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. Although it has been shown that cellular signaling stimulates the disruption of 7SK snRNP, releasing dephosphorylated and catalytically inactive P-TEFb, little is known about how the inactive released P-TEFb is reactivated. Here, we show that the Cdk9/CycT1 heterodimer released from 7SK snRNP is completely dissociated into monomers in response to stress. Brd4 or SEC then recruits monomerized Cdk9 and CycT1 to reassemble the core P-TEFb. Meanwhile, the binding of monomeric dephosphorylated Cdk9 to either Brd4 or SEC induces the autophosphorylation of T186 of Cdk9. Finally, the same mechanism is employed during nocodazole released entry into early G1 phase of cell cycle. Therefore, our studies demonstrate a novel mechanism by which Cdk9 and CycT1 monomers are reassembled on chromatin to form active P-TEFb by its interaction with Brd4 or SEC to regulate transcription.

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. Upon stimulation, Brd4 recruits P-TEFb to Spt5/DSIF via a recruitment pathway consisting of Med1, Med23 and Tat-SF1, whereas SEC recruits P-TEFb to NELF-A and NELF-E via Paf1c and Med26, respectively. P-TEFb-mediated phosphorylation of Spt5, NELF-A and NELF-E results in the dissociation of NELF from Pol II, thereby transiting transcription from pausing to elongation. Additionally, we demonstrate that P-TEFb-mediated Ser2 phosphorylation of Pol II is dispensable for pause release. Therefore, our studies reveal a co-regulatory mechanism of Brd4 and SEC in modulating the transcriptional pause release by recruiting multiple P-TEFbs via a Mediator- and Paf1c-coordinated recruitment network.

meHOLMES: A CRISPR-cas12a-based method for rapid detection of DNA methylation in a sequence-independent manner.

Aberrant DNA methylation is closely associated with various diseases, particularly cancer, and its precise detection plays an essential role in disease diagnosis and monitoring. In this study, we present a novel DNA methylation detection method (namely meHOLMES), which integrates both the TET2/APOBEC-mediated cytosine deamination step and the CRISPR-Cas12a-based signal readout step. TET2/APOBEC efficiently converts unmethylated cytosine to uracil, which is subsequently changed to thymine after PCR amplification. Utilizing a rationally designed crRNA, Cas12a specifically identifies unconverted methylated cytosines and generates detectable signals using either fluorescent reporters or lateral flow test strips. meHOLMES quantitatively detects methylated CpG sites with or without Protospacer Adjacent Motif (PAM) sequences in both artificial and real biological samples. In addition, meHOLMES can complete the whole detection process within 6 h, which is much faster than traditional bisulfite-based sample pre-treatment method. Above all, meHOLMES provides a simpler, faster, more accurate, and cost-effective approach for quantitation of DNA methylation levels in a sequence-independent manner.

CRISPR-HOLMES-based NAD+ detection.

Studies have indicated that the intracellular nicotinamide adenine dinucleotide (NAD+) level is associated with the occurrence and development of many diseases. However, traditional nicotinamide adenine dinucleotide (NAD+) detection techniques are time-consuming and may require large and expensive instruments. We recently found that the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a protein can be inactivated by AcrVA5-mediated acetylation and reactivated by CobB, using NAD+ as the co-factor. Therefore, in this study, we created a CRISPR-Cas12a-based one-step HOLMES(NAD+) system for rapid and convenient NAD+ detection with the employment of both acetylated Cas12a and CobB. In HOLMES(NAD+), acetylated Cas12a loses its trans-cleavage activities and can be reactivated by CobB in the presence of NAD+, cutting ssDNA reporters to generate fluorescence signals. HOLMES(NAD+) shows both sensitivity and specificity in NAD+ detection and can be used for quantitative determination of intracellular NAD+ concentrations. Therefore, HOLMES(NAD+) not only provides a convenient and rapid approach for target NAD+ quantitation but also expands the application scenarios of HOLMES to non-nucleic acid detection.

Gene editing and its applications in biomedicine.

The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.

Visualization of GLUT1 Trafficking in Live Cancer Cells by the Use of a Dual-Fluorescence Reporter.

