproliferative disorders. The Runx1(Q307X) homozygous mouse exhibits embryonic lethality at E12.5 due to central nervous system hemorrhages and a complete lack of hematopoietic stem cell function. While able to bind DNA, Runx1(Q307X) is unable to activate target genes, resulting in deregulation of various hematopoietic markers. Thus, we demonstrate that the subnuclear targeting and transcriptional regulatory activities of the Runx1 C-terminus are critical for hematopoietic development. We propose that compromising the C-terminal functions of Runx1 is a common mechanism for the pathological consequences of a variety of somatic mutations and Runx1-related leukemic fusion proteins observed in human patients.

The histone gene activator HINFP is a nonredundant cyclin E/CDK2 effector during early embryonic cell cycles

Competency for DNA replication is functionally coupled to the activation of histone gene expression at the onset of S phase to form chromatin. Human histone nuclear factor P (HiNF-P; gene symbol HINFP) bound to its cyclin E/cyclin-dependent kinase 2 (CDK2) responsive coactivator p220NPAT is a key regulator of multiple human histone H4 genes that encode a major subunit of the nucleosome. Induction of the histone H4 transcription factor (HINFP)/p220NPAT coactivation complex occurs in parallel with the CDK-dependent release of pRB from E2F at the restriction point. Here, we show that the downstream CDK-dependent cell cycle effector HINFP is genetically required and, in contrast to the CDK2/cyclin E complex, cannot be compensated. We constructed a mouse Hinfp-null mutation and found that heterozygous Hinfp mice survive, indicating that 1 allele suffices for embryogenesis. Homozygous loss-of-function causes embryonic lethality: No homozygous Hinfp-null mice are obtained at or beyond embryonic day (E) 6.5. In blastocyst cultures, Hinfp-null embryos exhibit a delay in hatching, abnormal growth, and loss of histone H4 gene expression. Our data indicate that the CDK2/cyclin E/p220NPAT/HINFP/histone gene signaling pathway at the G1/S phase transition is an essential, nonredundant cell cycle regulatory mechanism that is established early in embryogenesis.

Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

ABSTRACT Fidelity of chromatin organization is crucial for normal cell cycle progression, and perturbations in packaging of DNA may predispose to transformation. Histone H4 protein is the most highly conserved chromatin protein, required for nucleosome assembly, with multiple histone H4 gene copies encoding identical protein. There is a long-standing recognition of the linkage of histone gene expression and DNA replication. A fundamental and unresolved question is the mechanism that couples histone biosynthesis with DNA replication and fidelity of cell cycle control. Here, we conditionally ablated the obligatory histone H4 transcription factor HINFP to cause depletion of histone H4 in mammalian cells. Deregulation of histone H4 results in catastrophic cellular and molecular defects that lead to genomic instability. Histone H4 depletion increases nucleosome spacing, impedes DNA synthesis, alters chromosome complement, and creates replicative stress. Our study provides functional evidence that the tight coupling between DNA replication and histone synthesis is reciprocal.

CDK inhibitors selectively diminish cell cycle controlled activation of the histone H4 gene promoter by p220NPAT and HiNF-P

Cell cycle progression into S phase requires the induction of histone gene expression to package newly synthesized DNA as chromatin. Cyclin E stimulation of CDK2 at the Restriction point late in G1 controls both histone gene expression by the p220NPAT/HiNF‐P pathway and initiation of DNA replication through the pRB/E2F pathway. The three CDK inhibitors (CKIs) p21CIP1/WAF1, p27KIP1, and p57KIP2 attenuate CDK2 activity. Here we find that γ‐irradiation induces p21CIP1/WAF1 but not the other two CKIs, while reducing histone H4 mRNA levels but not histone H4 gene promoter activation by the p220NPAT/HiNF‐P complex. We also show that p21CIP1/WAF1 is less effective than p27KIP1 and p57KIP2 in inhibiting the CDK2 dependent phosphorylation of p220NPAT at subnuclear foci and transcriptional activation of histone H4 genes. The greater effectiveness of p57KIP2 in blocking the p220NPAT/HiNF‐P pathway is attributable in part to its ability to form a specific complex with p220NPAT that may suppress CDK2/cyclin E phosphorylation through direct substrate inhibition. We conclude that CKIs selectively control stimulation of the histone H4 gene promoter by the p220NPAT/HiNF‐P complex. J. Cell. Physiol. 219: 438–448, 2009.