CDK9 inhibition blocks the initiation of PINK1-PRKN-mediated mitophagy by regulating the SIRT1-FOXO3-BNIP3 axis and enhances the therapeutic effects involving mitochondrial dysfunction in hepatocellular carcinoma.

Mitophagy is a type of selective macroautophagy/autophagy that degrades dysfunctional or excessive mitochondria. Regulation of this process is critical for maintaining cellular homeostasis and has been closely implicated in acquired drug resistance. However, the regulatory mechanisms and influences of mitophagy in cancer are still unclear. Here, we reported that inhibition of CDK9 blocked PINK1-PRKN-mediated mitophagy in HCC (hepatocellular carcinoma) by interrupting mitophagy initiation. We demonstrated that CDK9 inhibitors promoted dephosphorylation of SIRT1 and promoted FOXO3 protein degradation, which was regulated by its acetylation, leading to the transcriptional repression of FOXO3-driven BNIP3 and impairing the BNIP3-mediated stability of the PINK1 protein. Lysosomal degradation inhibitors could not rescue mitophagy flux blocked by CDK9 inhibitors. Thus, CDK9 inhibitors inactivated the SIRT1-FOXO3-BNIP3 axis and PINK1-PRKN pathway to subsequently block mitophagy initiation. Moreover, CDK9 inhibitors facilitated mitochondrial dysfunction. The dual effects of CDK9 inhibitors resulted in the destruction of mitochondrial homeostasis and cell death in HCC. Importantly, a novel CDK9 inhibitor, oroxylin A (OA), from Scutellaria baicalensis was investigated, and it showed strong therapeutic potential against HCC and a striking capacity to overcome drug resistance by downregulating PINK1-PRKN-mediated mitophagy. Additionally, because of the moderate and controlled inhibition of CDK9, OA not led to extreme repression of general transcription and appeared to overcome the inconsistent anti-HCC efficacy and high normal tissue toxicity that was associated with existing CDK9 inhibitors. All of the findings reveal that mitophagy disruption is a promising strategy for HCC treatment and OA is a potential candidate for the development of mitophagy inhibitors.Abbreviations: BNIP3: BCL2 interacting protein 3; CCCP: carbonyl cyanide p-trichloromethoxy-phenylhydrazone; CDK9: cyclin dependent kinase 9; CHX: cycloheximide; CQ, chloroquine; DFP: deferiprone; DOX: doxorubicin; EBSS: Earle's balanced salt solution; E64d: aloxistatin; FOXO3: forkhead box O3; HCC: hepatocellular carcinoma; HepG2/ADR: adriamycin-resistant HepG2 cells; MMP: mitochondrial membrane potential; mito-Keima: mitochondria-targeted and pH-sensitive fluorescent protein; MitoSOX: mitochondrial reactive oxygen species; OA: oroxylin A; PB: phosphate buffer; PDX: patient-derived tumor xenograft; PINK1: PTEN induced kinase 1; POLR2A: RNA polymerase II subunit A; p-POLR2A-S2: Ser2 phosphorylation of RNA polymerase II subunit A; PRKN: parkin RBR E3 ubiquitin protein ligase; SIRT1: sirtuin 1.

Heat shock proteins Hsp70-1 and Hsp70-3 Are necessary and sufficient to prevent arsenite-induced dysmorphology in mouse embryos.

Heat Shock Proteins (HSPs) represent a variety of protein families that are induced by stressors such as heat and toxicants, and the induction of HSPs in the organogenesis stage rodent embryo is well established. It has been proposed that thermotolerance and chemotolerance result from expression of the HSPs. However, whether these proteins function to prevent dysmorphogenesis and which family members serve this function are unknown. Therefore, we evaluated the specific ability of stress-inducible Hsp70-1 and Hsp70-3 to prevent arsenite-induced dysmorphology in the cultured mouse embryo using gain- and loss-of-function models. Loss of HSP function was accomplished by injecting antisense oligonucleotides directed against hsp70-1 and hsp 70-3 mRNAs into the amniotic cavity of cultured Day 9 mouse embryos. Suppression of hsp70-1 and hsp70-3 expression resulted in an up to six-fold increase in the incidence of arsenite-induced neural tube defects. Gain of HSP function was accomplished by microinjecting a transgene with a constitutive promotor driving expression of the hsp70-1 coding region, and resulted in a decreased incidence of arsenite-induced neural tube defects. These results indicate that Hsp70-1 and Hsp70-3 are both necessary and sufficient for preventing arsenite-induced dysmorphology in early-somite staged mouse embryos. Mol. Reprod. Dev. 59:285-293, 2001.

