Periodic Reporting for period 1 - DNAmethAML (Investigation of aberrant DNA methylation in malignant haematopoiesis)
Reporting period: 2018-01-01 to 2019-12-31
Acute myeloid leukaemia (AML) is a rapidly progressing haematopoietic malignancy, which is caused by diverse genetic aberrations. It is one of the most common myeloid malignancies with poor prognosis: the 5-year survival rate is only 19% for AML patients in Europe. The mutations in AML mainly occur in combinations and affect signaling molecules as well as important transcriptional and epigenetic regulators. High percentage of AML patients carries mutations in enzymes regulating DNA methylation and/or demethylation. Since DNA methylation is a reversible mark, it is a promising target for treatment of myeloid malignancies. However, to develop more efficient therapies there is a need to investigate potential functions of genome methylation and how its misregulation contributes to disease on mechanistic level.
TET2 is one of the most-frequently mutated genes in AML (12-32%) and other haematopoietic disorders. It catalyses removal of DNA methylation mark. Several studies have shown that loss of TET2 function impedes the normal haematopoietic program and promotes self-renewal. Enhancers are specialized DNA regions that are bound by transcription factors that remotely target genes. Active enhancers do not usually have DNA methylation, suggesting that demethylation can be crucial for their function. However, the mechanism of how TET2 influences enhancers’ function is not known. Thus, the goal of my proposal is to investigate aberrant DNA methylation on enhancers in malignant haematopoiesis and to explore its functional role. To attain it, I have set the following objectives: 1) Identification and functional validation of methylation-sensitive enhancers; 2) Identification and characterization of methylation-sensitive transcription factors involved in stem cell self-renewal in leukaemogenesis.
TET2 is one of the most-frequently mutated genes in AML (12-32%) and other haematopoietic disorders. It catalyses removal of DNA methylation mark. Several studies have shown that loss of TET2 function impedes the normal haematopoietic program and promotes self-renewal. Enhancers are specialized DNA regions that are bound by transcription factors that remotely target genes. Active enhancers do not usually have DNA methylation, suggesting that demethylation can be crucial for their function. However, the mechanism of how TET2 influences enhancers’ function is not known. Thus, the goal of my proposal is to investigate aberrant DNA methylation on enhancers in malignant haematopoiesis and to explore its functional role. To attain it, I have set the following objectives: 1) Identification and functional validation of methylation-sensitive enhancers; 2) Identification and characterization of methylation-sensitive transcription factors involved in stem cell self-renewal in leukaemogenesis.
The proposal relied on several genomics techniques (bisulfite sequencing and ATAC-seq) as well as computational analysis of CRISPR screens that required extensive optimization in order to achieve satisfactory results using rare cell types. Moreover, I obtained relevant knock-out cell lines (without DNA methylation and without DNA hydroxymethylation) that allowed me to assess all methylation-sensitive techniques and verify project outcomes.
To achieve optimal results and adequate coverage for whole-genome bisulfite sequencing, I tested several commercial kits for the conversion step as well as for library generation. I showed that each of those steps had great impact on mapping efficiencies, thus directly influenced the cost of sequencing. My optimized protocol allows efficient generation of sequencing libraries from 50-300 ng of genomic DNA with 70-80% mapping efficiencies.
TET enzymes catalyze conversion of methylated cytosines (5meC) to hydroxymethylated cytosines (5hmeC). Meaning, that amount and genomic distribution 5hmeC is a direct functional read-out of TET proteins activity unlike 5meC. However, hydroxymethylation detection techniques are not as widely used as DNA methylation assays. I adopted oxidative bisulfite sequencing (oxBS-seq) that differs from a traditional bisulfate sequencing (BS-seq) by one step (in vitro oxidation of 5meC to 5hmeC). Sensitivity and specificity of the assay was assessed by using knock-out cells with no DNA methylation as well as cells with no DNA hydromethylation.
Data processing and analysis are considered one of the bottlenecks in biological research. I created a pipeline for CRISPR data mapping and analysis as well as other NGS data workflows, thus making data investigation and interpretation faster and more productive.
To achieve optimal results and adequate coverage for whole-genome bisulfite sequencing, I tested several commercial kits for the conversion step as well as for library generation. I showed that each of those steps had great impact on mapping efficiencies, thus directly influenced the cost of sequencing. My optimized protocol allows efficient generation of sequencing libraries from 50-300 ng of genomic DNA with 70-80% mapping efficiencies.
TET enzymes catalyze conversion of methylated cytosines (5meC) to hydroxymethylated cytosines (5hmeC). Meaning, that amount and genomic distribution 5hmeC is a direct functional read-out of TET proteins activity unlike 5meC. However, hydroxymethylation detection techniques are not as widely used as DNA methylation assays. I adopted oxidative bisulfite sequencing (oxBS-seq) that differs from a traditional bisulfate sequencing (BS-seq) by one step (in vitro oxidation of 5meC to 5hmeC). Sensitivity and specificity of the assay was assessed by using knock-out cells with no DNA methylation as well as cells with no DNA hydromethylation.
Data processing and analysis are considered one of the bottlenecks in biological research. I created a pipeline for CRISPR data mapping and analysis as well as other NGS data workflows, thus making data investigation and interpretation faster and more productive.
The main impact of the work is the generation and dissemination of knowledge. I tested and optimized state-of-the-art approaches as well as established computational pipelines. The intermediate results were disseminated via presentations and data/protocols sharing within the laboratory and the institute.