Quantitative Proteomics of the CDK9 Interactome Reveals a Function of the HSP90-CDC37-P-TEFb Complex for BETi-Induced HIV-1 Latency Reactivation.

Brd4 has been intensively investigated as a promising drug target because of its implicated functions in oncogenesis, inflammation, and HIV-1 transcription. The formation of the Brd4-P-TEFb (CDK9/Cyclin T1) complex and its regulation of transcriptional elongation are critical for HIV latency reactivation and expression of many oncogenes. To further investigate the mechanism of the Brd4-P-TEFb complex in controlling elongation, mass spectrometry-based quantitative proteomics of the CDK9 interactome was performed. Upon treatment with the selective BET bromodomain inhibitor JQ1, 352 proteins were successfully identified with high confidence as CDK9-interacting proteins. Among them, increased bindings of HSP90 and CDC37 to CDK9 were particularly striking, and our data suggest that the HSP90-CDC37-P-TEFb complex is involved in controlling the dynamic equilibrium of the P-TEFb complex during BETi-induced reactivation of HIV-1 latency. Furthermore, the HSP90-CDC37-P-TEFb complex directly regulates HIV-1 transcription and relies on recruitment by heat shock factor 1 (HSF1) for binding to the HIV-1 promoter. These results advance the understanding of HSP90-CDC37-P-TEFb in HIV-1 latency reversal and enlighten the development of potential strategies to eradicate HIV-1 using a combination of targeted drugs.

Brd4-dependent CDK9 expression induction upon sustained pharmacological inhibition of P-TEFb kinase activity.

CDK9 is the kinase subunit of P-TEFb (positive transcription elongation factor b), which is crucial for effective transcriptional elongation. The activity of P-TEFb is well maintained, mainly through dynamic association with several larger protein complexes. Here, we show that CDK9 expression is induced upon inhibition of P-TEFb activity, a process dependent on Brd4 as later revealed. Brd4 inhibition synergizes with CDK9 inhibitor to suppress P-TEFb activity and tumor cell growth. Our study suggests that combined inhibition of Brd4 and CDK9 can be evaluated as a potential therapeutic strategy.

Curcumin Derivative C66 Suppresses Pancreatic Cancer Progression through the Inhibition of JNK-Mediated Inflammation.

Pancreatic adenocarcinoma is by far the deadliest type of cancer. Inflammation is one of the important risk factors in tumor development. However, it is not yet clear whether deterioration in pancreatic cancer patients is related to inflammation, as well as the underlying mechanism. In addition, JNK is abnormally activated in pancreatic cancer cells and the JNK inhibitor C66 reduces the inflammatory microenvironment in the tumor. Therefore, the aim of this study was to evaluate the role of C66 in the proliferation and migration of pancreatic cancer. Our results showed that various inflammatory cytokines, such as IL-1ß, IL-6, IL-8, and IL-15, were more expressed in pancreatic cancer than in the matching normal tissue. Furthermore, C66, a curcumin analogue with good anti-inflammatory activity, inhibited the proliferation and migration of pancreatic cancer cells in a dose-dependent manner, and effectively inhibited the expression of the above inflammatory factors. Our previous research demonstrated that C66 prevents the inflammatory response by targeting JNK. Therefore, in this study, JNK activity in pancreatic cancer cells was investigated, revealing that JNK was highly activated, and the treatment with C66 inhibited the phosphorylation of JNK. Next, shJNK was used to knockdown JNK expression in pancreatic cancer cells to further confirm the role of JNK in the proliferation and migration of this tumor, as well as in the inflammatory tumor microenvironment (TME). The results demonstrated that JNK knockdown could significantly inhibit the proliferation and migration of pancreatic cancer. Moreover, the low JNK expression in pancreatic cancer cells significantly inhibited the expression of various inflammatory factors. These results indicated that C66 inhibited the progression of pancreatic cancer through the inhibition of JNK-mediated inflammation.

Poly(ADP-ribosylation) of P-TEFb by PARP1 disrupts phase separation to inhibit global transcription after DNA damage.

