The elongation of transcription is a highly regulated process that requires negative and positive effectors. By binding the double-stranded stem in the transactivation response (TAR) element, RD protein from the negative transcription elongation factor (NELF) inhibits basal transcription from the long terminal repeat of the human immunodeficiency virus type 1 (HIVLTR). Tat and its cellular cofactor, the positive transcription elongation factor b (P-TEFb), overcome this negative effect. Cdk9 in P-TEFb also phosphorylates RD at sites next to its RNA recognition motif. A mutant RD protein that mimics its phosphorylated form no longer binds TAR nor represses HIV transcription. In sharp contrast, a mutant RD protein that cannot be phosphorylated by P-TEFb functions as a dominant-negative effector and inhibits Tat transactivation. These results better define the transition from abortive to productive transcription and thus replication of HIV.The elongation of transcription from the human immunodeficiency virus type 1 long terminal repeat (HIVLTR) is regulated negatively and positively by cellular factors and the viral transactivator Tat (22). In the absence of Tat, the elongating RNA polymerase II (RNAPII) is arrested by the negative transcriptional elongation factor (N-TEF), which includes the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF), resulting in the accumulation of short transcripts (12,26,31). However, in the presence of Tat, the positive transcription elongation factor b (P-TEFb), consisting of the cyclin-dependent kinase 9 (Cdk9) and cyclin T1 (CycT1), is recruited to the transactivation response (TAR) element, which forms a stable RNA stem-loop at the 5Ј end of all viral transcripts (15,22,28,32). Cdk9 then phosphorylates DSIF and the C-terminal domain (CTD) of RNAPII, which is essential for the productive elongation of transcription (19,23).Both DSIF and NELF are found on the HIVLTR after the initiation of viral transcription (18). DSIF is composed of Spt4 and Spt5 (26). Spt5 binds the unphosphorylated but not the phosphorylated form of the CTD (CTDa of RNAPIIa but not CTDo from RNAPIIo) (10, 27). Thus, P-TEFb directly regulates the interaction between DSIF and RNAPII. NELF is comprised of four subunits, NELF-A or WHSC, NELF-B, alternatively spliced NELF-C/D, and NELF-E or RD (17,29,31). NELF-A and RD contain RNA recognition motifs (RRM) and bind a number of RNA elements, which are required for the inhibitory effect of NELF on transcription (17, 29, 31). Of importance, RD binds TAR via its RRM (30). This interaction could contribute to low basal levels of viral transcription, and therefore, to the proviral transcriptional latency in infected cells (1,14). Although P-TEFb can alleviate negative effects of NELF in vitro (25,27), no mechanism exists for this transition from negative to positive regulation of transcriptional elongation.In this study, we provide such a mechanism, taken from HIV. First, by binding the bottom stem in TAR, RD from NELF and Spt5 from DSIF coopera...

The recent identification of the genes responsible for several human genetic diseases affecting bone homeostasis and the characterization of mouse models for these diseases indicated that canonical Wnt signaling plays a critical role in the control of bone mass. Here, we report that the osteoblast-specific transcription factor Osterix (Osx), which is required for osteoblast differentiation, inhibits Wnt pathway activity. First, in calvarial cells of embryonic day (E)18.5 Osx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. Moreover, our studies demonstrated that Osx bound to and activated the Dkk1 promoter. In addition, Osx inhibited ␤-catenin-induced Topflash reporter activity and ␤-catenin-induced secondary axis formation in Xenopus embryos. Importantly, in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, ␤-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo. Our data further showed that Osx disrupted binding of Tcf to DNA, providing a likely mechanism for the inhibition by Osx of ␤-catenin transcriptional activity. We also showed that Osx decreased osteoblast proliferation. Indeed, E18.5 Osx-null calvaria showed greater BrdU incorporation than wildtype calvaria and that Osx overexpression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, we speculate that Osxmediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/␤-catenin activity. Our results add a layer of control to Wnt/␤-catenin signaling in bone.

