Sheng-Chung Lee

Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication

Chromatin is suppressive in nature to cellular enzymes that metabolize DNA, mainly due to the inherent inaccessibility of the DNA template. Despite extensive understanding of the involvement of chromatin‐modifying factors in transcription, roles of related activities in DNA replication remain largely elusive. Here, we show that the heterodimeric transcriptional elongation factor FACT (facilitates chromatin transcription) is functionally linked to DNA synthesis. Its involvement in DNA replication is partly mediated by the stable association with the replicative helicase complex, MCM, and further by the coexistence with MCM on replication origin. Furthermore, relying on its nucleosome‐reorganizing activity, FACT can facilitate chromatin unwinding by the MCM complex, which is otherwise inert on the nucleosomal template. As a consequence, the physical and functional interaction between FACT and MCM is an important determinant in the proper initiation of DNA replication and S phase in vivo. Together, our findings identify FACT as an integral and conserved component of the endogenous replication machinery, and support a model in which the concerted action of helicase and chromatin‐modifying activities promotes chromosome replication.

Clonal origin of recurrent hepatocellular carcinomas.

Recurrence of hepatocellular carcinoma after treatment is frequent. To study the clonal origin of the recurrent tumors, we examined five pairs of hepatocellular carcinomas resected from individual hepatitis B surface antigen carriers. Using integrated hepatitis B virus DNA as a marker, tumor clonality was determined by Southern blot analysis. In 2 cases the second tumor contained the same integrated viral DNA as the first one. In the other 3 cases, the clonality of the second cancer differed. We conclude that recurrent hepatocellular carcinomas originate from the first tumor in some cases but represent de novo neoplasms in others.

Purification and characterization of nucleolin and its identification as a transcription repressor.

Expression of the acute-phase response genes, such as that for alpha-1 acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor.

The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins

The chromosomes of the soil bacteria Streptomyces, unlike those of most other bacteria, are linear DNA molecules. Their telomeres contain long‐terminal inverted repeats and covalently bound terminal proteins (TPs). These bacteria also harbour linear plasmids that share the same structural features. In this study, we demonstrated that the TP was covalently bound to the 5′ ends as proposed previously. A linear plasmid with chromosomal telomeres was constructed and used to purify the TPs of the Strep‐tomyces coelicolor A3(2) chromosome. A 20 kDa protein and its 10 kDa degradation product were isolated and their sequences determined by mass spectrometry. The coding sequence (tpgC) was about 100 kb from the right end of the chromosome. Two tpg homologues were identified by sequencing the 50 kb linear plasmid SLP2 of Streptomyces lividans: tpgSLP2 at 6 kb from the left end and a putative tpg pseudogene at 8 kb from the right. The latter was in a terminal repeat shared by the right end of SLP2 and both ends of the S. lividans chromosome. The lack of the typical Streptomyces codon preference in this open reading frame suggests that it is a pseudogene. The close physical linkage between the tpg genes and their cognate telomeres would favour their co‐segregation and co‐evolution. All the Tpg polypeptides are similar in length (184–185 amino acids) and sequences, which include a putative helix domain that is homologous to part of the DNA‐binding ‘thumb’ domain of HIV reverse transcriptase, and a putative amphiphilic beta‐sheet that may be involved in the observed self‐aggregation of the TP and/or the proposed membrane binding.

FACT facilitates chromatin transcription by RNA polymerases I and III

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, co‐purify and co‐immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA‐mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre‐rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III‐transcribed genes.

Coactivator TIF1β Interacts with Transcription Factor C/EBPβ and Glucocorticoid Receptor To Induce α1-Acid Glycoprotein Gene Expression

ABSTRACT The transcription of the α1-acid glycoprotein gene is induced by inflammatory cytokines and glucocorticoids. C/EBPβ is a major transcription factor involved in the induction of the agpgene by some cytokines. In this report, we have identified a novel transcriptional intermediary factor, TIF1β, which could enhance the transcription of the agp gene by the glucocorticoid receptor (GR) and C/EBPβ. TIF1β belongs to a subgroup of RING (really interesting new gene) finger proteins that contain a RING finger preceding two B box-type fingers and a putative coiled-coil domain (RBCC domain). Immunoprecipitation experiments showed that the interaction between GR and TIF1β is ligand independent. The overexpression of the TIF1β gene enhances GR-regulated expression in a ligand- and glucocorticoid-responsive element (GRE)-dependent manner. TIF1β can also augment C/EBPβ-mediated activity on wild-type and GRE-mutated agp genes, but this augmentation is diminished when all three C/EBPβ-binding elements are mutated. Functional and biochemical characterizations indicated that the bZIP domain of C/EBPβ and the RBCC domain, plant homeodomain finger, and bromodomain of TIF1β are crucial for the interactions of these proteins. Taken together, these results suggest that TIF1β serves as a converging mediator of signal transduction pathways of glucocorticoids and some inflammatory cytokines.

