High-resolution promoterome mining for insights transcriptional regulation of the lineage-biased hematopoietic stem cells

Supervisors:  Dr Supat Thongjuea and Prof Claus Nerlov

Synopsis

Promoters are genomic regions located in front of the transcribed part of protein-coding and non-coding RNAs. They are critical for gene regulation because they contain crucial DNA elements (i.e. TATA box), which are the sites of binding and positioning of regulatory inputs from general transcription factors required to initiate transcription. The cap analysis of gene expression (CAGE) has been introduced and successfully used for profiling active promoters in high-resolution and genome-wide scales. CAGE captures the 5’end of the transcribed and capped RNAs [Kodzius et al., Nature Methods, 2006]. It has been used to profile promoteromes of a large collection of human and mouse primary cell types known as the FANTOM5 mammalian expression atlas [Forrest et al., Nature, 2014]. Profiling of CAGE peaks reveals promoter genomic locations and expression level of protein-coding mRNAs, short and long non-coding RNAs (e.g. lincRNAs, primary miRNAs, antisense RNAs, and bidirectional enhancer RNAs), and active transposable elements. In addition, identified CAGE peaks would be able to reveal binding sites of transcription factors, which are essential to building up the transcriptional network for gene regulation. 

This project will mainly analyse the promoterome of a tiny population of hematopoietic stem cells (HSCs), a type of cells that is necessary and sufficient for replenishing all mature blood cells. We will focus on the HSCs subpopulation that has a strong platelet bias [Sanjuan-Pla et al., Nature 2013]. We aim to understand mechanisms at the molecular level about why this subpopulation of cells produces only platelets, a type of blood cells required for preventing bleeding and important for many kinds of cancer therapies. We wish to use the knowledge from this study to molecularly increase the production of platelets when needed for the cancer treatment.

We will use CAGE to profile the promoterome of platelet-biased HSCs, their counterpart control types of HSCs (i.e. balanced HSCs), and the downstream lineage committed progenitor cells. In addition, a highly sensitive profiling of histone modifications for promoters and enhancers (e.g. H3K4me3, H3K4me1, and H3K27ac) will be developed and performed. Furthermore, genome-wide chromatin accessible regions will be detected using ATAC-seq [Buenrostro et al., Nature Methods, 2013], a method for mapping chromatin accessibility genome-wide. Finally, computational approaches and a visualisation system will be developed to analyse and to integrate data from both promoteromes and epigenomes to answering key biological questions that are related to the biology of platelet-biased HSCs. The approaches described above will allow to identifying key regulatory mechanisms that control the production of platelet-biased HSCs at the transcriptional level.

To achieve the goals of the project, we aim to provide a great opportunity for training in a broad range of different techniques from the expert in the research field. These include the stem cell biology, the use of a transgenic mouse, gene regulation and transcription (in both protein-coding and non-coding RNAs biology), epigenetics, and computational biology of the high-throughput sequencing data.
This project will be based in the MRC Molecular Hematology Unit at the Weatherall Institute of Molecular Medicine, with access to state-of-the-art facilities. The project will give an opportunity for the collaboration with leaders in the field of stem cell, promoterome and epigenome analyses and will have an opportunity to be trained in the WIMM Centre for Computational Biology.

In addition to training opportunities through the University, in the WIMM, we run a course on basic techniques for new students of approximately 20 lectures. There are also courses on Immunology and Bioinformatics and others may be added. Institute seminars are held on a weekly basis and regularly attract world-class scientists in haematopoiesis research. Informal exchange of ideas in the coffee area is encouraged and is an attractive feature of the WIMM.

For further information, please contact Dr Supat Thongjuea