Diego A. Rojas

Rrn7 Protein, an RNA Polymerase I Transcription Factor, Is Required for RNA Polymerase II-dependent Transcription Directed by Core Promoters with a HomolD Box Sequence

The region in promoters that specifies the transcription machinery is called the core promoter, displaying core promoter elements (CPE) necessary for establishment of a preinitiation complex and the initiation of transcription. A classical CPE is the TATA box. In fission yeast, Schizosaccharomyces pombe, a new CPE, called HomolD box, was discovered. Collectively, 141 ribosomal protein genes encoding the full set of 79 different ribosomal proteins and more than 60 other housekeeping genes display a HomolD box in the core promoter. Here, we show that transcription directed by the HomolD box requires the RNA polymerase II machinery, including the general transcription factors. Most intriguingly, however, we identify, by DNA affinity purification, Rrn7 as the protein binding to the HomolD box. Rrn7 is an evolutionary conserved member of the RNA polymerase I machinery involved in transcription initiation of core ribosomal DNA promoters. ChIP shows that Rrn7 cross-links to a ribosomal protein gene promoter containing the HomolD box but not to a promoter containing a TATA box. Taken together, our results suggest that Rrn7 is an excellent candidate to be involved in the coordination of ribosomal DNA and ribosomal gene transcription during ribosome synthesis and, therefore, offer a new perspective to study conservation and evolvability of regulatory networks in eukaryotes.

Identification of Ski as a target for Aurora A kinase

Ski is a negative regulator of the transforming growth factor-β and other signalling pathways. The absence of SKI in mouse fibroblasts leads to chromosome segregation defects and genomic instability, suggesting a role for Ski during mitosis. At this stage, Ski is phosphorylated but to date little is known about the kinases involved in this process. Here, we show that Aurora A kinase is able to phosphorylate Ski in vitro. In vivo, Aurora A and Ski co-localized at the centrosomes and co-immunoprecipitated. Conversely, a C-terminal truncation mutant of Ski (SkiΔ491-728) lacking a coiled-coil domain, displayed decreased centrosomal localization. This mutant no longer co-immunoprecipitated with Aurora-A in vivo, but was still phosphorylated in vitro, indicating that the Ski-Aurora A interaction takes place at the centrosomes. These data identify Ski as a novel target of Aurora A and contribute to an understanding of the role of these proteins in the mitotic process.

Fungal colonization with Pneumocystis correlates to increasing chloride channel accessory 1 (hCLCA1) suggesting a pathway for up-regulation of airway mucus responses, in infant lungs

Fungal colonization with Pneumocystis is associated with increased airway mucus in infants during their primary Pneumocystis infection, and to severity of COPD in adults. The pathogenic mechanisms are under investigation. Interestingly, increased levels of hCLCA1 – a member of the calcium-sensitive chloride conductance family of proteins that drives mucus hypersecretion – have been associated with increased mucus production in patients diagnosed with COPD and in immunocompetent rodents with Pneumocystis infection. Pneumocystis is highly prevalent in infants; therefore, the contribution of Pneumocystis to hCLCA1 expression was examined in autopsied infant lungs. Respiratory viruses that may potentially increase mucus, were also examined. hCLCA1 expression was measured using actin-normalized Western-blot, and the burden of Pneumocystis organisms was quantified by qPCR in 55 autopsied lungs from apparently healthy infants who died in the community. Respiratory viruses were diagnosed using RT-PCR for RSV, metapneumovirus, influenza, and parainfluenza viruses; and by PCR for adenovirus. hCLCA1 levels in virus positive samples were comparable to those in virus-negative samples. An association between Pneumocystis and increased hCLCA1 expression was documented (P=0.028). Additionally, increasing Pneumocystis burden correlated with increasing hCLCA1 protein expression levels (P=0.017). Results strengthen the evidence of Pneumocystis-associated up-regulation of mucus-related airway responses in infant lungs. Further characterization of this immunocompetent host-Pneumocystis-interaction, including assessment of potential clinical significance, is warranted.

