Role of Rad54, Rad54b and Snm1 in DNA damage repair

The aim of this thesis is to investigate the function of a number of genes involved in mammalian DNA damage repair, in particular in repair of DNA double-strand breaks (DSBs). Among a large number of different damages that can be introduced to DNA, DSBs are especially toxic. If left unrepaired, DSBs can trigger apoptosis or induce chromosomal rearrangements that can lead to carcinogenesis. Two main pathways are responsible for repair of DSBs: homologous recombination (HR) and nonhomologous end joining (NHEJ). HR is generally an error-free mechanism that restores missing information on the basis of homologous sequence obtained from sister chromatid or homologous chromosome. By contrast, NHEJ is generally error-prone. During repair by NHEJ the DNA ends can be directly ligated or short stretches of homology at the ends can be used, leading to deletions or insertions at the site of the break. A number of genes have been identified as players in DSB repair, both in prokaryotes and eukaryotes. Many of them are found in all the kingdoms of life and are similar in aspects of their sequence and function, although there are genes characteristic only for prokaryotes or eukaryotes. Many subtle differences in function also emerge from studies on HR proteins in different species. These differences might have appeared because of diverse functions repair genes have to perform in more complexed organisms, or because the initial function(s) of a gene has been distributed over multiple paralogues. Herein I concentrate on genes involved mainly in DSB repair via HR in mammalian systems. Two members of the group of HR genes are studied: Rad54 and its paralogue Rad548. Using mice and cells deficient in these genes we try to define the role of both Rad54 and Rad54B in HR and their contribution to other cellular processes. Additionally, we investigate the link between Rad54 and Snm1, a gene originally identified as being important for interstrand cross link (ICL) repair, with regard to ionizing radiation-induced DNA damage repair

The epigenetic landscape of clear-cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of all kidney tumors. During the last few years, epigenetics has emerged as an important mechanism in ccRCC pathogenesis. Recent reports, involving large-scale methylation and sequencing analyses, have identified genes frequently inactivated by promoter methylation and recurrent mutations in genes encoding chromatin regulatory proteins. Interestingly, three of detected genes (PBRM1, SETD2 and BAP1) are located on chromosome 3p, near the VHL gene, inactivated in over 80% ccRCC cases. This suggests that 3p alterations are an essential part of ccRCC pathogenesis. Moreover, most of the proteins encoded by these genes cooperate in histone H3 modifications. The aim of this review is to summarize the latest discoveries shedding light on deregulation of chromatin machinery in ccRCC. Newly described ccRCC-specific epigenetic alterations could potentially serve as novel diagnostic and prognostic biomarkers and become an object of novel therapeutic strategies

Signal Integration of IFN-I and IFN-II With TLR4 Involves Sequential Recruitment of STAT1-Complexes and NFkB to Enhance Pro-inflammatory Transcription

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Genome-wide inhibition of pro-atherogenic gene expression by multi-STAT targeting compounds as a novel treatment strategy of CVDs