DNA damage shuts down genome-wide transcription to prevent transcriptional mutagenesis and to initiate repair signalling, but the mechanism to stall elongating RNA polymerase II (Pol II) is not fully understood. Central to the DNA damage response, poly(ADP-ribose) polymerase 1 (PARP1) initiates DNA repair by translocating to the lesions where it catalyses protein poly(ADP-ribosylation). Here we report that PARP1 inhibits Pol II elongation by inactivating the transcription elongation factor P-TEFb, a CDK9-cyclin T1 (CycT1) heterodimer. After sensing damage, the activated PARP1 binds to transcriptionally engaged P-TEFb and modifies CycT1 at multiple positions, including histidine residues that are rarely used as an acceptor site. This prevents CycT1 from undergoing liquid-liquid phase separation that is required for CDK9 to hyperphosphorylate Pol II and to stimulate elongation. Functionally, poly(ADP-ribosylation) of CycT1 promotes DNA repair and cell survival. Thus, the P-TEFb-PARP1 signalling plays a protective role in transcription quality control and genomic stability maintenance after DNA damage.

Rosmarinic Acid Decreases the Malignancy of Pancreatic Cancer Through Inhibiting Gli1 Signaling.

BACKGROUND: Rosmarinic acid (RA) has been shown to exert anti-tumor effects on various types of cancer. However, its roles in the treatment of pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanisms remain elusive. PURPOSE: The present study aimed to investigate the therapeutic effects of RA on PDAC as well as the underlying mechanisms. STUDY DESIGN: Evaluation of the effects of RA on PDAC malignancy both in vitro and in vivo. METHODS: Cell counting kit 8 (CCK8) assay, colony formation assay, 5-Ethynyl-2'-deoxyuridine (EDU) incorporation assay, cell cycle analysis, and apoptosis assay were conducted to assess the inhibitory effect of RA on PDAC cell proliferation. Meanwhile, western blotting and RT-qPCR assay were performed to detect the target gene expression at protein and mRNA levels, respectively. Moreover, the in vivo anti-tumor activities of RA were assayed in an xenograft mouse model of PDAC. RESULTS: RA dramatically down-regulated Gli1 and its downstream targets. Further studies showed that RA prevents the nuclear translocation of Gli1, while promoting the degradation of cytosolic Gli1 via the proteasome pathway. Moreover, we observed that RA induced G1/S cell cycle arrest and apoptosis in the PDAC cells through regulating the expression of P21, P27, CDK2, Cyclin E, Bax, and Bcl-2, it inhibited the PDAC cell migration and invasion via E-cadherin and MMP-9. Notably, Gli1 overexpression markedly reversed the above RA-induced effects on PDAC cells, whereas Gli1 knockdown enhanced the effects. Additionally, the in vivo assays demonstrated that RA suppresses the tumor growth of PDAC presumably by inhibiting Gli1. CONCLUSION: We provided evidence that RA restrained the nuclear translocation of Gli1 and facilitates Gli1 degradation via proteasome pathway, reducing the malignancy of PDAC cells. These findings implicated RA as a therapeutic agent for PDAC.

ELL-associated factors EAF1/2 negatively regulate HIV-1 transcription through inhibition of Super Elongation Complex formation.

The ELL (ELL1 and ELL2)-containing Super Elongation Complex (SEC) is required for efficient HIV-1 transactivation by the viral-encoded Tat protein. EAF1 and EAF2 are ELL-associated factors and considered as positive regulators of ELL. However, their role in HIV-1 transcriptional control is unknown. In this study, we show that EAF1/2 inhibit the SEC-dependent and Tat-activated HIV-1 transcription. EAF1/2 are found to interact with the SEC components in an ELL1/2-dependent manner. Surprisingly, the depletion of EAF1/2 increases the SEC formation and occupancy on the HIV-1 proviral DNA, thereby stimulating Tat transactivation of HIV-1. Although EAF1/2 interact with members of the SEC in a ELL-dependent manner, this interaction competes with the binding of the scaffolding subunit AFF1 with ELL, thus reducing the SEC formation. Together, these data reveal how EAF1/2 regulate the SEC formation to control HIV-1 transcription.

