Disrupting the Cdk9/Cyclin T1 heterodimer of 7SK snRNP for the Brd4 and AFF1/4 guided reconstitution of active P-TEFb.

P-TEFb modulates RNA polymerase II elongation through alternative interaction with negative and positive regulation factors. While inactive P-TEFbs are mainly sequestered in the 7SK snRNP complex in a chromatin-free state, most of its active forms are in complex with its recruitment factors, Brd4 and SEC, in a chromatin-associated state. Thus, switching from inactive 7SK snRNP to active P-TEFb (Brd4/P-TEFb or SEC/P-TEFb) is essential for global gene expression.

H. pylori infection confers resistance to apoptosis via Brd4-dependent BIRC3 eRNA synthesis.

H. pylori infection is one of the leading causes of gastric cancer and the pathogenicity of H. pylori infection is associated with its ability to induce chronic inflammation and apoptosis resistance.

Apigenin exhibits protective effects in a mouse model of d-galactose-induced aging via activating the Nrf2 pathway.

The aim of the present research was to study the protective effects and underlying mechanisms of apigenin on d-galactose-induced aging mice. Firstly, apigenin exhibited a potent antioxidant activity in vitro. Secondly, d-galactose was administered by subcutaneous injection once daily for 8 weeks to establish an aging mouse model to investigate the protective effect of apigenin.

P-TEFb: Finding its ways to release promoter-proximally paused RNA polymerase II.

The release of a paused Pol II depends on the recruitment of P-TEFb. Recent studies showed that both active P-TEFb and inactive P-TEFb (7SK snRNP) can be recruited to the promoter regions of global genes by different mechanisms. Here, we summarize the recent advances on these distinct recruitment mechanisms.

HMBA Enhances Prostratin-Induced Activation of Latent HIV-1 via Suppressing the Expression of Negative Feedback Regulator A20/TNFAIP3 in NF-κB Signaling.

In the past decade, much emphasis has been put on the transcriptional activation of HIV-1, which is proposed as a promised strategy for eradicating latent HIV-1 provirus. Two drugs, prostratin and hexamethylene bisacetamide (HMBA), have shown potent effects as inducers for releasing HIV-1 latency when used alone or in combination, although their cellular target(s) are currently not well understood, especially under drug combination. Here, we have shown that HMBA and prostratin synergistically release HIV-1 latency via different mechanisms.

Multiple P-TEFbs cooperatively regulate the release of promoter-proximally paused RNA polymerase II.

The association of DSIF and NELF with initiated RNA Polymerase II (Pol II) is the general mechanism for inducing promoter-proximal pausing of Pol II. However, it remains largely unclear how the paused Pol II is released in response to stimulation. Here, we show that the release of the paused Pol II is cooperatively regulated by multiple P-TEFbs which are recruited by bromodomain-containing protein Brd4 and super elongation complex (SEC) via different recruitment mechanisms.

Competitive Inhibition of Lysine Acetyltransferase 2B by a Small Motif of the Adenoviral Oncoprotein E1A.

The adenovirus early region 1A (E1A) oncoprotein hijacks host cells via direct interactions with many key cellular proteins, such as KAT2B, also known as PCAF (p300/CBP associated factor). E1A binds the histone acetyltransferase (HAT) domain of KAT2B to repress its transcriptional activation. However, the molecular mechanism by which E1A inhibits the HAT activity is not known.

BET Inhibition Attenuates Helicobacter pylori-Induced Inflammatory Response by Suppressing Inflammatory Gene Transcription and Enhancer Activation.

Helicobacter pylori infection causes chronic gastritis and peptic ulceration. H. pylori-initiated chronic gastritis is characterized by enhanced expression of many NF-κB-regulated inflammatory cytokines.

CaMKII: do not work too hard in the failing heart.

