Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the MGMT Gene Expression in Human Glioblastoma Cells

Background: The therapeutically important DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [Int. J. Mol. Sci.2021, 22(5), 2492], we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types.

DksA-dependent regulation of RpoS contributes to Borrelia burgdorferi tick-borne transmission and mammalian infectivity

ABSTRACTThroughout its enzootic cycle, the Lyme disease spirochete Borreliella (Borrelia) burgdorferi, senses and responds to changes in its environment by using a small repertoire of transcription factors which coordinate the expression of genes required for infection of Ixodes ticks and various mammalian hosts. Among these transcription factors, the DnaK suppressor protein (DksA) plays a pivotal role in regulating gene expression in B. burgdorferi during periods of nutrient limitation and is required for mammalian infectivity. In many pathogenic bacteria, the gene regulatory activity of DksA along with the alarmone guanosine penta- and tetra-phosphate ((p)ppGpp) coordinates the stringent response to various environmental stresses including nutrient limitation. In this study, we sought to characterize the role of DksA in regulating the transcriptional activity of RNA polymerase and in the regulation of RpoS-dependent gene expression required for B. burgdorferi infectivity. Using in vitro transcription assays, we observed recombinant DksA inhibits RpoD-dependent transcription by B. burgdorferi RNA polymerase independent of ppGpp Additionally, we determined the pH-inducible expression of RpoS-dependent genes relies on DksA, but is independent of (p)ppGpp produced by Relbbu. Subsequent transcriptomic and western blot assays indicated DksA regulates the expression of BBD18, a protein previously implicated in the post-transcriptional regulation of RpoS. Moreover, we observed DksA was required for infection of mice following intraperitoneal inoculation or for transmission of B. burgdorferi by Ixodes scapularis nymphs. Together, these data suggest DksA plays a central role in coordinating transcriptional responses of B. burgdorferi required for infectivity through its interactions with RNA polymerase and post-transcriptional control of RpoS.Author SummaryLyme disease, caused by the spirochetal bacteria Borrelia burgdorferi, is the most common vector-borne illness in North America. The ability of B. burgdorferi to establish infection is predicated by its ability to coordinate the expression of virulence factors in response to diverse environmental stimuli encountered within Ixodes ticks and mammalian hosts. Previous studies have shown an essential role for the alternative sigma factor RpoS in regulating the expression of genes required for the successful transmission of B. burgdorferi by Ixodes ticks and infection of mammalian hosts. The DnaK suppressor protein (DksA) is a global gene regulator in B. burgdorferi that also contributes to the expression of RpoS-dependent genes. In this study, we determined DksA exerts its gene regulatory function through direct interactions with the B. burgdorferi RNA polymerase using in vitro transcription assays and controls the expression of RpoS-dependent genes required for mammalian infection by post-transcriptionally regulating cellular levels of RpoS. Our results demonstrate the utility of in vitro transcription assays to determine how gene regulatory proteins like DksA control gene expression in B. burgdorferi, and reveal a novel role for DksA in the infectious cycle of B. burgdorferi.

Monitoring nearshore ecosystem health using Pacific razor clams (Siliqua patula) as an indicator species

An emerging approach to ecosystem monitoring involves the use of physiological biomarker analyses in combination with gene transcription assays. For the first time, we employed these tools to evaluate the Pacific razor clam (Siliqua patula), which is important both economically and ecologically, as a bioindicator species in the northeast Pacific. Our objectives were to (1) develop biomarker and gene transcription assays with which to monitor the health of the Pacific razor clam, (2) acquire baseline biomarker and gene transcription reference ranges for razor clams, (3) assess the relationship between physiological and gene transcription assays and (4) determine if site-level differences were present. Pacific razor clams were collected in July 2015 and 2016 at three sites within each of two national parks in southcentral Alaska. In addition to determining reference ranges, we found differences in biomarker assay and gene transcription results between parks and sites which indicate variation in both large-scale and local environmental conditions. Our intent is to employ these methods to evaluate Pacific razor clams as a bioindicator of nearshore ecosystem health. Links between the results of the biomarker and gene transcription assays were observed that support the applicability of both assays in ecosystem monitoring. However, we recognize the need for controlled studies to examine the range of responses in physiology and gene transcripts to different stressors.

IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis

The HIV capsid is semipermeable and covered in electropositive pores that are essential for viral DNA synthesis and infection. Here, we show that these pores bind the abundant cellular polyanion IP6, transforming viral stability from minutes to hours and allowing newly synthesised DNA to accumulate inside the capsid. An arginine ring within the pore coordinates IP6, which strengthens capsid hexamers by almost 10°C. Single molecule measurements demonstrate that this renders native HIV capsids highly stable and protected from spontaneous collapse. Moreover, encapsidated reverse transcription assays reveal that, once stabilised by IP6, the accumulation of new viral DNA inside the capsid increases >100 fold. Remarkably, isotopic labelling of inositol in virus-producing cells reveals that HIV selectively packages over 300 IP6 molecules per infectious virion. We propose that HIV recruits IP6 to regulate capsid stability and uncoating, analogous to picornavirus pocket factors. HIV-1/IP6/capsid/co-factor/reverse transcription.