Effect of NK-5962 on Gene Expression Profiling of Retina in a Rat Model of Retinitis Pigmentosa

Purpose: NK-5962 is a key component of photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis (OUReP&trade;). Previously, we found that NK-5962 solution could reduce the number of apoptotic photoreceptors in the eyes of the Royal College of Surgeons (RCS) rats by intravitreal injection under a 12 h light/dark cycle. This study aimed to explore possible molecular mechanisms underlying the anti-apoptotic effect of NK-5962 in the retina of RCS rats. Methods: RCS rats received intravitreal injections of NK-5962 solution in the left eye at the age of 3 and 4 weeks, before the age of 5 weeks when the speed in the apoptotic degeneration of photoreceptors reaches its peak. The vehicle-treated right eyes served as controls. All rats were housed under a 12 h light/dark cycle, and the retinas were dissected out at the age of 5 weeks for RNA sequence (RNA-seq) analysis. For the functional annotation of differentially expressed genes (DEGs), the Metascape and DAVID databases were used. Results: In total, 55 up-regulated DEGs, and one down-regulated gene (LYVE1) were found to be common among samples treated with NK-5962. These DEGs were analyzed using Gene Ontology (GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. We focused on the up-regulated DEGs that were enriched in extracellular matrix organization, extracellular exosome, and PI3K–Akt signaling pathways. These terms and pathways may relate to mechanisms to protect photoreceptor cells. Moreover, our analyses suggest that SERPINF1, which encodes pigment epithelium-derived factor (PEDF), is one of the key regulatory genes involved in the anti-apoptotic effect of NK-5962 in RCS rat retinas. Conclusions: Our findings suggest that photoelectric dye NK-5962 may delay apoptotic death of photoreceptor cells in RCS rats by up-regulating genes related to extracellular matrix organization, extracellular exosome, and PI3K–Akt signaling pathways. Overall, our RNA-seq and bioinformatics analyses provide insights in the transcriptome responses in the dystrophic RCS rat retinas that were induced by NK-5962 intravitreal injection and offer potential target genes for developing new therapeutic strategies for patients with retinitis pigmentosa.</jats:p

The Effect of Cyanine Dye NK-4 on Photoreceptor Degeneration in a Rat Model of Early-Stage Retinitis Pigmentosa

The present study aimed to evaluate the effects of NK-4 on the apoptosis of photoreceptors in a rat model of retinitis pigmentosa and explore the mechanism underlying anti-apoptosis activity. The Royal College of Surgeons (RCS) rats received an intravitreous injection of NK-4 solution in the left eye and vehicle control in the right eye. Apoptosis was detected by TUNEL method in frozen sections of the eyes. The retinal tissues of the rats were dissected for RNA-seq analysis. Functional and pathway enrichment analyses of differentially expressed genes (DEGs) were performed by using Metascape and DAVID software. The expression levels of DEGs were confirmed by real-time quantitative PCR (RT-qPCR). The number of apoptotic cells decreased in the outer nuclear layer (ONL) and the thickness of the ONL was significantly thicker in the retina of NK-4-injected eyes, compared with control eyes. Five DEGs were identified by RNA-seq analysis, and Hmox1, Mt1, Atf5, Slc7a11, and Bdh2 were confirmed to be up-regulated by RT-qPCR. Functional and pathway enrichment analysis of the up-regulated genes showed that anti-apoptosis effects of NK-4 in the retina of RCS rats may be related to the pathways of metal ion homeostasis, negative regulation of neuron death, response to toxic substance, and pigment metabolic process. We found a potential mechanism of NK-4, providing a new viewpoint for the development of more therapeutic uses of NK-4 in the future

Candidate Genes in Testing Strategies for Linkage Analysis and Bioinformatic Sorting of Whole Genome Sequencing Data in Three Small Japanese Families with Idiopathic Superior Oblique Muscle Palsy