Plasmids for the in vitro analysis of RNA polymerase II-dependent transcription based on a G-free template.

Described are a series of plasmids containing combinations of adenovirus-2 major late promoter elements, including consensus TATA box and initiator, upstream of G-free transcription cassettes of various lengths. These provide an assortment of tools for investigating both basal and regulated transcription mechanisms by in vitro transcription methods.

Location of subunit-subunit contact sites on RNA polymerase II subunit 3 from the fission yeast Schizosaccharomyces pombe.

RNA polymerase II from the fission yeast Schizosaccharomyces pombe consists of 10 putative subunits. Subunit 3 (Rpb3) is a homologue of prokaryotic alpha subunit, which plays a key role in the assembly of core enzyme subunits. Previously we indicated that Rpb3 also plays an essential role in subunit assembly because it interacts with at least four subunits, two large subunits (Rpb1 and Rpb2) and two medium-sized subunits (Rpb3 and Rpb5) (1), and it constitutes a core subassembly consisting of Rpb2, Rpb3, and Rpb11 (2). Using a synthetic mixture of equimolar amounts of individual subunits, which were all purified from cDNA-expressed Escherichia coli, we found here that Rpb3 also interacts with Rpb11, another alpha homologue. By making a set of Rpb3 deletion derivatives, we carried out mapping of the Rpb5- and Rpb11-contact sites on Rpb3. By far-Western blot and GST pull-down assays, we found that the amino acid sequence between residues 105-263 of Rpb3 is involved in binding Rpb5, and the sequence between residues 105-297 is required for binding Rpb11. Although the Rpb5- and Rpb11-contact sites on Rpb3 overlap each other, both subunits are able to associate with Rpb3 simultaneously. The binding of Rpb5 stabilizes the Rpb3-Rpb11 heterodimer.

New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB.

A sequence element located immediately upstream of the TATA element, and having the consensus sequence 5'-G/C-G/C-G/A-C-G-C-C-3', affects the ability of transcription factor IIB to enter transcription complexes and support transcription initiation. The sequence element is recognized directly by the transcription factor IIB. Recognition involves alpha-helices 4' and 5' of IIB, which comprise a helix-turn-helix DNA-binding motif. These observations establish that transcription initiation involves a fourth core promoter element, the IIB recognition element (BRE), in addition to the TATA element, the initiator element, and the downstream promoter element, and involves a second sequence-specific general transcription factor, IIB, in addition to transcription factor IID.

The photoactivated cross-linking of recombinant C-terminal domain to proteins in a HeLa cell transcription extract that comigrate with transcription factors IIE and IIF.

The C-terminal domain (CTD) of RNA polymerase II (RNAP II) is essential for the assembly of RNAP II into preinitiation complexes on some promoters such as the dihydrofolate reductase (DHFR) promoter. In addition, during the transition from a preinitiation complex to a stable elongation complex, the CTD becomes heavily phosphorylated. In this report, interactions involving the CTD have been examined by protein-protein cross-linking. As a prelude to the study of CTD interactions, the effect of recombinant CTD on in vitro transcription was examined. The presence of recombinant CTD inhibits in vitro transcription from both the DHFR and adenovirus 2 major late promoters, suggesting that the CTD is involved in essential interactions with a general transcription factor(s). Factors in the transcription extract that interact with the CTD were identified by protein-protein cross-linking. Recombinant CTD was phosphorylated at its casein kinase II site, at the C terminus of the CTD, in the presence of [35S]adenosine 5'-O-(thiotriphosphate) and alkylated with azidophenacyl bromide. Incubation of azido-modified 35S-labeled CTD with a HeLa transcription extract followed by ultraviolet irradiation results in the covalent cross-linking of the CTD to proteins in contact with the CTD at the time of irradiation. Subsequent incubation with phenylmercuric acetate results in the transfer of 35S from the CTD to the protein to which it was cross-linked. The two major photolabeled bands have a M(r) of 34,000 and 74,000. The specificity of CTD interactions was demonstrated by a reduction in photolabeling in the presence of unmodified CTD or RNAP II containing an intact CTD (RNAP IIA) but not in the presence of a CTD-less RNAP II (RNAP IIB). The 35S-labeled 34- and 74-kDa proteins comigrate on SDS-polyacrylamide gel electrophoresis with the beta subunit of transcription factor IIE and the 74-kDa subunit of transcription factor IIF, respectively. Moreover, some of the minor 35S-labeled bands comigrate with other subunits of the general transcription factors.

Transcription elongation of the murine ornithine decarboxylase (ODC) gene is regulated in vitro at two downstream elements by different attenuation mechanisms.