Cardiovascular diseases (CVDs), including atherosclerosis, are globally the leading cause of death. Key factors contributing to onset and progression of atherosclerosis include the pro-inflammatory cytokines Interferon (IFN)a and IFN? and the Pattern Recognition Receptor (PRR) Toll-like receptor 4 (TLR4). Together, they trigger activation of Signal Transducer and Activator of Transcription (STAT)s. Searches for compounds targeting the pTyr-SH2 interaction area of STAT3, yielded many small molecules, including STATTIC and STX-0119. However, many of these inhibitors do not seem STAT3-specific. We hypothesized that multi-STAT-inhibitors that simultaneously block STAT1, STAT2, and STAT3 activity and pro-inflammatory target gene expression may be a promising strategy to treat CVDs. Using comparative in silico docking of multiple STAT-SH2 models on multi-million compound libraries, we identified the novel multi-STAT inhibitor, C01L-F03. This compound targets the SH2 domain of STAT1, STAT2, and STAT3 with the same affinity and simultaneously blocks their activity and expression of multiple STAT-target genes in HMECs in response to IFNa. The same in silico and in vitro multi-STAT inhibiting capacity was shown for STATTIC and STX-0119. Moreover, C01L-F03, STATTIC and STX-0119 were also able to affect genome-wide interactions between IFN? and TLR4 by commonly inhibiting pro-inflammatory and pro-atherogenic gene expression directed by cooperative involvement of STATs with IRFs and/or NF-κB. Moreover, we observed that multi-STAT inhibitors could be used to inhibit IFN?+LPS-induced HMECs migration, leukocyte adhesion to ECs as well as impairment of mesenteric artery contractility. Together, this implicates that application of a multi-STAT inhibitory strategy could provide great promise for the treatment of CVDsThis publication was supported by grants UMO-2015/17/B/NZ2/00967 (HB) and UMO-2015/16/T/NZ2/00055 (MS) from National Science Centre Poland. This work was supported by the KNOW RNA Research Centre in Poznan (No. 01/KNOW2/2014) and in part by PL-Grid Infrastructure (MS

STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1

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Direct Inhibition of IRF-Dependent Transcriptional Regulatory Mechanisms Associated With Disease

Interferon regulatory factors (IRFs) are a family of homologous proteins that regulate the transcription of interferons (IFNs) and IFN-induced gene expression. As such they are important modulating proteins in the Toll-like receptor (TLR) and IFN signaling pathways, which are vital elements of the innate immune system. IRFs have a multi-domain structure, with the N-terminal part acting as a DNA binding domain (DBD) that recognizes a DNA-binding motif similar to the IFN-stimulated response element (ISRE). The C-terminal part contains the IRF-association domain (IAD), with which they can self-associate, bind to IRF family members or interact with other transcription factors. This complex formation is crucial for DNA binding and the commencing of target-gene expression. IRFs bind DNA and exert their activating potential as homo or heterodimers with other IRFs. Moreover, they can form complexes (e.g., with Signal transducers and activators of transcription, STATs) and collaborate with other co-acting transcription factors such as Nuclear factor-κB (NF-κB) and PU.1. In time, more of these IRF co-activating mechanisms have been discovered, which may play a key role in the pathogenesis of many diseases, such as acute and chronic inflammation, autoimmune diseases, and cancer. Detailed knowledge of IRFs structure and activating mechanisms predisposes IRFs as potential targets for inhibition in therapeutic strategies connected to numerous immune system-originated diseases. Until now only indirect IRF modulation has been studied in terms of antiviral response regulation and cancer treatment, using mainly antisense oligonucleotides and siRNA knockdown strategies. However, none of these approaches so far entered clinical trials. Moreover, no direct IRF-inhibitory strategies have been reported. In this review, we summarize current knowledge of the different IRF-mediated transcriptional regulatory mechanisms and how they reflect the diverse functions of IRFs in homeostasis and in TLR and IFN signaling. Moreover, we present IRFs as promising inhibitory targets and propose a novel direct IRF-modulating strategy employing a pipeline approach that combines comparative in silico docking to the IRF-DBD with in vitro validation of IRF inhibition. We hypothesize that our methodology will enable the efficient identification of IRF-specific and pan-IRF inhibitors that can be used for the treatment of IRF-dependent disorders and malignancies

Genome-Wide Inhibition of Pro-atherogenic Gene Expression by Multi-STAT Targeting Compounds as a Novel Treatment Strategy of CVDs