Balanced between order and disorder: a new phase in transcription elongation control and beyond.

We recently reported that the cyclin T1 histidine-rich domain creates a phase-separated environment to promote hyperphosphorylation of RNA polymerase II C-terminal domain and robust transcriptional elongation by P-TEFb. Here, we discuss this and several other recent discoveries to demonstrate that phase separation is important for controlling various aspects of transcription.

Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II.

Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing1-3. The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation1,4,5. The sequence determinant on P-TEFb that facilitates this action is currently unknown. Here we identify a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase-DYRK1A 6 -also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH1-3. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II.

The PARP1-Siah1 Axis Controls HIV-1 Transcription and Expression of Siah1 Substrates.

Recent studies have revealed a key role of PARP1 that catalyzes the poly-ADP-ribosylation (PARylation) of substrates in regulating gene transcription. We show here that HIV-1 transcriptional activation also requires PARP1 activity. Because efficient HIV-1 transactivation is known to depend on the ELL2-containing super elongation complex (SEC), we investigated the functional relationship between PARP1 and ELL2-SEC in HIV-1 transcriptional control. We show that PARP1 elevates ELL2 protein levels to form more ELL2-SEC in cells. This effect is caused by PARP1's suppression of expression of Siah1, an E3 ubiquitin ligase for ELL2, at both mRNA and protein levels. At the mRNA level, PARP1 coordinates with the co-repressor NCoR to suppress Siah1 transcription. At the protein level, PARP1 promotes Siah1 proteolysis, likely through inducing PARylation-dependent ubiquitination (PARdU) of Siah1. Thus, a PARP1-Siah1 axis activates HIV-1 transcription and controls the expression of ELL2 and other Siah1 substrates.

Membrane associated protein flotillin-2 in Litopenaeus vannamei plays a role in WSSV infection.

Flotillin-2, an important protein of vesicular endocytosis, plays an essential role in a large number of cellular processes, including viruses and pathogen infection. In the present study, a flotillin-2 homolog in Litopenaeus vannamei, designed as Lvflotillin-2, was cloned and characterized. To analyze the putative role of Lvflotillin-2 during white spot syndrome virus (WSSV) infection, real-time quantitative PCR was performed. The result showed that the transcriptional level of Lvflotillin-2 was up-regulated significantly after virus challenge. Furthermore, upon WSSV stimulation, Lvflotillin-2 in shrimp cells could translocate from the plasma membrane to intracellular compartments, and unexpectedly, also into nucleus. Additionally, depletion of Lvflotillin-2 inhibited WSSV gene ie1 transcription. It suggested that Lvflotillin-2 could be hijacked by WSSV. These observations indicated that Lvflotillin-2 was involved in WSSV infection, and presented here should be useful for gaining insight into shrimp immunity and WSSV pathogenesis.

Mechanism and factors that control HIV-1 transcription and latency activation.

After reverse transcription, the HIV-1 proviral DNA is integrated into the host genome and thus subjected to transcription by the host RNA polymerase II (Pol II). With the identification and characterization of human P-TEFb in the late 1990 s as a specific host cofactor required for HIV-1 transcription, it is now believed that the elongation stage of Pol II transcription plays a particularly important role in regulating HIV-1 gene expression. HIV-1 uses a sophisticated scheme to recruit human P-TEFb and other cofactors to the viral long terminal repeat (LTR) to produce full-length HIV-1 transcripts. In this process, P-TEFb is regulated by the reversible association with various transcription factors/cofactors to form several multi-subunit complexes (e.g., 7SK snRNP, super elongation complexes (SECs), and the Brd4-P-TEFb complex) that collectively constitute a P-TEFb network for controlling cellular and HIV-1 transcription. Recent progresses in HIV-1 transcription were reviewed in the paper, with the emphasis on the mechanism and factors that control HIV-1 transcription and latency activation.