Ornithine decarboxylase (ODC) plays an important role in cell proliferation. Its expression is tightly regulated at the mRNA and protein levels and is found to be deregulated in various malignancies. The rapid and dramatic induction of cellular ODC mRNA upon serum addition raised the possibility that a transcriptional attenuation mechanism may be involved in the regulation of ODC gene expression. Using transcription in HeLa nuclear extract and isolated transcription complexes, we have identified two sites of transcription arrest downstream to the transcription start site: Attenuator 1 (Att.1) located at +220, near two repeats of a USF/Myc-Max binding consensus sequence and attenuator 2 (Att.2) located at +1590 near a long stretch of T-residues. The two attenuators exhibit distinct properties as revealed by elongation of briefly initiated and partially purified transcription complexes: Att.1 serves as a transient pause site while arrest at Att.2 is more prolonged. The arrest at both attenuators is modulated by the general elongation factor TFIIS. In a promoter independent transcription system, using partially purified RNA polymerase II, only Att.2 was recognized efficiently. This suggests that the recognition of Att.2 is an intrinsic property of the polymerase while Att.1 recognition has to be facilitated by an auxiliary factor/s.

Role of differential mRNA stability in the regulated expression of IgM and IgD.

The expression of IgM and IgD heavy chain mRNA in resting vs activated B cells offers a unique tool for the assessment of the effect of translation on mRNA stability because mu and delta mRNA have identical VDJ sequences but differ substantially in the rest of the molecule. We have shown that despite the 5' UTR identity that allows equal access to the translation machinery, mu mRNA has a significantly higher turnover rate than delta mRNA. However, the short t1/2 of mu mRNA increases significantly after B cell activation. Furthermore, the induction of microS mRNA after B cell activation provides yet another related molecule for comparison. Thus, despite the fact that microS and microM mRNA differ at their 3' ends, they have identical turnover rates in activated B cells. In addition, because the turnover rates of delta mRNA and beta 2 and GAPDH mRNA remain unchanged, these experiments suggest that B cell activation results in the induction of regulatory factor(s) that target specific sequences within mRNA-mu to confer greater stability. They also argue against a more passive regulation of mRNA stability that is a consequence of alterations in the secretory machinery.

Aromatic stacking and bending of the DNA helix by the individual repeat units of the carboxy-terminal domain of RNA polymerase II.

The carboxy-terminal domain (CTD) of RNA polymerase II consists of multiple repeats of the unique heptad sequence -(Ser-Pro-Thr-Ser-Pro-Ser-Tyr)- which may interact with DNA through the intercalation of adjacent tyrosine aromatic rings. We have examined details of the interaction of this motif with calf thymus DNA through analysis of peptide analogues that contain (1) an amino-terminal tyrosine which mimics the presence of an adjacent heptad repeat and (2) positively-charged lysine residues which facilitate the initial contact between peptide and DNA. Results of fluorescence experiments, NMR titrations, and viscometric analyses indicate that these peptides bind to the DNA helix through a non-classical intercalation mode involving partial aromatic stacking of the tyrosine rings with the Watson-Crick base pairs.

RNA polymerase II-directed gene transcription by rat skeletal muscle nuclear extracts.

A cell-free transcription system was developed using nuclear extracts of rat skeletal muscle to examine the transcription of specific genes involved in ribosome biogenesis and histone synthesis. Isolation and purification of muscle tissue nuclei were required prior to obtaining a transcriptionally active extract. The transcriptional abilities of myoblast, myotube, and muscle tissue nuclear extracts were then compared using the adenovirus major late promoter as a reporter gene. Transcription of r-protein L32 and histone H4 gene templates remained high in all extracts while histone H3 gene transcription was reduced in both myotube and muscle tissue extracts. These data indicate that transcription of these genes in myotubes and muscle tissue nuclear extracts is similar. Therefore, the L6 myoblast system accurately reflects the ability of intact muscle tissue to transcribe the genes concerned with histone production and ribosome biogenesis.

Trypanosoma brucei contains two RNA polymerase II largest subunit genes with an altered C-terminal domain.

We have identified and cloned four trypanosomal RNA polymerase largest subunit genes. Here, we present the molecular analysis of two genes, Trp4.8 and Trp5.9. The sequence of these genes shows that they are almost identical to each other and indicates that they encode the largest subunit of RNA polymerase II. Both genes contain a C-terminal extension that is clearly distinct from that of other eukaryotic RNA polymerase II genes, because it lacks the common tandemly repeated heptapeptide sequence and is rich in acidic amino acids. It shares many potential phosphorylation sites, however, with the C-terminal extension of other eukaryotic RNA polymerase II large subunits. The presence of two RNA polymerase II loci suggests that a fourth RNA polymerase could be formed. Interestingly, the fourth gene is only found in species exhibiting antigenic variation.