Idiopathic superior oblique muscle palsy is a major type of paralytic, non-comitant strabismus and presents vertical and cyclo-torsional deviation of one eye against the other eye, with a large vertical fusion range and abnormal head posture such as head tilt. Genetic background is considered to play a role in its development, as patients with idiopathic superior oblique muscle palsy have varying degrees of muscle hypoplasia and, rarely, the complete absence of the muscle, that is, aplasia. In this study, whole genome sequencing was performed, and single nucleotide variations and short insertions/deletions (SNVs/InDels) were annotated in two patients each in three small families (six patients in total) with idiopathic superior oblique muscle palsy, in addition to three normal individuals in one family. At first, linkage analysis was carried out in the three families and SNVs/InDels in chromosomal loci with negative LOD scores were excluded. Next, SNVs/InDels shared by the six patients, but not by the three normal individuals, were chosen. SNVs/InDels were further narrowed down by choosing low-frequency (&lt;1%) or non-registered SNVs/InDels in four databases for the Japanese population, and then by choosing SNVs/InDels with functional influence, leading to one candidate gene, SSTR5-AS1 in chromosome 16. The six patients were heterozygous for 13-nucleotide deletion in SSTR5-AS1, except for one homozygous patient, while the three normal individuals were wild type. Targeted polymerase chain reaction (PCR) and direct sequencing of PCR products confirmed the 13-nucleotide deletion in SSTR5-AS1. In the face of newly-registered SSTR5-AS1 13-nucleotide deletion at a higher frequency in a latest released database for the Japanese population, the skipping of low-frequency and non-registration sorting still resulted in only 13 candidate genes including SSTR5-AS1 as common variants. The skipping of linkage analysis also led to the same set of 13 candidate genes. Different testing strategies that consisted of linkage analysis and simple unintentional bioinformatics could reach candidate genes in three small families with idiopathic superior oblique muscle palsy

Topoisomerase II beta targets DNA crossovers formed between distant homologous sites to induce chromatin opening

Type II DNA topoisomerases (topo II) flip the spatial positions of two DNA duplexes, called G- and T- segments, by a cleavage-passage-resealing mechanism. In living cells, these DNA segments can be derived from distant sites on the same chromosome. Due to lack of proper methodology, however, no direct evidence has been described so far. The beta isoform of topo II (topo II beta) is essential for transcriptional regulation of genes expressed in the final stage of neuronal differentiation. Here we devise a genome-wide mapping technique (eTIP-seq) for topo II beta target sites that can measure the genomic distance between G- and T-segments. It revealed that the enzyme operates in two distinctive modes, termed proximal strand passage (PSP) and distal strand passage (DSP). PSP sites are concentrated around transcription start sites, whereas DSP sites are heavily clustered in small number of hotspots. While PSP represent the conventional topo II targets that remove local torsional stresses, DSP sites have not been described previously. Most remarkably, DSP is driven by the pairing between homologous sequences or repeats located in a large distance. A model-building approach suggested that topo II beta acts on crossovers to unknot the intertwined DSP sites, leading to chromatin decondensation

Histone acetylation-independent transcription stimulation by a histone chaperone

Histone chaperones are thought to be important for maintaining the physiological activity of histones; however, their exact roles are not fully understood. The physiological function of template activating factor (TAF)-I, one of the histone chaperones, also remains unclear; however, its biochemical properties have been well studied. By performing microarray analyses, we found that TAF-I stimulates the transcription of a sub-set of genes. The transcription of endogenous genes that was up-regulated by TAF-I was found to be additively stimulated by histone acetylation. On performing an experiment with a cell line containing a model gene integrated into the chromosome, TAF-I was found to stimulate the model gene transcription in a histone chaperone activity-dependent manner additively with histone acetylation. TAF-I bound to the core histones and remodeled the chromatin structure independent of the N-terminal histone tail and its acetylation level in vitro. These results suggest that TAF-I remodel the chromatin structure through its interaction with the core domain of the histones, including the histone fold, and this mechanism is independent of the histone acetylation status

Nuclear dynamics of topoisomerase II beta reflects its catalytic activity that is regulated by binding of RNA to the C-terminal domain