Glucose metabolism is an essential process for energy production and cell survival for both normal and abnormal cellular metabolism. Several glucose transporter/solute carrier 2A (GLUT/SLC2A) superfamily members, including glucose transporter 1 (GLUT1), have been shown to mediate the cellular uptake of glucose in diverse cell types. GLUT1-mediated glucose uptake is a transient and rapid process; thus, the real-time monitoring of GLUT1 trafficking is pivotal for a better understanding of GLUT1 expression and GLUT1-dependent glucose uptake. In the present study, we established a rapid and effective method to visualize the trafficking of GLUT1 between the plasma membrane (PM) and endolysosomal system in live cells using an mCherry-EGFP-GLUT1 tandem fluorescence tracing system. We found that GLUT1 localized at the PM exhibited both red (mCherry) and green (EGFP) fluorescence (yellow when overlapping). However, a significant increase in red punctate fluorescence (mCherry is resistant to acidic pH), but not green fluorescence (EGFP is quenched by acidic pH), was observed upon glucose deprivation, indicating that the mCherry-EGFP-GLUT1 functional protein was trafficked to the acidic endolysosomal system. Besides, we were able to calculate the relative ratio of mCherry to EGFP by quantification of the translocation coefficient, which can be used as a readout for GLUT1 internalization and subsequent lysosomal degradation. Two mutants, mCherry-EGFP-GLUT1-S226D and mCherry-EGFP-GLUT1-ΔC4, were also constructed, which indirectly confirmed the specificity of mCherry-EGFP-GLUT1 for monitoring GLUT1 trafficking. By using a series of endosomal (Rab5, Rab7, and Rab11) and lysosomal markers, we were able to define a model of GLUT1 trafficking in live cells in which upon glucose deprivation, GLUT1 dissociates from the PM and experiences a pH gradient from 6.8-6.1 in the early endosomes to 6.0-4.8 in the late endosomes and finally pH 4.5 in lysosomes, which is appropriate for degradation. In addition, our proof-of-concept study indicated that the pmCherry-EGFP-GLUT1 tracing system can accurately reflect endogenous changes in GLUT1 in response to treatment with the small molecule, andrographolide. Since targeting GLUT1 expression and GLUT1-dependent glucose metabolism is a promising therapeutic strategy for diverse types of cancers and certain other glucose addiction diseases, our study herein indicates that pmCherry-EGFP-GLUT1 can be utilized as a biosensor for GLUT1-dependent functional studies and potential small molecule screening.

Combination BET Family Protein and HDAC Inhibition Synergistically Elicits Chondrosarcoma Cell Apoptosis Through RAD51-Related DNA Damage Repair.

BACKGROUND: Chondrosarcoma is the second-most common type of bone tumor and has inherent resistance to conventional chemotherapy. Present study aimed to explore the therapeutic effect and specific mechanism(s) of combination BET family protein and HDAC inhibition in chondrosarcoma. METHODS: Two chondrosarcoma cells were treated with BET family protein inhibitor (JQ1) and histone deacetylase inhibitors (HDACIs) (vorinostat/SAHA or panobinostat/PANO) separately or in combination; then, the cell viability was determined by Cell Counting Kit-8 (CCK-8) assay, and the combination index (CI) was calculated by the Chou method; cell proliferation was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation and colony formation assay; cell apoptosis and reactive oxygen species (ROS) level were determined by flow cytometry; protein expressions of caspase-3, Bcl-XL, Bcl-2, γ-H2AX, and RAD51 were examined by Immunoblotting; DNA damage was determined by comet assay; RAD51 and γ-H2AX foci were observed by immunofluorescence. RESULTS: Combined treatment with JQ1 and SAHA or PANO synergistically suppressed the growth and colony formation ability of the chondrosarcoma cells. Combined BET and HDAC inhibition also significantly elevated the ROS level, followed by the activation of cleaved-caspase-3, and the downregulation of Bcl-2 and Bcl-XL. Mechanistically, combination treatment with JQ1 and SAHA caused numerous DNA double-strand breaks (DSBs), as evidenced by the comet assay. The increase in γ-H2AX expression and foci formation also consistently indicated the accumulation of DNA damage upon cotreatment with JQ1 and SAHA. Furthermore, RAD51, a key protein of homologous recombination (HR) DNA repair, was found to be profoundly suppressed. In contrast, ectopic expression of RAD51 partially rescued SW 1353 cell apoptosis by inhibiting the expression of cleaved-caspase-3. CONCLUSION: Taken together, our results disclose that BET and HDAC inhibition synergistically inhibit cell growth and induce cell apoptosis through a mechanism that involves the suppression of RAD51-related HR DNA repair in chondrosarcoma cells.