Transcription directed by human core promoters with a HomolD box sequence requires DDB1, RECQL and RNA polymerase II machinery

TATA box is the most studied core promoter element and has a well-described transcription mechanism. However, most metazoan promoters lack TATA box and contain other core promoter elements. One of such elements is HomolD box, which was first described in promoters of ribosomal protein genes in Schizosaccharomyces pombe, and studies performed in this model showed that transcription directed by HomolD box is dependent on RNAPII machinery, and the HomolD-binding protein was Rrn7, a component of RNAPI core factor. Nevertheless, the mechanisms that underlie HomolD-dependent transcription are still unknown. The purpose of this study is to determine the mechanism of transcription directed by human HomolD box. By stepwise purification through different ion exchange columns and affinity chromatography, we purified two proteins: DDB1 and RECQL (DNA damage-binding protein 1 and ATP-dependent DNA helicase Q1 respectively). These proteins showed specific HomolD-binding activity and were required for in vitro HomolD-directed transcription. Recombinant RECQL, but not DDB1, presented HomolD-binding activity in vitro. Both proteins bound to HomolD box in vivo, which could be explained because these proteins co-immunoprecipitated. Additionally, RNAPII machinery was also required to transcription. Collectively, these data suggest that HomolD-containing promoters require the RNAPII machinery and the proteins DDB1 and RECQL for an accurate transcription.

Estrés oxidativo e inflamación en insuficiencia cardiaca: Mecanismos de daño y alternativas terapéuticas

Despite advances in treatment, chronic heart failure still is associated with a poor prognosis and remains a leading cause of cardiovascular death. Cumulating evidence suggests that imbalances in redox state lead to a higher generation of reactive oxygen species. This phenomenon, along with pro-inflammatory cytokine activation and extra cellular matrix alterations with reactive fibrosis, play an important role in the pathogenesis and progression of heart failure, through the development of endothelial and myocardial dysfunction. The understanding of the underlying phenomena and the metabolic pathways involved will allow further development of therapies aiming to change the natural history of heart failure.

Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1–6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids.

The Ski Protein is Involved in the Transformation Pathway of Aurora Kinase A

Oncogenic kinase Aurora A (AURKA) has been found to be overexpresed in several tumors including colorectal, breast, and hematological cancers. Overexpression of AURKA induces centrosome amplification and aneuploidy and it is related with cancer progression and poor prognosis. Here we show that AURKA phosphorylates in vitro the transcripcional co‐repressor Ski on aminoacids Ser326 and Ser383. Phosphorylations on these aminoacids decreased Ski protein half‐life. Reduced levels of Ski resulted in centrosomes amplification and multipolar spindles formation, same as AURKA overexpressing cells. Importantly, overexpression of Ski wild type, but not S326D and S383D mutants inhibited centrosome amplification and cellular transformation induced by AURKA. Altogether, these results suggest that the Ski protein is a target in the transformation pathway mediated by the AURKA oncogene. J. Cell. Biochem. 117: 334–343, 2016.

Casein kinase 2 inhibits HomolD-directed transcription by Rrn7 in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA analogue element called the HomolD box. The HomolD‐binding protein Rrn7 forms a complex with the RNA polymerase II machinery. Despite the importance of ribosome biogenesis to cell survival, the mechanisms involved in the regulation of transcription of eukaryotic RPGs are unknown. In this study, we identified Rrn7 as a new substrate of the pleiotropic casein kinase 2 (CK2), which is a regulator of basal transcription. Recombinant Rrn7 from S. pombe, which is often used as a model organism for studying eukaryotic transcription, interacted with CK2 in vitro and in vivo. Furthermore, CK2‐mediated phosphorylation of Rrn7 inhibited its HomolD‐directed transcriptional activity and ability to bind to an oligonucleotide containing a HomolD box in vitro. Mutation of Rrn7 at Thr67 abolished these effects, indicating that this residue is a critical CK2 phosphorylation site. Finally, Rrn7 interacted with the regulatory subunit of CK2 in vivo, inhibition of CK2 in vivo potentiated ribosomal protein gene transcription, and chromatin immunoprecipitation analyses identified that the catalytic subunit of CK2 was associated with the rpk5 gene promoter in S. pombe. Taken together, these data suggest that CK2 inhibits ribosomal protein gene transcription in S. pombe via phosphorylation of Rrn7 at Thr67.

Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.

RNA polymerase II components and Rrn7 form a pre-initiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA‐binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross‐linked to HomolD‐containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD‐directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross‐linked to the promoter region of HomolD‐containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box‐containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.