CaMKIIδ, a calcium/calmodulin-dependent protein kinase, plays pivotal roles in the development of heart disease. In this issue of The Journal of Pathology, Salma Awad and colleagues demonstrate that CaMKIIδ is engaged in both pathological hypertrophy and heart failure. By analysis of mouse and human heart samples, they found that the level of CaMKIIδ is increased in both pathological processes.

Protein kinase D3 is essential for prostratin-activated transcription of integrated HIV-1 provirus promoter via NF-κB signaling pathway.

Prostratin has been proposed as a promising reagent for eradicating the latent HIV-1 provirus by inducing HIV-1 transcription activation. The molecular mechanism of this activation, however, is far from clear. Here, we show that the protein kinase D3 (PKD3) is essential for prostratin-induced transcription activation of latent HIV-1 provirus.

Histone cross-talk connects protein phosphatase 1α (PP1α) and histone deacetylase (HDAC) pathways to regulate the functional transition of bromodomain-containing 4 (BRD4) for inducible gene expression.

Transcription elongation has been recognized as a rate-limiting step for the expression of signal-inducible genes. Through recruitment of positive transcription elongation factor P-TEFb, the bromodomain-containing protein BRD4 plays critical roles in regulating the transcription elongation of a vast array of inducible genes that are important for multiple cellular processes. The diverse biological roles of BRD4 have been proposed to rely on its functional transition between chromatin targeting and transcription regulation.

Abnormal response to the anorexic effect of GHS-R inhibitors and exenatide in male Snord116 deletion mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a genetic disease characterized by persistent hunger and hyperphagia. The lack of the Snord116 small nucleolar RNA cluster has been identified as the major contributor to PWS symptoms. The Snord116 deletion (Snord116del) mouse model manifested a subset of PWS symptoms including hyperphagia and hyperghrelinemia.

Brd4 and HEXIM1: multiple roles in P-TEFb regulation and cancer.

Bromodomain-containing protein 4 (Brd4) and hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) are two opposing regulators of the positive transcription elongation factor b (P-TEFb), which is the master modulator of RNA polymerase II during transcriptional elongation. While Brd4 recruits P-TEFb to promoter-proximal chromatins to activate transcription, HEXIM1 sequesters P-TEFb into an inactive complex containing the 7SK small nuclear RNA. Besides regulating P-TEFb's transcriptional activity, recent evidence demonstrates that both Brd4 and HEXIM1 also play novel roles in cell cycle progression and tumorigenesis.

Dimeric structure of p300/CBP associated factor.

Background: p300/CBP associating factor (PCAF, also known as KAT2B for lysine acetyltransferase 2B) is a catalytic subunit of megadalton metazoan complex ATAC (Ada-Two-A containing complex) for acetylation of histones. However, relatively little is known about the regulation of the enzymatic activity of PCAF.

Results: Here we present two dimeric structures of the PCAF acetyltransferase (HAT) domain.

Mediator MED23 regulates basal transcription in vivo via an interaction with P-TEFb.

The Mediator is a multi-subunit complex that transduces regulatory information from transcription regulators to the RNA polymerase II apparatus. Growing evidence suggests that Mediator plays roles in multiple stages of eukaryotic transcription, including elongation. However, the detailed mechanism by which Mediator regulates elongation remains elusive.

Signal-induced Brd4 release from chromatin is essential for its role transition from chromatin targeting to transcriptional regulation.

Bromodomain-containing protein Brd4 is shown to persistently associate with chromosomes during mitosis for transmitting epigenetic memory across cell divisions. During interphase, Brd4 also plays a key role in regulating the transcription of signal-inducible genes by recruiting positive transcription elongation factor b (P-TEFb) to promoters. How the chromatin-bound Brd4 transits into a transcriptional regulation mode in response to stimulation, however, is largely unknown.

An efficient approach for site-directed mutagenesis using central overlapping primers.

QuikChange is a popular method for site-directed mutagenesis in structural and functional studies of proteins and nucleic acids. However, the standard protocol is often inefficient in producing the desired mutations. Here we present a novel strategy for primer design, central overlapping primers (COP), which employs a pair of bipartite primers of different lengths, with the short primer complementary to the middle region of the long primer.

