In this episode of the Epigenetics Podcast, we caught up with Dr. Hodaka Fujii, Professor of Biochemistry and Genome Biology at Hirosaki University Graduate School of Medicine and School of Medicine, to talk about his work on the development of locus-specific ChIP technologies.
The goal of conventional chromatin immunoprecipitation (ChIP) assays is to find genomic locations of transcription factor binding or genome-wide profiles of histone tail modifications. In contrast to that, the guest of this episode, Dr. Fujii, has developed methods such as insertional chromatin immunoprecipitation (iChIP) and engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) to identify the factors that are binding to specific sites on the genome.
In iChIP, LexA binding sites are inserted into the genomic region of interest. In parallel, the DNA-binding domain of LexA, fused with FLAG epitope tags and a nuclear localization signal, is expressed in the same cells. After crosslinking and chromatin preparation, the resulting chromatin is immunoprecipitated with an antibody against the tag. This allows proteins or RNA interacting with the region of interest to be analyzed with the appropriate downstream application. The enChIP takes a similar approach, but does not require insertion of the LexA binding sites. Instead, a FLAG-tagged dCas9 protein together with the respective guide RNA are used to target the region of the genome of interest. After the IP and the purification DNA, RNA, or proteins can be analyzed accordingly. The lack of the requirement of to insert the LexA binding sites into the genome makes enChIP much more straightforward than iChIP.
In this interview, we discuss the story behind how Dr. Fujii got into the field of epigenetics, how he developed iChIP, and how the method was improved over the years. Furthermore, we discuss the development of enChIP and how this can be used as an alternate method to Hi-C.
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Characterizing Chromatin at the Nuclear Lamina (Bas van Steensel)
Nucleosome Positioning in Cancer Diagnostics (Vladimir Teif)
The Effect of Histone Demethylases on Gene Expression and Cancer Cell Stability (Johnathan Whetstine)
Stem Cell Transcriptional Regulation in Naive vs. Primed Pluripotency (Christa Buecker)
Gene Expression Control and Intricacies of X-chromosome Inactivation (Claire Rougeulle)
Epigenetic Mechanisms in Genome Regulation and Developmental Programming (James Hackett)
Advanced Optical Imaging in 3D Nuclear Organisation (Lothar Schermelleh)
DNA Replication, Transcription and R-loops (Stephan Hamperl)
Mutations of Gene Regulatory Elements in Human Disease (Nadav Ahituv)
Using Single-Cell Multiomics to Characterize Human Developmental Hematopoiesis (Ana Cvejic)
The Impact of Sequence Variation on Transcription Factor Binding (Sven Heinz)
Comparing CUT&Tag to ENCODE ChIP-Seq in Alzheimer's Disease Samples (Sarah Marzi)
The Role of Hat1p in Chromatin Assembly (Mark Parthun)
The Impact of Paternal Diet on Offspring Metabolism (Upasna Sharma)
H3K36me3, H4K16ac and Cryptic Transcription in Ageing (Weiwei Dang)
Split-Pool Recognition of Interactions by Tag Extension (SPRITE) (Mitch Guttman)
MLL Proteins in Mixed-Lineage Leukemia (Yali Dou)
Sex-biased Imprinting and DNA Regulatory Landscapes During Reprogramming (Sam Buckberry)
BET Proteins and Their Role in Chromosome Folding and Compartmentalization (Kyle Eagen)
Epigenetic Underpinnings of Human Addiction (Francesca Telese & Jessica Zhou)
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