RAPID-SELEX identifies high-affinity RNA targets for chromatin binding proteins PARP1 and MeCP2
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ABSTRACT:
PROVIDER: PRJNA1122221 | ENA |
REPOSITORIES: ENA
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Project description:Recent studies showed that some chromatin-binding proteins also interact with RNA to regulate gene expression. Poly (ADP-ribose) polymerase 1 (PARP1) and Methyl-CpG Binding Protein 2 (MeCP2) are two chromatin-associated, DNA-binding proteins with central roles in gene expression, DNA damage response, and epigenetic regulation. PARP1 and MeCP2 both have RNA binding properties, but it is still unclear how PARP1 and MeCP2 are recruited to or recognize RNA binding sites. Here, we used a RAPID-SELEX procedure, a random 70-mer RNA library, and 12 rounds of selection to identify high-affinity PARP1 and MeCP2 RNA targets. PARP1 prefers RNA targets enriched in guanine, while MeCP2 shows a preference for targets enriched in cytosine. However, neither protein seems to have a consensus (linear) RNA sequence motif. Instead, high-affinity PARP1 targets contain intrinsic secondary structures characterized by a relatively long stem and small loop. High affinity MeCP2 targets are also intrinsically structured, characterized by a large loop (or bulge) and multiple stems. These data suggest that both proteins target RNAs based on (unique) structural features rather than nucleotide sequence, and that many different RNA sequences can adopt similar structures.
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2022-02-23 | GSE197262 | GEO
Project description:BACKGROUND & AIMS: The immune system comprises an innate and an adaptive immune response to combat pathogenic agents. The human enteropathogen Salmonella enterica serovar Typhimurium invades the intestinal mucosa and triggers an early innate pro-inflammatory host gene response, which results in diarrheal disease. Several host factors are involved in the acute early response to Salmonella infection. Transcription factors and transcription co-regulators have an especially important function, because they are required for the expression and synthesis of pro-inflammatory cytokines, chemokines and adhesion molecules. A central transcription factor involved in inflammation is NF-κB, which requires the nuclear protein PARP1 as co-factor for the expression of some of its target genes. Here, we investigated the role of PARP1 during Salmonella infection using a mouse model for Salmonella-induced colitis. METHODS: To study enterocolitis by Salmonella Typhimurium, an established mouse model system, which relies on streptomycin-pretreatment prior to Salmonella infection, was employed. Histopathologic signs of inflammation and cecum colonization at various time-points after infection of wild type and PARP1 knockout mice were analyzed. PARP1 expression in the gut mucosa was studied by quantitative RT-PCR, Western blot and immunofluorescence. Gene expression profiles of infected and control infected mice in the wild type or PARP1 knockout background were obtained by whole mouse genome arrays and confirmed by quantitative RT-PCR. 2 genotypes (wildtype, PARP1 knockout), 2 treatments (Salmonella SB300 infection, Salmonella SB161 control infection), 2 time-points (6h, 10h). 2-3 replicates/condition.
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Project description:MECP2 is critical for proper brain development and expressed at near-histone levels in neurons, but the mechanism of its genomic localization remains poorly understood. Using high-resolution MeCP2 binding data, we show that genetic features alone can predict binding with 88% accuracy. Integrating MeCP2 binding and DNA methylation in a probabilistic graphical model, we demonstrate that previously reported methylation preferences may be due to MeCP2âs affinity to GC-rich chromatin, a result replicated using published data. Furthermore, MeCP2 co-localized with nucleosomes, and Mecp2 deletion led to nucleosome repositioning. Finally, MeCP2 binding downstream of promoters correlated with increased expression in Mecp2 deficient neurons. Study of genetic and epigenetic determinants of MeCP2 binding using MeCP2 ChIP-seq, MNase-seq, Bisulfite-seq and RNA-seq. Please see individual sample record for details on experimental design.
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Project description:MECP2 is critical for proper brain development and expressed at near-histone levels in neurons, but the mechanism of its genomic localization remains poorly understood. Using high-resolution MeCP2 binding data, we show that genetic features alone can predict binding with 88% accuracy. Integrating MeCP2 binding and DNA methylation in a probabilistic graphical model, we demonstrate that previously reported methylation preferences may be due to MeCP2’s affinity to GC-rich chromatin, a result replicated using published data. Furthermore, MeCP2 co-localized with nucleosomes, and Mecp2 deletion led to nucleosome repositioning. Finally, MeCP2 binding downstream of promoters correlated with increased expression in Mecp2 deficient neurons.
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Project description:Trabectedin is a DNA-damaging agent with a peculiar mechanism of action; it traps the DNA repair machinery leading to DNA single- and double-strand breaks, particularly in BRCA1/2-deficient tumors. We hypothesized that trabectedin-induced DNA damage might activate PARP1 (a DNA-repair machinery key player), and consequently, PARP1 inhibition would perpetuate trabectedin-induced DNA damage. In several tumor histotypes, we demonstrated a different degree of synergism between trabectedin and PARP1 inhibitors (PARP1-Is). Independent of BRCA1/2 status, PARP1 expression dictated the degree of synergism. Namely, silenced PARP1 reduced trabectedin-PARP1-Is synergism, whereas overexpressed PARP1 increased combination efficacy. High-PARP1 expression and specific gene signatures associated with DNA damage response and repair (DDR-R) were predictive of trabectedin+PARP1-I synergy. These findings pave the way for the clinical development of this novel combination therapy, as well as evaluation of PARP1 expression and DDR-R signatures in tumor samples as predictive biomarkers of response.
2017-06-12 | GSE76981 | GEO
Project description:Poly(ADP-ribose) polymerase 1 (PARP1) is a highly conserved nuclear protein in multicellular organisms that modulates chromatin opening, and by doing so, it plays a significant role in gene expression regulation during development and differentiation. Despite its significance, all reported Parp1 total knockout mice strains are viable and fertile with no developmental anomalies. It was previously believed that functional redundancy with other PARP family members explain such a controversy. PARP2 is the main candidate to fulfill PARP1 functions in Parp1 knockout mice. Indeed, Parp1 Parp2 double knockout mice are arrested early in development and unable to proceed through gastrulation. However, while PARP2 has similar catalytic domain to PARP1, it lacks other domains that play important regulatory roles, thus making the lack of developmental problems in Parp1 mice total knockouts highly unlikely. To address this question, we checked the best investigated and publicly available Parp1 knockout mice strain. We found that Parp1 is still expressed in these mice, based on the presence of its mRNA transcript. Contrary to prior assumptions, we identified persistent mRNA expression in knockout mice, albeit at reduced levels. Detailed transcript analysis revealed an alternatively spliced PARP1 variant lacking exon two with introduced artificial cassette. Subsequent protein analysis confirmed the existence of a truncated PARP1 protein in knockout mice. We established embryonic stem cell lines from knockout mice and revealed the presence of pADPr. The decreased level of pADPr was detected in knockout ES cells with Western Blotting analysis, but immunofluorescence staining didn’t detect any difference in distribution or level of pADPr in nuclei of knockout ES cells compared to control ones. Moreover, in double Parp1 Parg mutant ES cells the accumulation of pADPr greatly exceeded its amount in normal and even in hypomorph Parg mutant ES cells, suggesting the presence of functionally active PARP1. Therefore, our findings challenge the conventional understanding of PARP1 depletion effects.
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