DNA topoisomerase II (topo II) changes DNA topology by cleavage/re-ligation cycle(s) and thus contributes to various nuclear DNA transactions. It is largely unknown how the enzyme is controlled in a nuclear context. Several studies have suggested that its C-terminal domain (CTD), which is dispensable for basal relaxation activity, has some regulatory influence. In this work, we examined the impact of nuclear localization on regulation of activity in nuclei. Specifically, human cells were transfected with wild-type and mutant topo II beta tagged with EGFP. Activity attenuation experiments and nuclear localization data reveal that the endogenous activity of topo II beta is correlated with its subnuclear distribution. The enzyme shuttles between an active form in the nucleoplasm and a quiescent form in the nucleolus in a dynamic equilibrium. Mechanistically, the process involves a tethering event with RNA. Isolated RNA inhibits the catalytic activity of topo II beta in vitro through the interaction with a specific 50-residue region of the CTD (termed the CRD). Taken together, these results suggest that both the subnuclear distribution and activity regulation of topo II beta are mediated by the interplay between cellular RNA and the CRD

ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen

ATM遺伝子変異による乳癌発症機構を解明 --HBOC症候群の乳腺特異的発癌機構の解明に貢献--. 京都大学プレスリリース. 2023-02-09.ATM gene mutation carriers are predisposed to estrogen-receptor-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis after ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. After estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes, including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis

Topoisomerase IIβ Activates a Subset of Neuronal Genes that Are Repressed in AT-Rich Genomic Environment

DNA topoisomerase II (topo II) catalyzes a strand passage reaction in that one duplex is passed through a transient brake or gate in another. Completion of late stages of neuronal development depends on the presence of active β isoform (topo IIβ). The enzyme appears to aid the transcriptional induction of a limited number of genes essential for neuronal maturation. However, this selectivity and underlying molecular mechanism remains unknown. Here we show a strong correlation between the genomic location of topo IIβ action sites and the genes it regulates. These genes, termed group A1, are functionally biased towards membrane proteins with ion channel, transporter, or receptor activities. Significant proportions of them encode long transcripts and are juxtaposed to a long AT-rich intergenic region (termed LAIR). We mapped genomic sites directly targeted by topo IIβ using a functional immunoprecipitation strategy. These sites can be classified into two distinct classes with discrete local GC contents. One of the classes, termed c2, appears to involve a strand passage event between distant segments of genomic DNA. The c2 sites are concentrated both in A1 gene boundaries and the adjacent LAIR, suggesting a direct link between the action sites and the transcriptional activation. A higher-order chromatin structure associated with AT richness and gene poorness is likely to serve as a silencer of gene expression, which is abrogated by topo IIβ releasing nearby genes from repression. Positioning of these genes and their control machinery may have developed recently in vertebrate evolution to support higher functions of central nervous system

Selective DNA-binding of SP120 (rat ortholog of human hnRNP U) is mediated by arginine-glycine rich domain and modulated by RNA

A human protein heterogeneous ribonucleoprotein U (hnRNP U) also known as Scaffold attachment factor A (SAF-A) and its orthologous rat protein SP120 are abundant and multifunctional nuclear protein that directly binds to both DNA and RNA. The C-terminal region of hnRNP U enriched with arginine and glycine is essential for the interaction with RNA and the N-terminal region of SAF-A termed SAP domain has been ascribed to the DNA binding. We have reported that rat hnRNP U specifically and cooperatively binds to AT-rich DNA called nuclear scaffold/matrix-associated region (S/MAR) although its detailed mechanism remained unclear. In the present study analysis of hnRNP U deletion mutants revealed for the first time that a C-terminal domain enriched with Arg-Gly (defined here as 'RG domain') is predominantly important for the S/MAR-selective DNA binding activities. RG domain alone directly bound to S/MAR and coexistence with the SAP domain exerted a synergistic effect. The binding was inhibited by netropsin, a minor groove binder with preference to AT pairs that are enriched in S/MAR, suggesting that RG domain interacts with minor groove of S/MAR DNA. Interestingly, excess amounts of RNA attenuated the RG domain-dependent S/MAR-binding of hnRNP U. Taken together, hnRNP U may be the key element for the RNA-regulated recognition of S/MAR DNA and thus contributing to the dynamic structural changes of chromatin compartments