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...

At the G 1 /S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G 1 /S phase transition.The G 1 /S phase transition represents the critical stage during the somatic cell cycle that defines cellular commitment to replicate the genome and to progress towards mitotic division. Passage beyond the G 1 /S boundary depends initially on the activation of a cyclin/cyclin-dependent kinase (CDK) cascade by growth factors and induction of the cyclin E/CDK2 kinase complex at the restriction (R) point (9,14,29). Subsequently, at the onset of S phase, de novo synthesis of histone proteins is required to package nascent DNA into chromatin immediately upon initiation of DNA synthesis (28,35,36). The exquisitely stringent coupling between histone biosynthesis and DNA replication necessitates the transcriptional activation of the 14 distinct human genes encoding histone H4, the most highly conserved nucleosomal protein (1,13,19,28).Control of histone genes provides a paradigm for gene expression that is temporally and functionally linked to DNA synthesis. It has long been postulated but not yet experimentally validated that the multiple histone H4 genes are coordinately regulated by a single histone H4 gene subtype-specific factor. However, the identity of this protein has never been established. We and others have previously demonstrated that histone H4 genes are regulated by multiple elements and cognate DNA binding activities (6,7,16,30,31,44). The histone gene proximal promoter element site II interacts with three factors (HiNF-M, -D, and -P) and mediates cell cycle control of transcription at the onset of S phase (3,6,7,16,30,31,36,(43)(44)(45)(46)(47). Site II encompasses the H4 subtype-specific element that is phylogenetically conserved among multiple histone H4 genes in metazoan species. The cell cycle regulatory mechanisms operative at site II at the onset of S phase function independently of...

The replication of eukaryotic genomes necessitates the coordination of histone biosynthesis with DNA replication at the onset of S phase. The multiple histone H4 genes encode identical proteins, but their regulatory sequences differ. The contributions of these individual genes to histone H4 mRNA expression have not been described. We have determined, by real-time quantitative PCR and RNase protection, that the human histone H4 genes are not equally expressed and that a subset contributes disproportionately to the total pool of H4 mRNA. Differences in histone H4 gene expression can be attributed to observed unequal activities of the H4 gene promoters, which exhibit variations in gene regulatory elements. The overall expression pattern of the histone H4 gene complement is similar in normal and cancer cells. However, H4 genes that are moderately expressed in normal cells are sporadically silenced in tumor cells with compensation of expression by other H4 gene copies. Chromatin immunoprecipitation analyses and in vitro DNA binding assays indicated that 11 of the 15 histone H4 genes interact with the cell cycle regulatory histone nuclear factor P, which forms a complex with the cyclin E/CDK2-responsive co-regulator p220 NPAT . These 11 H4 genes account for 95% of the histone H4 mRNA pool. We conclude that the cyclin E/CDK2/p220 NPAT /histone nuclear factor P signaling pathway is the principal regulator of histone H4 biosynthesis.Histones have crucial roles in replication, transcription, repair, and recombination (1-3). There is a fundamental requirement for coordinated de novo synthesis of the core histone proteins H2A, H2B, H3, and H4 as well as the linker H1 protein during S phase to package nascent genomic DNA (1, 2). Replication of a complete mammalian genome requires 10 8 of each of the individual histone proteins. Efficient production of this vast quantity of proteins necessitates that transcription of multiple histone genes at multiple loci be coordinately regulated with the onset and progression of genome replication during the cell cycle (4).Histone biosynthesis is a unique process involving transcription initiation from compact promoters to form primary transcripts that lack introns and that contain a highly conserved stem-loop structure that forms the 3Ј-end of the mature non-polyadenylated mRNA (4, 5). Histone genes are organized into clusters, and this organization has persisted throughout the course of evolution from yeast to human (2, 4). The majority of the 74 known and characterized human histone genes is located in two major clusters at chromosomes 6p21 and 1q21, respectively (TABLE ONE) (1, 6 -8). It is now known that the human genome contains 15 histone H4 genes that encode identical proteins. H4 genes in lower eukaryotes (e.g. sea urchin and Drosophila) are organized with the other histone gene types (i.e. H2A, H2B, H3, and H1) into units that are tandemly repeated, and all H4 genes in these organisms have virtually identical promoters and coding regions. Although the coding regions of the human...

The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα+) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7 kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.

BackgroundNatural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host.ResultsAs an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5–7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology.ConclusionThe technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds

Seven transmembrane G protein-coupled receptors (GPCRs) have gained much interest in recent years as it is the largest class among cell surface receptors. G proteins lie in the heart of GPCRs signalling and therefore can be therapeutically targeted to overcome complexities in GPCR responses and signalling. G proteins are classified into four families (G, G, G and G); G is further subdivided into four classes. Among them G and G isoforms are most crucial and ubiquitously expressed; these isoforms are almost 88% similar at their amino acid sequence but may exhibit functional divergences. However, uncertainties often arise about G and G inhibitors, these G proteins might also have suitability to the invention of novel-specific inhibitors for each isoforms. YM-254890 and UBO-QIC are discovered as potent inhibitors of G functions and also investigated in thrombin protease-activated receptor (PAR)-1 inhibitors and platelet aggregation inhibition. The most likely G protein involved in PAR-1 stimulates responses is one of the G family isoforms. In this review, we highlight the molecular structures and pharmacological responses of G family which may reflect the biochemical and molecular role of G and G. The advanced understanding of G and G role in GPCR signalling may shed light on our understanding on cell biology, cellular physiology and pathophysiology and also lead to the development of novel therapeutic agents for a number of diseases.

Our previous studies showed that MYB is required for proliferation of, and confers protection against apoptosis on, estrogen receptor-positive (ER+ve) breast cancer cells, which are almost invariably also MYB+ve. We have also shown that MYB expression in ER+ve breast cancer cells is regulated at the level of transcriptional elongation and as such, is suppressed by CDK9i. Here we examined the effects of CDK9i on breast cancer cells and the involvement of MYB in these effects. ER+ve breast cancer cell lines including MCF-7 were much more sensitive (> 10 times) to killing by CDK9i than ER−ve/MYB−ve cells. Moreover, surviving cells showed a block at the G2/M phase of the cell cycle. Importantly, ectopic MYB expression conferred resistance to apoptosis induction, cell killing and G2/M accumulation. Expression of relevant MYB target genes including BCL2 and CCNB1 was suppressed by CDK9 inhibition, and this too was reversed by ectopic MYB expression. Nevertheless, inhibition of BCL2 alone either by MYB knockdown or by ABT-199 treatment was insufficient for significant induction of apoptosis. Further studies implied that suppression of MCL-1, a well-documented target of CDK9 inhibition, was additionally required for apoptosis induction, while maximal levels of apoptosis induced by CDK9i are likely to also involve inhibition of BCL2L1 expression. Taken together these data suggest that MYB regulation of BCL2 underlies the heightened sensitivity of ER+ve compared to ER−ve breast cancer cells to CDK9 inhibition, and that these compounds represent a potential therapeutic for ER+ve breast cancers and possibly other MYB-dependent cancers.

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.blastocyst ͉ development ͉ embryogenesis ͉ p220 NPAT ͉ human embryonic stem cells