Identification of Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) as a Repressor of C/EBPβ-mediated Gene Activation

Transcription factor C/EBPβ has been known to regulate a wide array of genes including those involved in the acute-phase response. One of the molecular mechanisms underlying transcription activation by C/EBPβ is through protein-protein interaction with other transcription factors. Here we report the identification and characterization of physical and functional interactions between C/EBPβ and heterogeneous nuclear ribonucleoprotein (hnRNP) K. This interaction results in the repression of C/EBPβ-dependent trans-activation of theagp gene. Footprinting assays indicate that hnRNP K cannot bind to the promoter region of agp gene or interfere with the binding of C/EBPβ to its cognate DNA site. Furthermore,agp gene activation by the synergistic interaction of Nopp140 and C/EBPβ is abolished by hnRNP K. The kinetics of appearance of C/EBPβ-hnRNP K complex in the nuclear extract after initiation of acute-phase reaction indicates that hnRNP K functions as a negative regulator of C/EBPβ-mediated activation of agpgene.

Transcriptional induction of the alpha-1 acid glycoprotein (AGP) gene by synergistic interaction of two alternative activator forms of AGP/enhancer-binding protein (C/EBP beta) and NF-kappaB or Nopp140.

Alpha-1 acid glycoprotein/enhancer-binding protein (AGP/EBP) (C/EBPbeta), a member of the C/EBP family, is one of the key transcription factors responsible for the induction of a wide array of genes, some of which are expressed during the acute-phase response. Both activator and repressor were shown to be encoded by the intronless agp/ebp or its rat and human homologs, which contain a common bZIP domain at their C-terminal regions. Expression of the AGP gene (agp) is regulated by AGP/EBP in liver during the acute-phase response. However, the molecular mechanism for this regulation is poorly understood. The experiments reported here demonstrate that two activator forms of AGP/EBP, one of which has an additional 21 amino acids at its N-terminal region, are expressed in liver as well as in a number of cell lines. We have also demonstrated that NF-kappaB and a phosphoprotein of 140 kDa, Nopp140, interact with different AGP/EBP activators synergistically, which results in induction of the agp gene in an AGP/EBP-binding-motif-dependent manner. Furthermore, extracellular stimuli that are known to be NF-kappaB inducers can selectively activate the agp gene by cooperating with one of the two activator forms of AGP/EBP. The physiological significance of differential regulation for the function of two activator forms of AGP/EBP through selective interaction with different transcription factors is discussed.

Nucleophosmin acts as a novel AP2α‐binding transcriptional corepressor during cell differentiation

Nucleophosmin (NPM) is an important nucleolar phosphoprotein with pleiotropic functions in various cellular processes. In this study, we have further examined the largely uncharacterized role of NPM in transcriptional regulation by uncovering novel NPM‐binding transcriptional factors. Among potential interactors, we found that activating protein transcription factor 2 (AP2)α forms a complex with NPM during retinoic‐acid‐induced cell differentiation. We show that this complex is recruited to the promoters of certain retinoic‐acid‐responsive genes, including NPM itself. Such binding of AP2α, and consequent recruitment of NPM, is selective and dependent on a consensus AP2α‐binding sequence. Remarkably, suppression of NPM by RNA interference alleviates the repression of gene expression mediated by retinoic acid and AP2α. Our findings further show that, on promoter binding, NPM probably exerts its repressive effect by inducing a change in local chromatin structure that also engages histone deacetylases. This study unveils a hitherto unrecognized transcriptional corepressor function of the NPM protein, and highlights a novel mechanism by which NPM regulates cell growth and differentiation.

Identification and Characterization of a Nucleolar Phosphoprotein, Nopp140, as a Transcription Factor

Expression of the alpha-1 acid glycoprotein (AGP) gene (agp) is activated by a key transcription factor, AGP/enhancer-binding protein (AGP/EBP, commonly called C/EBP beta), in the liver during the acute-phase response. In addition to this positive regulation, agp is negatively regulated by nucleolin (T. H. Yang et al., Mol. Cell. Biol. 14:6068-6074, 1994). Other factors involve in positive regulation of the agp gene are poorly characterized. In a systematic search for factors that may interact with AGP/EBP, we have identified Nopp 140, a phosphoprotein of 140 kDa, by immunoaffinity chromatography. Nopp 140 not only functions as a transcriptional activator per se but also interacts with AGP/EBP to synergistically activate the agp gene in an AGP/EBP-binding motif-dependent manner. In addition to interacting with AGP/EBP, Nopp140 interacts specifically with TFIIB. Distinct regions of Nopp140 that interact with AGP/EBP and TFIIB have been characterized. The sequence of Nopp140 contains several stretches of serine- and acidic amino acid-rich sequences which are also found in ICP4 of herpes simplex virus type 1, a known transcription factor that interacts with TFIIB. The physical interaction between TFIIB and wild-type Nopp140 or several deletion mutants of Nopp140 correlates with the ability of Nopp140 to activate the agp gene synergistically with AGP/EBP. Thus, the molecular mechanism for agp gene activation may involve the interaction of AGP/EBP and TFIIB mediated by coactivator Nopp140.