Quikgene: a gene synthesis method integrated with ligation-free cloning.

Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step.

Isolation and functional characterization of P-TEFb-associated factors that control general and HIV-1 transcriptional elongation.

Originally identified as a factor crucial for RNA polymerase (Pol) II transcriptional elongation of cellular genes, the P-TEFb kinase was subsequently shown to also serve as a specific host co-factor required for HIV-1 transcription. Recruited by either the bromodomain protein Brd4 to cellular promoters for general transcription or the HIV-1 Tat protein to the viral LTR for activated HIV-1 transcription, P-TEFb stimulates the processivity of Pol II through phosphorylating the C-terminal domain of Pol II and a pair of negative elongation factors, leading to the synthesis of full-length transcripts. However, abundant evidence indicates that P-TEFb does not act alone in the cell and that all of its known biological functions are likely mediated through the interactions with various regulators.

A capping-independent function of MePCE in stabilizing 7SK snRNA and facilitating the assembly of 7SK snRNP.

The 7SK snRNP represents a major reservoir of activity where P-TEFb, a general transcription factor key for RNA polymerase II elongation, can be withdrawn to promote gene expression, cell growth and development. Within this complex, 7SK snRNA is a central scaffold that coordinates key protein-protein interactions and maintains P-TEFb in an inactive state. Although the stability of 7SK directly affects the amount of active P-TEFb in vivo, relatively little is known about how it is maintained and how the 7SK methylphosphate capping enzyme MePCE and LARP7, a La-related protein associated with the 3'-poly(U) of 7SK, contribute to this process.

PP2B and PP1alpha cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling.

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown.

Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription.

Human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (Tat) recruits the positive transcription elongation factor b (P-TEFb) to the viral promoter. Consisting of cyclin dependent kinase 9 (Cdk9) and cyclin T1, P-TEFb phosphorylates RNA polymerase II and the negative transcription elongation factor to stimulate the elongation of HIV-1 genes. A major fraction of nuclear P-TEFb is sequestered into a transcriptionally inactive 7SK small nuclear ribonucleoprotein (snRNP) by the coordinated actions of the 7SK small nuclear RNA (snRNA) and hexamethylene bisacetamide (HMBA) induced protein 1 (HEXIM1).

Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4.

The cyclinT1/Cdk9 heterodimer that constitutes core P-TEFb is generally presumed to be the transcriptionally active form for stimulating RNA polymerase II elongation. About half of cellular P-TEFb also exists in an inactive complex with the 7SK snRNA and the HEXIM1 protein. Here, we show that the remaining half associates with the bromodomain protein Brd4.

Compensatory contributions of HEXIM1 and HEXIM2 in maintaining the balance of active and inactive positive transcription elongation factor b complexes for control of transcription.

Human positive transcriptional elongation factor b (P-TEFb), consisting of a cyclin-dependent kinase 9-cyclin T heterodimer, stimulates general and disease-specific transcriptional elongation by phosphorylating RNA polymerase II. The HEXIM1 protein, aided by the 7SK snRNA, sequesters P-TEFb into an inactive 7SK.HEXIM1.

A human immunodeficiency virus type 1 Tat-like arginine-rich RNA-binding domain is essential for HEXIM1 to inhibit RNA polymerase II transcription through 7SK snRNA-mediated inactivation of P-TEFb.

The HEXIM1 protein inhibits the kinase activity of P-TEFb (CDK9/cyclin T) to suppress RNA polymerase II transcriptional elongation in a process that specifically requires the 7SK snRNA, which mediates the interaction of HEXIM1 with P-TEFb. In an attempt to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, we have identified the first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 as both necessary and sufficient for binding to 7SK in vivo and in vitro. This 7SK-binding motif was essential for HEXIM1's inhibitory action, as the HEXIM1 mutants with this motif replaced with a foreign NLS failed to interact with 7SK and P-TEFb and hence were unable to inactivate P-TEFb.