Development of Bivalent mRNA Vaccines against SARS-CoV-2 Variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected billions of individuals and is the cause of the current global coronavirus disease 2019 (COVID-19) pandemic. We previously developed an mRNA vaccine (LVRNA009) based on the S protein of the Wuhan-Hu-1 strain; the phases I and II clinical trials showed that LVRNA009 has a promising safety and immunogenicity profile. In order to counteract the immune escape by SARS-CoV-2 variants of concern, a panel of mRNA vaccines was developed based on the S proteins of the Wuhan-Hu-1, Delta, Omicron BA.1, BA.2, and BA.5 strains, and each vaccine's protective potency against the virus variants was evaluated. Furthermore, to achieve excellent neutralization against SARS-CoV-2 variants, bivalent vaccines were developed and tested against the variants. We found that the monovalent Wuhan-Hu-1 or the Delta vaccines could induce high level of neutralization antibody and protect animals from the infection of the SARS-CoV-2 Wuhan-Hu-1 or Delta strains, respectively. However, serum samples from mice immunized with monovalent Delta vaccine showed relatively low virus neutralization titers (VNTs) against the pseudotyped virus of the Omicron strains. Serum samples from mice immunized with bivalent Delta/BA.1 vaccine had high VNTs against the pseudotyped Wuhan-Hu-1, Delta, and BA.1 strains but low VNTs against BA.2 and BA.5 (p < 0.05). Serum samples from mice immunized with Delta/BA.2 vaccine had high VNTs against the pseudotyped Wuhan-Hu-1, Delta, BA.1 and BA.2 strains but low VNTs against BA.5. Finally, serum samples from mice immunized with Delta/BA.5 vaccine had high VNTs against all the tested pseudotyped SARS-CoV-2 strains including the Wuhan-Hu-1, Delta, and Omicron variants (p > 0.05). Therefore, a bivalent mRNA vaccine with Delta/BA.5 combination is promising to provide broad spectrum immunity against all VOCs.

In Vivo Expansion and Antitumor Activity of Coinfused CD28- and 4-1BB-Engineered CAR-T Cells in Patients with B Cell Leukemia.

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.

Maternal expression and early induction of histone gene transcription factor Hinfp sustains development in pre-implantation embryos.

Fidelity of histone gene expression is important for normal cell growth and differentiation that is stringently controlled during development but is compromised during tumorigenesis. Efficient production of histones for packaging newly replicated DNA is particularly important for proper cell division and epigenetic control during the initial pre-implantation stages of embryonic development. Here, we addressed the unresolved question of when the machinery for histone gene transcription is activated in the developing zygote to accommodate temporal demands for histone gene expression. We examined induction of Histone Nuclear Factor P (HINFP), the only known transcription factor required for histone H4 gene expression, that binds directly to a unique H4 promoter-specific element to regulate histone H4 transcription. We show that Hinfp gene transcripts are stored in oocytes and maternally transmitted to the zygote. Transcripts from the paternal Hinfp gene, which reflect induction of zygotic gene expression, are apparent at the 4- to 8-cell stage, when most maternal mRNA pools are depleted. Loss of Hinfp expression due to gene ablation reduces cell numbers in E3.5 stage embryos and compromises implantation. Reduced cell proliferation is attributable to severe reduction in histone mRNA levels accompanied by reduced cell survival and genomic damage as measured by cleaved Caspase 3 and phospho-H2AX staining, respectively. We conclude that transmission of maternal Hinfp transcripts and zygotic activation of the Hinfp gene together are necessary to control H4 gene expression in early pre-implantation embryos in order to support normal embryonic development.

p53 checkpoint ablation exacerbates the phenotype of Hinfp dependent histone H4 deficiency.

Histone Nuclear Factor P (HINFP) is essential for expression of histone H4 genes. Ablation of Hinfp and consequential depletion of histones alter nucleosome spacing and cause stalled replication and DNA damage that ultimately result in genomic instability. Faithful replication and packaging of newly replicated DNA are required for normal cell cycle control and proliferation. The tumor suppressor protein p53, the guardian of the genome, controls multiple cell cycle checkpoints and its loss leads to cellular transformation. Here we addressed whether the absence of p53 impacts the outcomes/consequences of Hinfp-mediated histone H4 deficiency. We examined mouse embryonic fibroblasts lacking both Hinfp and p53. Our data revealed that the reduced histone H4 expression caused by depletion of Hinfp persists when p53 is also inactivated. Loss of p53 enhanced the abnormalities in nuclear shape and size (i.e. multi-lobed irregularly shaped nuclei) caused by Hinfp depletion and also altered the sub-nuclear organization of Histone Locus Bodies (HLBs). In addition to the polyploid phenotype resulting from deletion of either p53 or Hinfp, inactivation of both p53 and Hinfp increased mitotic defects and generated chromosomal fragility and susceptibility to DNA damage. Thus, our study conclusively establishes that simultaneous loss of both Hinfp and the p53 checkpoint is detrimental to normal cell growth and may predispose to cellular transformation.

Mitochondria apoptosis pathway synergistically activated by hierarchical targeted nanoparticles co-delivering siRNA and lonidamine.

