Biology subjects

Ohishi, H.

Publications and source records attributed to Ohishi, H..

3 recordsLinked to original sources

bioRxiv2023

Precise immunofluorescence canceling for highly multiplexed imaging capturing specific cell state

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and inter-cellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we developed Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

cell biology↗

bioRxiv2023

Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

DNA methyltransferase 3A (DNMT3A) and its catalytically inactive cofactor DNA methyltransferase 3-Like (DNMT3L) proteins form functional heterotetramers to deposit DNA methylation in mammalian germ cells. While both proteins have an ATRX-DNMT3-DNMT3L (ADD) domain that recognizes histone H3 tail unmethylated at lysine-4 (H3K4me0), the combined and differential roles of the domains in the two proteins have not been fully defined in vivo. Here we investigate DNA methylation landscapes in female and male germ cells derived from mice with loss-of-function amino acid substitutions in the ADD domains of DNMT3A and/or DNMT3L. Mutations in either the DNMT3A-ADD or the DNMT3L-ADD domain moderately decreases global CG methylation levels, but to different degrees, in both germ cells. Furthermore, when the ADD domains of both DNMT3A and DNMT3L lose their functions, the CG methylation levels are much more reduced, especially in oocytes, comparable to the impact of the Dnmt3a/3L knockout. In contrast, aberrant accumulation of non-CG methylation occurs at thousands of genomic regions in the double mutant oocytes and spermatozoa. These results highlight the critical role of the ADD-H3K4me0 binding in proper CG and non-CG methylation in germ cells and the various impacts of the ADD domains of the two proteins.

cell biology↗

bioRxiv2022

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

molecular biology↗