Implementation of SLIC-CAGE and ATAC-seq genome-wide assays to map active regulatory regions in breast cancer cell lines

INTRODUCTION
Gene expression is a tightly regulated process with a coordinated action of various players at different layers [1,2]. This process is known to be dysregulated in several diseases such as cancers [3]. Among the various players that orchestrate the proper regulation of gene expression are transcription factors (TFs), which are proteins that interact with DNA in a sequence specific manner. TFs control gene regulation by specifically binding to non-coding regulatory regions such as (1) promoters, proximal to the transcription start sites of genes, and (2) enhancers, distal to the genes they control [4].

AIM
The aim of the master project is to identify regulatory regions specifically active in selected breast cancer cell lines. The Master candidate(s) will implement a recent state-of-the art molecular method, SLIC-CAGE (Super-Low Input Carrier-CAGE) developed by our collaborators [5], to capture active promoters and enhancers in breast cancer cell lines. In addition, the Master project aims at profiling the chromatin accessibility landscape of the same cell lines using ATAC-sequencing [6,7] to analyze TF binding footprints at active enhancer and promoter regions. This project is part of a larger scientific endeavor with the ultimate goal to map active regulatory regions in breast cancer patient samples to provide an improved stratification of breast cancer patients for better treatment outcomes.

METHODOS
The student(s) will be involved in the implementation of the recent molecular assay:
SLIC-CAGE, which allows for capturing the 5¡¯ end of capped RNAs in cells from a small amount of input material (as low as 5-10 ng of RNAs) [5]. As such, this recent technique captures active promoters and enhancers at base pair resolution. The assay will be applied on total RNA isolated from a panel of breast cancer and normal breast cell lines. The selected Master student(s) will be responsible for generating the SLIC-CAGE library in two selected breast cancer lines along with a normal breast cell line. In addition to generating SLIC-CAGE, the student(s) will perform ATAC-seq (Assay for Transposase Accessible Chromatin followed by high-throughput sequencing). Finally, there will be the opportunity to get a bioinformatics training to analyze the ATAC-seq and CAGE data generated. This project can accommodate two master students who will work in parallel.

LEARNING OUTCOME
The student(s), in addition to being exposed to an interdisciplinary research environment, where computational and experimental methods harmoniously work together to answer important scientific questions, will be trained with the following set of knowledge and skills:

• Buffer preparation, laboratory safety, safe handling of chemicals and reagents
• RNA isolation, cDNA synthesis, PCR (Polymerase Chain Reaction)
• Improved understanding of gene expression regulation
• Eukaryotic cell culture techniques and principles of cell culture and cell biology
• Library preparation skills along with state of the art genome-wide method for precisely mapping active promoter and enhancer regions (nAnT-iCAGE and SLIC-CAGE)
• Library preparation skill for profiling chromatin accessibility using ATAC-seq
• High throughput sequencing principles
• Other routine molecular biology and general laboratory techniques
• Bioinformatics analysis of ATAC-seq and SLIC-CAGE data

HOST ENVIRONMENT
The selected candidate(s) will be part of the Computational Biology and Gene Regulation group, Centre for Molecular Medicine Norway (NCMM), UiO, led by Dr. Anthony Mathelier. The group is a combined dry and wet laboratory that studies gene expression regulation and how it is disrupted in diseases like cancers. Main supervision will be provided by Dr. Mathelier with close day-to-day supervision from Dr. Roza Berhanu Lemma and Dina Aronsen, molecular biology experts. Moreover, internal co-supervision will be provided by Prof. Rein Aasland at IBV.

REFERENCES
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