The mitochondria-mediated apoptosis pathway is an effective option for cancer therapy due to the presence of cell-suicide weapons in mitochondria. However, anti-apoptotic proteins that are over-expressed in the mitochondria of many malignant tumors, such as Bcl-2 protein, could allow the cancer cells to evade apoptosis, greatly reducing the efficacy of this type of chemotherapy. Here, we constructed a hierarchical targeted delivery system that can deliver siRNA and chemotherapeutic agents sequentially to tumor cells and mitochondria. In detail, the copolymer TPP-CP-LND (TCPL) was synthesized by the mitochondria-targeting ligand triphenylphosphine (TPP) and therapeutic drug lonidamine (LND) conjugated to the polyethyleneimine in chitosan-graft-PEI (CP), and then complexed with siRNA. Followed, the complexes were coated with poly(acrylic acid)-polyethylene glycol-folic acid (PPF) copolymer to form a hierarchical targeted co-delivery system (TCPL/siRNA/PPF NPs). The TCPL/siRNA/PPF NPs had a neutral surface charge, were stable in plasma and exhibited pH-responsive shell separation. Remarkably, the TCPL/siRNA/PPF NPs simultaneously released siBcl-2 into the cytoplasm and delivered LND to mitochondria in the same cancer cell after FA-directed internalization, and even synergistically activated mitochondria apoptosis pathway. This work demonstrated the potential of RNA-interference and mitochondria-targeted chemotherapeutics to collaboratively stimulate the mitochondria apoptosis pathway for cancer therapy.

A novel potential biocompatible hyperbranched polyspermine for efficient lung cancer gene therapy.

The clinical successful application of gene therapy critically depends upon the development of non-toxic and efficient delivery system. Although polycationic non-viral vectors hold great promise in nanomedicine, the exploring of application in clinics still remains a big challenge. To develop a non-toxic and efficient non-viral gene delivery system, two kinds of endogenous substance, citric acid (CA) and spermine (SPE), were used to prepare a new low charge density hyperbranched polyspermine (HPSPE) by one-pot polymerization. The biocompatibility evaluated by hemolytic activity and red blood cell (RBC) aggregation indicated that HPSPE was highly biocompatible without causing hemolysis and RBC aggregation compared with PEI as well as SPE. The MTS assay also demonstrated that the cell viability of HPSPE was above 90% even at 200 µg/mL at different time (24 and 72 h), which much higher than PEI 25K. Besides, HPSPE showed high transfection efficiency without any toxic effect after aerosol delivery to the mice. Moreover, aerosol delivery of HPSPE/Akt1 shRNA significantly reduced tumor size and numbers and efficiently suppressed lung tumorigenesis ultimately in K-ras(LA1) lung cancer model mice. These results suggest that low charge density as well as endogenous substance skeleton endow HPSPE with great potential for toxicity-free and efficient gene therapy.

Chemical modification of chitosan for efficient gene therapy.

Gene therapy involves the introduction of foreign genetic material into cells in order to exert a therapeutic effect. Successful gene therapy relies on effective vector system. Viral vectors are highly efficient in transfecting cells, but the undesirable complications limit their therapeutic applications. As a natural biopolymer, chitosan has been considered to be a good gene carrier candidate due to its ideal character which combines biocompatibility, low toxicity with high cationic density together. However, the low cell specificity and low transfection efficiency of chitosan as a gene carrier need to be overcome before undertaking clinical trials. This chapter is principally on those endeavors such as chemical modifications using cell-specific ligands and stimuli-response groups as well as penetrating modifications that have been done to increase the performances of chitosan in gene therapy.

Fidelity of histone gene regulation is obligatory for genome replication and stability.

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.

Control of mesenchymal lineage progression by microRNAs targeting skeletal gene regulators Trps1 and Runx2.

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.

A program of microRNAs controls osteogenic lineage progression by targeting transcription factor 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.

Functional coupling of transcription factor HiNF-P and histone H4 gene expression during pre- and post-natal mouse development.

Transcription factor Histone Nuclear Factor P (HiNF-P; gene symbol Hinfp) mediates cell cycle control of histone H4 gene expression to support the packaging of newly replicated DNA as chromatin. The HiNF-P/p220(NPAT) complex controls multiple H4 genes in established human cell lines and is critical for cell proliferation. The mouse Hinfp(LacZ) null allele causes early embryonic lethality due to a blastocyst defect. However, neither Hinfp function nor its temporal expression relative to histone H4 genes during fetal development has been explored. Here, we establish that expression of Hinfp is biologically coupled with expression of twelve functional mouse H4 genes during pre- and post-natal tissue-development. Both Hinfp and H4 genes are robustly expressed at multiple embryonic (E) days (from E5.5 to E15.5), coincident with ubiquitous LacZ staining driven by the Hinfp promoter. Five highly expressed mouse H4 genes (Hist1h4d, Histh4f, Hist1h4m and Hist2h4) account for >90% of total histone H4 mRNA throughout development. Post-natal expression of H4 genes in mice is most evident in lung, spleen, thymus and intestine, and with few exceptions (e.g., adult liver) correlates with Hinfp gene expression. Histone H4 gene expression decreases butHinfp levels remain constitutive upon cell growth inhibition in culture. The in vivo co-expression of Hinfp and histone H4 genes is consistent with the biological function of Hinfp as a principal transcriptional regulator of histone H4 gene expression during mouse development.