Cardiovascular diseases (CVDs), including atherosclerosis, are globally the leading cause of death. Key factors contributing to onset and progression of atherosclerosis include the pro-inflammatory cytokines Interferon (IFN)α and IFNγ and the Pattern Recognition Receptor (PRR) Toll-like receptor 4 (TLR4). Together, they trigger activation of Signal Transducer and Activator of Transcription (STAT)s. Searches for compounds targeting the pTyr-SH2 interaction area of STAT3, yielded many small molecules, including STATTIC and STX-0119. However, many of these inhibitors do not seem STAT3-specific. We hypothesized that multi-STAT-inhibitors that simultaneously block STAT1, STAT2, and STAT3 activity and pro-inflammatory target gene expression may be a promising strategy to treat CVDs. Using comparative in silico docking of multiple STAT-SH2 models on multi-million compound libraries, we identified the novel multi-STAT inhibitor, C01L_F03. This compound targets the SH2 domain of STAT1, STAT2, and STAT3 with the same affinity and simultaneously blocks their activity and expression of multiple STAT-target genes in HMECs in response to IFNα. The same in silico and in vitro multi-STAT inhibiting capacity was shown for STATTIC and STX-0119. Moreover, C01L_F03, STATTIC and STX-0119 were also able to affect genome-wide interactions between IFNγ and TLR4 by commonly inhibiting pro-inflammatory and pro-atherogenic gene expression directed by cooperative involvement of STATs with IRFs and/or NF-κB. Moreover, we observed that multi-STAT inhibitors could be used to inhibit IFNγ+LPS-induced HMECs migration, leukocyte adhesion to ECs as well as impairment of mesenteric artery contractility. Together, this implicates that application of a multi-STAT inhibitory strategy could provide great promise for the treatment of CVDs

Suppressor of cytokine signaling and accelerated atherosclerosis in kidney disease

The prevalence of cardiovascular disease in patients with renal failure is extremely high and accounts for a large part of the morbidity and mortality. Inflammation participates importantly in host defense against infectious agents and injury, but also contributes to the pathophysiology of many diseases, including cardiovascular atherosclerosis, which is a main problem in patients with renal failure. Recruitment of blood leukocytes to the injured vascular endothelium characterizes the initiation and progression of atherosclerosis and involves many inflammatory mediators, modulated by cells of both innate and adaptive immunity. Excessive inflammatory and immune responses, communicated by these different cell types, are driven by inflammatory cytokines that promote associated tissue damage if cytokine signaling pathways remain unregulated. Thus, pathways capable of suppressing proinflammatory cytokine signaling hold the potential to limit life-threatening cardiovascular events caused by atherogenesis. Suppressor of cytokine signaling (SOCS) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine-mediated homeostasis, including innate and adaptive immunity. Accumulating evidence supports the idea that dysregulation of cytokine signaling by differential SOCS expression is involved in the pathogenesis of various inflammatory, and immunological diseases, including atherosclerosis. Based on recent observations, in which SOCS expression levels are profoundly altered in kidney disease, we discuss the possibilities of SOCS as new intracellular markers of inflammation as well as their potential atherogenic properties in renal failure related cardiovascular disease

STAT activation and differential complex formation dictate selectivity of interferon responses

Interferons (IFNs) induce gene expression by phosphorylating latent transcription factors belonging to the signal transducer and activator of transcription (STAT) family, mediated by janus kinases (Jaks). STAT dimers directly activate genes containing the IFNγ activation site (GAS) DNA element, with different STAT proteins displaying slightly different intrinsic DNA binding specificities. The combinatorial association of STATs with the additional DNA binding adaptor protein interferon regulatory factor (IRF)9 expands the range of enhancer elements that can be targeted by the JAK-STAT pathway to interferon-stimulated response element (ISRE) and IRF response element (IRE). Based on the amino-acid sequence similarity within the IRF family and functional overlap with the STAT family, in this paper we hypothesize that other IRF members could serve as adapter proteins for the STATs during IFN responses to redirect them to subsets of ISRE, GAS and/or IRE-containing IFN-stimulated genes (ISGs). In addition, the fact that STAT2 homodimers are not capable of binding consensus GAS sites leaves the possibility for a novel type of DNA-binding site bound by STAT2 homodimers and potentially other STAT complexes