The cellular immune response to respiratory syncytial virus (RSV) is felt to contribute to viral clearance and/or the inflammation accompanying pulmonary infections with this virus. Both tumor necrosis factor (TNF) and prostaglandin E2 (PGE2) are important regulatory mediators of the cellular immune response. We examined the production of these mediators from purified human alveolar and blood mononuclear phagocytes (MP) after RSV infection in vitro and compared production induced by virus with that induced by lipopolysaccharide (LPS). RSV infection of alveolar MP did not alter PGE2 production but increased expression of TNF alpha mRNA paralleled by increased secretion of immunoreactive and biologically active TNF. TNF production by alveolar MP was dependent on the infectious dose of virus and occurred early in the viral replication cycle. In contrast, RSV had minimal effects on blood MP production of TNF and PGE2. However, blood MP (and not alveolar MP) infected with RSV and costimulated with LPS demonstrated a 1.7-fold increase in PGE2 levels compared with LPS alone (P less than 0.001). Therefore, RSV has differential effects on human alveolar and blood MP production of these immunoregulatory molecules.

B cyclins regulate G2-M transition. Because human somatic cells continue to cycle after reduction of cyclin B1 (cycB1) or cyclin B2 (cycB2) by RNA interference (RNAi), and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated HeLa BD™ Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were RNAi resistant. Cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy. This suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFPs did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous cyclin in single kd experiments, and either cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle, while cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet residual levels are sufficient to promote mitosis in the presence of the dispensible cyclin B2. We discuss a simple model that would explain most data if cyclin B1 is necessary.

The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyte enhancer factor 2 (MEF2) family of transcription factors, key regulatory factors for myocyte development. Knockdown of endogenous cyclin T1 in murine C2C12 cells abolishes MEF2-dependent reporter gene expression as well as transcription of endogenous MEF2 target genes, whereas overexpression of P-TEFb enhances MEF2-dependent transcription. P-TEFb interacts with MEF2 both in vitro and in vivo. Activation of MEF2-dependent transcription induced by serum starvation is mediated by a rapid dissociation of P-TEFb from its inhibitory subunit, HEXIM1, and a subsequent recruitment of P-TEFb to MEF2 binding sites in the promoter region of MEF2 target genes. These results indicate that recruitment of P-TEFb is a critical step for stimulation of MEF2-dependent transcription, therefore providing a fundamentally important regulatory mechanism underlying the transcriptional program in muscle cells.

No abstract

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide and is a leading cause of preventable blindness in underdeveloped areas as well as some developed countries. Chlamydia carries genes that encode a limited number of known transcription factors. While Euo is thought to be critical for early chlamydial development, the functions of GrgA and HrcA in the developmental cycle are unclear.

Chlamydiae are common, important pathogens for humans and animals alike. Despite recent advancement in genetics, scientists are still searching for efficient tools to knock out or knock down the expression of chromosomal genes. We attempted to adopt a dCas9-based CRISPR interference (CRISPRi) technology to conditionally knock down gene expression in Chlamydia trachomatis using an anhydrotetracycline (ATC)-inducible expression system. Surprisingly, expression of the commonly used Streptococcus pyogenes dCas9 in C. trachomatis causes strong inhibition in the absence of any guide RNA (gRNA). Staphylococcus aureus dCas9 also shows strong toxicity in the presence of only an empty gRNA scaffold. Toxicity of the S. pyogenes dCas9 is readily observed with as little as 0.2 nM ATC. Growth inhibition by S. aureus dCas9 is evident starting at 1.0 nM ATC. In contrast, C. trachomatis growth was not affected by methionine-tRNA ligase overexpression induced with 10 nM ATC. We conclude that S. pyogenes and S. aureus dCas9 proteins in their current forms have limited utility for chlamydial research and suggest strategies to overcome this problem.