Spatial Localization of Genes Determined by Intranuclear DNA Fragmentation with the Fusion Proteins Lamin KRED and Histone KRED und Visible Light

Abstract

The highly organized DNA architecture inside of the nuclei of cells is accepted in the scientific world. In the human genome about 3 billion nucleotides are organized as chromatin in the cell nucleus. In general, they are involved in gene regulation and transcription by histone modification. Small chromosomes are localized in a central nuclear position whereas the large chromosomes are peripherally positioned. In our experiments we inserted fusion proteins consisting of a component of the nuclear lamina (lamin B1) and also histone H2A, both combined with the light inducible fluorescence protein KillerRed (KRED). After activation, KRED generates reactive oxygen species (ROS) producing toxic effects and may cause cell death. We analyzed the spatial damage distribution in the chromatin after illumination of the cells with visible light. The extent of DNA damage was strongly dependent on its localization inside of nuclei.The ROS activity allowed to gain information about the location of genes and their functions via sequencing and data base analysis of the double strand breaks of the isolated DNA. A connection between the damaged gene sequences and some diseases was found.

Advances in the Genetically Engineered KillerRed for Photodynamic Therapy Applications

“…In addition to cellular senescence, oxidative stress caused by activated nuc-KR is also an efficient approach for tumor elimination. Previous studies have demonstrated that KillerRed fusing with histone 2A (H2A) or nuclear lamina protein B1 can trigger cell cycle arrest, increase the rupture of the DNA strand, and eventually kill the tumor cell [ 45 , 46 , 59 ]. Moreover, nuc-KR fusing with a tet-repressor (tetR) or transcription-activator (TA) in U2OS cells can produce ROS and cause heterochromatin or euchromatin damage after illumination.…”

Photodynamic Physiology—Photonanomanipulations in Cellular Physiology with Protein Photosensitizers

“…The fusion protein TRF1-KillerRed has been used to target the telomeres (Sun et al, 2015). KillerRed fusion proteins (laminB1-KillerRed/histone2A-KillerRed) have been used to determine the spatial localization of chromosomal genes in the cell nucleus (Waldeck et al, 2013). The histone fusion protein H2B-KillerRed can be used to exert photodynamic blockade of cell division (Serebrovskaya et al, 2011; Shirmanova et al, 2015).…”

“…KillerRed fusion-expressed either with the peripheral nuclear protein lamin B1 (KRed-Lamin B1) or with the diffusely distributed chromosomal protein H2A (H2A-KRed) was used to identify gene damages after photodynamic action. The extent of photo-oxidative damage was used to delineate the spatial localization of the respective genes and the gene-carrying chromosomes in the nucleus (Waldeck et al, 2013). KillerRed fused to a tet-repressor (tetR-KR) or a transcription activator (TA-KR) have been used to target oxidative stress to hetero- or euchromatin respectively in U2OS cells (Lan et al, 2014).…”

Cholecystokinin 1 Receptor – A Unique G Protein-Coupled Receptor Activated by Singlet Oxygen (GPCR-ABSO)

“…Genetically encoded KillerRed or miniSOG target-expressed with signal sequence tags at specific subcellular organelles ( Qi et al, 2012 ; Ryumina et al, 2013 ; Jarvela and Linstedt, 2014 ; Serebrovskaya et al, 2014 ) or by fusion-expression with target proteins ( Serebrovskaya et al, 2011 ; Lin et al, 2013 ; Waldeck et al, 2013 ; Zhou et al, 2013 ; Sun et al, 2015 ) in specific cell types under tissue-specific promoters ( Lin et al, 2013 ; Zhou et al, 2013 ) have been shown to be highly efficient precisely localized photosensitizers. Such cellular organelle-delimited protein photosensitizers after light irradiation would generate 1 O 2 locally in spatially defined fashion and photooxidize nanoscopically (<10 nm) target proteins such as plasma membrane CCK1R.…”