Methyl mercury stimulates chain elongation by purified HeLa RNA polymerase II.

Methyl mercury (MeHg) inhibited the overall RNA synthetic reaction of HeLa RNA polymerase II. However, when RNA chain initiation was allowed to occur in its absence, MeHg stimulated the rate of the subsequent elongation stage of the reaction. Chain elongation with both double-stranded and single-stranded DNA templates was stimulated. This stimulatory effect was specific for MeHg; both p-hydroxymercuribenzoate and HgCl2 inhibited chain elongation (to about the same degree as they inhibited the overall reaction). The stimulatory effect was also specific for the HeLa polymerase; with Escherichia coli RNA polymerase, MeHg inhibited elongation (to the same degree as it inhibited the overall reaction).

Polyadenylylation of an mRNA precursor occurs independently of transcription by RNA polymerase II in vivo.

Most eukaryotic messenger RNAs are transcribed as precursor molecules that must be processed by capping, splicing, 3' cleavage, and polyadenylylation to yield mature mRNAs. An important, unresolved issue is whether any of these reactions are linked either to transcription by RNA polymerase II or to each other. To address one aspect of this question, we constructed a chimeric gene containing an RNA polymerase III promoter (the adenovirus VAI promoter) fused to the body and 3'-flanking sequences of a protein-coding gene (the herpesvirus tk gene). Here we show that this hybrid gene was transcribed from the RNA polymerase III promoter following transfection of human 293 cells and that the transcripts produced were stable and efficiently transported to the cytoplasm. Although a significant proportion of the transcripts were prematurely terminated at specific sites within the gene, a high percentage of the full-length RNA was accurately cleaved and polyadenylylated. These results demonstrate that cleavage and polyadenylylation of mRNA precursors are not obligatorily coupled to transcription by RNA polymerase II in vivo.

Microinjection of RNA polymerase II corrects the temperature-sensitive defect of tsAF8 cells.

tsAF8 cells are a temperature-sensitive (ts) mutant of BHK cells that arrest in the G1 phase of the cell cycle at the non-permissive temperature of 40.6 degrees C. Previous reports had suggested that the temperature-sensitivity of these cells was based on a defect in either the synthesis, assembly or turnover of RNA polymerase II. We now show that the direct microinjection of purified RNA polymerase II into nuclei of tsAF8 cells corrects the ts defect and allows these cells to enter the S phase of the cell cycle.

Molecular biology of cell division.

Different domains of the SV40 A gene have different functions, such as viral DNA replication, cell DNA replication, and stimulation of cellular RNA synthesis. The sequences in the SV40 A gene that are critical for the induction of cell DNA synthesis lie on the map between nucleotide 4360 and nucleotide 4001, a stretch of 360 nucleotides coding for 120 of the 708 amino acids of the large T antigen. The sequences critical for stimulation of rRNA synthesis lie on the map further downstream, between nucleotides 3827 and 3506, thus indicating that the signals for growth in size and for cell DNA replication can be dissociated. Methylation of the SV40 A gene at multiple ECoRI* sites has no effect on its expression. However, methylation of the HSV-TK gene at one single ECoRI site 70 base pairs upstream from the cap site inhibits its expression. The results indicate that methylation of genes affects their expression, but only when methylation occurs at specific sites.

Non-histone proteins in fractionated chromatin before and after DNAse II treatment.

Chromatin from spleen cells of normal, non-immunized mice and from mice 3 days after immunization with human immunoglobulin G was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and a residual fraction, dissociated in 2 M NaCl/5 M urea. The residual fraction of chromatin, homogeneous by ultracentrifugation and containing only 25% of the total chromatin DNA, was associated with proteins strongly labeled with [3H]tryptophan, [3H]methionine and [3H]leucine. This fraction was more sensitive to DNAase II treatment than was native, non-fractionated chromatin and it contained approx. 40% Mg2+-soluble DNA sequences. The template activity of the residual fraction was 6--7-times higher than that of non-fractionated chromatin. Fraction A, characteristic for non-immunized spleen cells, was present in three chromatin fractions and after DNAase II treatment it remained only in the residual fraction, which suggests that this fraction is associated with genes non-transcribed in non-immunized mice. Fractions I and B1 were found mainly in the residual fraction, and only in smaller amounts in the 0.35 M NaCl-soluble fraction. After DNAase II treatment, fractions I and B1 in chromatin from immunized mice disappeared, which suggests that these fractions may be associated with active transcribed sequences during the immune reaction.