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 p220(NPAT) 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)/p220(NPAT) 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/p220(NPAT)/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.

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 p220(NPAT)/HiNF-P pathway and initiation of DNA replication through the pRB/E2F pathway. The three CDK inhibitors (CKIs) p21(CIP1/WAF1), p27(KIP1), and p57(KIP2) attenuate CDK2 activity. Here we find that gamma-irradiation induces p21(CIP1/WAF1) but not the other two CKIs, while reducing histone H4 mRNA levels but not histone H4 gene promoter activation by the p220(NPAT)/HiNF-P complex. We also show that p21(CIP1/WAF1) is less effective than p27(KIP1) and p57(KIP2) in inhibiting the CDK2 dependent phosphorylation of p220(NPAT) at subnuclear foci and transcriptional activation of histone H4 genes. The greater effectiveness of p57(KIP2) in blocking the p220(NPAT)/HiNF-P pathway is attributable in part to its ability to form a specific complex with p220(NPAT) 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 p220(NPAT)/HiNF-P complex.

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.

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.

The HiNF-P/p220NPAT cell cycle signaling pathway controls nonhistone target genes.

HiNF-P and its cofactor p220(NPAT) are principal factors regulating histone gene expression at the G(1)-S phase cell cycle transition. Here, we have investigated whether HiNF-P controls other cell cycle- and cancer-related genes. We used cDNA microarrays to monitor responsiveness of gene expression to small interfering RNA-mediated depletion of HiNF-P. Candidate HiNF-P target genes were examined for the presence of HiNF-P recognition motifs, in vitro HiNF-P binding to DNA, and in vivo association by chromatin immunoprecipitations and functional reporter gene assays. Of 177 proliferation-related genes we tested, 20 are modulated in HiNF-P-depleted cells and contain putative HiNF-P binding motifs. We validated that at least three genes (i.e., ATM, PRKDC, and CKS2) are HiNF-P dependent and provide data indicating that the DNA damage response is altered in HiNF-P-depleted cells. We conclude that, in addition to histone genes, HiNF-P also regulates expression of nonhistone targets that influence competency for cell cycle progression.

Transcriptional activation of the histone nuclear factor P (HiNF-P) gene by HiNF-P and its cyclin E/CDK2 responsive co-factor p220NPAT defines a novel autoregulatory loop at the G1/S phase transition.

Histone nuclear factor P (HiNF-P) activates histone H4 gene transcription at the G1/S phase transition upon association with its cyclin E/CDK2 responsive co-factor p220NPAT. Here we characterize the gene regulatory pathways that control the proliferation-related expression of HiNF-P. The HiNF-P locus contains a single TATA-less 0.6 kbp promoter with multiple phylogenetically conserved transcription factor recognition motifs. Transient reporter gene assays with HiNF-P promoter deletions show that there are at least three distinct activating regions (-387/-201, -201/-100 and -100/-1) that support maximal transcription. HiNF-P gene transcription is activated by SP1 through the -100/-1 domain and repressed by E2F1 through the -201/-100 domain. The multifunctional co-regulators CBP and p300 also stimulate HiNF-P gene transcription through the -201/-1 core promoter. Importantly, the HiNF-P promoter is activated by both HiNF-P and p220NPAT. This autoregulatory activation is further enhanced by cyclin E and CDK2, while blocked by CDK inhibition (i.e., p57KIP2 p27KIP1, p21CIP). Thus, the HiNF-P gene is a key non-histone target of p220NPAT and HiNF-P. The dependence of HiNF-P gene transcription on cyclin E/CDK2/p220NPAT signaling defines a novel feed-forward loop that may sustain HiNF-P expression in proliferating cells to support the cell cycle regulated synthesis of histone H4 proteins.

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 is a bifunctional regulator of cell cycle controlled histone H4 gene transcription.

Cell cycle progression beyond the G1/S phase transition requires the activation of a transcription complex containing histone nuclear factor P (HiNF-P) and nuclear protein mapped to ataxia telangiectasia (p220(NPAT)) in response to cyclin dependent kinase 2 (CDK2)/cyclin E signaling. We show here that the potent co-activating properties of HiNF-P/p220(NPAT) on the histone H4 gene promoter, which are evident in the majority of human cell types, are sporadically neutralized in distinct somatic cell lines. In cells where HiNF-P and p220(NPAT) do not activate the H4 gene promoter, HiNF-P instead represses transcription. Our data suggest that the cell type specific expression of the cyclin-dependent kinase inhibitory (CKI) protein p57(KIP2) inhibits the HiNF-P dependent activation of the histone H4 promoter. We propose that, analogous to E2F proteins and other cell cycle regulatory proteins, HiNF-P is a bifunctional transcriptional regulator that can activate or repress cell cycle controlled genes depending on the cellular context.