Genome-wide chromatin interactions in the mouse brain in vivo – establishment and validation of Hi-C protocol

Funding: DSM, Polish Ministry of Science and Higher Education
Project period: 2018 - 2019
Project leader: Agnieszka Kraft, Joanna Bem (Laboratory of Molecular Neurobiology, CeNT), Chaitali Chakraborty (Laboratory of Molecular Neurobiology, CeNT)

During embryonic development neurons and skin fibroblasts are derived from the same germ layer - ectoderm. Divergent destinies of these cell lineages are a result of separate gene expression programs, precisely regulated throughout development and further in the adult organisms. Neural cell differentiation, migration and establishment of connectivity network leads to the formation of highly specialized organ - the brain. Neurons in different brain regions differ substantially in gene expression patterns (Hobert, 2016). We are interested in genetic programs responsible for the development of the thalamus, a vertebrate brain structure that is essential for processing sensory and motor information and producing adaptive behavior in concert with the cerebral cortex. Perturbed development of the thalamus can lead to many mental diseases among them autism spectrum, bipolar disorder or schizophrenia. We are working on the identification of the components involved in proper thalamus development. The RNA-seq analysis performed in embryonic and adult thalamus allowed us recently to identify gene expression profiles involved in subsequent steps of thalamic development and a critical role of the TCF7L2 transcription factor in regulating thalamus-specific genes involved in neuronal excitability (Nagalski et al., 2013; Lipiec, in preparation).
Identification of gene expression profiles in various developmental stages of the thalamus opens new questions, e.g. about the exact regulatory mechanisms responsible for observed differences. We would like to address this question with analysing chromosomal landscapes. Chromosomes packed in the nucleus are organized in dynamic loops and knots that affect genomic expression. Distant DNA sequences can be brought into spatial proximity allowing promoter-enhancer interaction and induction of transcription. In contrary, looping away sequences required for gene expression will result in the inhibition of transcription (Mora, Sandve, Gabrielsen & Eskeland, 2016). In this project we are planning to use a Hi-C method that allows the identification of DNA sequences that are presumably interacting with each other (Lieberman-Aiden et al., 2009). This method combines proximity based ligation with massively parallel sequencing permitting identification of open and closed chromatin and chromosome territories. The interactions between regulatory sequences may be also validated, or new regulatory sequences discovered. Because tissues consist of many cells that may differ in gene expression and chromosome structure, performing the Hi-C on such samples is highly complex. Chromosome structure is known to be highly affected by cell cycle (Nagano et al., 2017). Neurons that are present in the adult mouse thalamus are postmitotic and the gene expression is more stable as compared to earlier developmental stages. This is why we have decided to optimize this protocol in the thalamus from the adult mouse brain.

 

Literature:

Hobert, O. (2016). Terminal Selectors of Neuronal Identity. Current Topics in Developmental Biology, 116, 455-475. https://doi.org/10.1016/BS.CTDB.2015.12.007