The genomic landscape of 8-oxodG reveals enrichment at specific inherently fragile promoters

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is the most common marker of oxidative stress and its accumulation within the genome has been associated with major human health issues such as cancer, aging, cardiovascular and neurodegenerative diseases. The characterization of the different genomic sites where 8-oxodG accumulates and the mechanisms underlying its formation are still poorly understood. Using OxiDIP-seq, we recently derived the genome-wide distribution of 8-oxodG in human non-tumorigenic epithelial breast cells (MCF10A). Here, we identify a subset of human promoters that accumulate 8-oxodG under steady-state condition. 8-oxodG nucleotides co-localize with double strand breaks (DSBs) at bidirectional and CG skewed promoters and their density correlate with RNA Polymerase II co-occupancy and transcription. Furthermore, by performing OxiDIP-seq in quiescent (G0) cells, we found a strong reduction of oxidatively-generated damage in the majority of 8-oxodG-positive promoters in the absence of DNA replication. Overall, our results suggest that the accumulation of 8-oxodG at gene promoters occurs through DNA replication-dependent or -independent mechanisms, with a possible contribution to the formation of cancer-associated translocation events

A novel member of the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts as a transcriptional repressor.

We have identified a novel human gene encoding a 59-kDa POZ-AT hook-zinc finger protein (PATZ) that interacts with RNF4, a mediator of androgen receptor activity, and acts as a transcriptional repressor. PATZ cDNA was isolated through a two-hybrid interaction screening using the RING finger protein RNF4 as a bait. In vitro and in vivo interaction between RNF4 and PATZ was demonstrated by protein-protein affinity chromatography and coimmunoprecipitation experiments. Such interaction occurred through a small region of PATZ containing an AT-hook DNA binding domain. Immunofluorescence staining and confocal microscopy showed that PATZ localizes in distinct punctate nuclear regions and colocalizes with RNF4. Functional analysis was performed by cotransfection assays: PATZ acted as a transcriptional repressor, whereas its partner RNF4 behaved as a transcriptional activator. When both proteins were overexpressed a strong repression of the basal transcription was observed, indicating that the association of PATZ with RNF4 switches activation to repression. In addition, RNF4 was also found to associate with HMGI(Y), a chromatin-modeling factor containing AT-hook domains

A Transcriptional Enhancer from the Coding Region of ADAMTS5

The revelation that the human genome encodes only approximately 25,000 genes and thus cannot account for phenotypic complexity has been one of the biggest surprises in the post-genomic era. However, accumulating evidence suggests that transcriptional regulation may be in large part responsible for this observed mammalian complexity. Consequently, there has been a strong drive to locate cis-regulatory regions in mammalian genomes in order to understand the unifying principles governing these regions, including their genomic distribution. Although a number of systematic approaches have been developed, these all discount coding sequence.Using the computational tool PRI (Pattern-defined Regulatory Islands), which does not mask coding sequence, we identified a regulatory region associated with the gene ADAMTS5 that encompasses the entirety of the essential coding exon 2. We demonstrate through a combination of chromatin immunoprecipitation and reporter gene studies that this region can not only bind the myogenic transcription factors MYOD and myogenin and the E-protein HEB but can also function as a very strong myogenic transcriptional enhancer.Thus, we report the identification and detailed characterization of an exonic enhancer. Ultimately, this leads to the interesting question of why evolution would be so parsimonious in the functional assignment of sequence

Caffeine Prevents Transcription Inhibition and P-TEFb/7SK Dissociation Following UV-Induced DNA Damage

Background: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. Methodology/Principal Findings: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. Conclusion/Significance: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibitio

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Autophagy roles in genome maintenance

In recent years, a considerable correlation has emerged between autophagy and genome integrity. A range of mechanisms appear to be involved where autophagy participates in preventing genomic instability, as well as in DNA damage response and cell fate decision. These initial findings have attracted particular attention in the context of malignancy; however, the crosstalk between autophagy and DNA damage response is just beginning to be explored and key questions remain that need to be addressed, to move this area of research forward and illuminate the overall consequence of targeting this process in human therapies. Here we present current knowledge on the complex crosstalk between autophagy and genome integrity and discuss its implications for cancer cell survival and response to therapy