A computational multiomics method for quantifying activity and regulatory mode of transcription factors and its application in leukemia

Recent breakthroughs in sequencing technologies allowed researchers to generate extensive amounts of data characterizing cellular regulation at many levels. Consequently, this boosted our understanding of gene regulatory networks responsible for different biological processes and highlighted the overall importance of transcription factors (TFs). TFs are dynamic mediators that react to both intra- and extracellular changes in order to ultimately transmit signals and execute genetically inherited gene regulatory programs in a time- and location-specific manner. However, it is still challenging to quantify in vivo TF specific binding occupancy and dynamics due to the high complexity of the regulatory part of the genome. Modern technologies measuring chromatin changes (e.g., chromatin accessibility, DNA methylation, histone modifications) can now generate testable hypotheses about the effects of TF binding on gene regulation. In this thesis, I mainly describe the novel computational tool diffTF, a multiomics data integration tool for globally assessing differential TF activity and classifying TFs into transcriptional activators and repressors (by integrating chromatin accessibility and gene expression data). We applied it to a recently published ATAC-seq dataset from a cohort of chronic lymphocytic leukemia (CLL) patients and identified dozens of differential active TFs representing two different CLL subtypes that are inherently linked to tumour progression. In addition, we integrated gene expression data from corresponding RNA-seq and were able to globally predict an activating or repressive role for 40% of the expressed TFs. We validated the approach on an independent CLL dataset and showed that the majority of TFs does not change their mode of action upon genetic or environmental perturbations. Finally, we extensively tested and benchmarked diffTF to validate its technical robustness. We also applied diffTF to a multiomics dataset from the mouse hematopoietic differentiation system and targeted potential TFs that are disturbed upon epigenetic dysregulation driven by a Tet methylcytosine dioxygenase 2 (TET2) knockout in acute myeloid leukemia (AML). TET2 plays an essential role in the cellular DNA methylation balance and is known to be frequently mutated in leukemia. We used the first high-quality TET2 binding map to identify TF families that can facilitate TET2 binding in the genome. In summary, we developed a novel hypothesis-generation computational tool that can, in a data-driven way, identify key regulators of cellular biological processes based on chromatin and expression data

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis.

Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.The study was supported by European Research Council project 677501 – ZF_Blood (to A.C. and A.M.R), EMBO small grant (to A.C.) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells.

The epigenetic regulator TET2 is frequently mutated in hematological diseases. Mutations have been shown to arise in hematopoietic stem cells early in disease development and lead to altered DNA methylation landscapes and an increased risk of hematopoietic malignancy. Here, we show by genome-wide mapping of TET2 binding sites in different cell types that TET2 localizes to regions of open chromatin and cell-type-specific enhancers. We find that deletion of Tet2 in native hematopoiesis as well as fully transformed acute myeloid leukemia (AML) results in changes in transcription factor (TF) activity within these regions, and we provide evidence that loss of TET2 leads to attenuation of chromatin binding of members of the basic helix-loop-helix (bHLH) TF family. Together, these findings demonstrate that TET2 activity shapes the local chromatin environment at enhancers to facilitate TF binding and provides an example of how epigenetic dysregulation can affect gene expression patterns and drive disease development.K.D.R. was supported by a postdoctoral fellowship from the Danish Medical Research Council (FSS 1333-00120B), K.N. was supported by Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers, JSPS (S2704), and L.S.-C. was supported by a Marie Sklodowska-Curie individual fellowship (Horizon 2020 Framework Programme, grant agreement no. H2020-MSCA-IF-2017-796341). The work in the Helin laboratory was supported by grants to K.H. from The European Research Council (294666_DNAMET), the Danish Cancer Society, the Danish National Research Foundation (DNRF82), and through a center grant from the Novo Nordisk Foundation (NNF17CC0027852). This work was also supported by the National Institutes of Health (NIH, P30 CA008748)

Drug-microenvironment perturbations reveal resistance mechanisms and prognostic subgroups in CLL

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited and systematic analyses are lacking. Using chronic lymphocytic leukaemia (CLL) as a model disease, we investigated the influence of 17 microenvironmental stimuli on 12 drugs in 192 genetically characterised patient samples. Based on microenvironmental response, we identified four subgroups with distinct clinical outcomes beyond known prognostic markers. Response to multiple microenvironmental stimuli was amplified in trisomy 12 samples. Trisomy 12 was associated with a distinct epigenetic signature. Bromodomain inhibition reversed this epigenetic profile and could be used to target microenvironmental signalling in trisomy 12 CLL. We quantified the impact of microenvironmental stimuli on drug response and their dependence on genetic alterations, identifying interleukin 4 (IL4) and Toll-like receptor (TLR) stimulation as the strongest actuators of drug resistance. IL4 and TLR signalling activity was increased in CLL-infiltrated lymph nodes compared with healthy samples. High IL4 activity correlated with faster disease progression. The publicly available dataset can facilitate the investigation of cell-extrinsic mechanisms of drug resistance and disease progression

Integration of scATAC-Seq with scRNA-Seq Data

: Single-cell studies are enabling our understanding of the molecular processes of normal cell development and the onset of several pathologies. For instance, single-cell RNA sequencing (scRNA-Seq) measures the transcriptome-wide gene expression at a single-cell resolution, allowing for studying the heterogeneity among the cells of the same population and revealing complex and rare cell populations. On the other hand, single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-Seq) can be used to define transcriptional and epigenetic changes by analyzing the chromatin accessibility at the single-cell level. However, the integration of multi-omics data still remains one of the most difficult tasks in bioinformatics. In this chapter, we focus on the combination of scRNA-Seq and scATACSeq data to perform an integrative analysis of the single-cell transcriptome and chromatin accessibility of human fetal progenitors

Integrative single-cell RNA-seq and ATAC-seq analysis of human developmental hematopoiesis

Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine. Ranzoni et al. provide a detailed transcriptional and chromatin accessibility map of fetal liver and bone marrow hematopoietic stem cells (HSCs). Within HSCs, they revealed extensive epigenetic but not transcriptional priming. They identified transcriptional and functional differences between HSCs from liver and bone marrow

Drug-microenvironment perturbations reveal resistance mechanisms and prognostic subgroups in CLL.

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited and systematic analyses are lacking. Using chronic lymphocytic leukaemia (CLL) as a model disease, we investigated the influence of 17 microenvironmental stimuli on 12 drugs in 192 genetically characterised patient samples. Based on microenvironmental response, we identified four subgroups with distinct clinical outcomes beyond known prognostic markers. Response to multiple microenvironmental stimuli was amplified in trisomy 12 samples. Trisomy 12 was associated with a distinct epigenetic signature. Bromodomain inhibition reversed this epigenetic profile and could be used to target microenvironmental signalling in trisomy 12 CLL. We quantified the impact of microenvironmental stimuli on drug response and their dependence on genetic alterations, identifying interleukin 4 (IL4) and Toll-like receptor (TLR) stimulation as the strongest actuators of drug resistance. IL4 and TLR signalling activity was increased in CLL-infiltrated lymph nodes compared with healthy samples. High IL4 activity correlated with faster disease progression. The publicly available dataset can facilitate the investigation of cell-extrinsic mechanisms of drug resistance and disease progression

Representations of rational Cherednik algebras with minimal support and torus knots

In this paper we obtain several results about representations of rational Cherednik algebras, and discuss their applications. Our first result is the Cohen-Macaulayness property (as modules over the polynomial ring) of Cherednik algebra modules with minimal support. Our second result is an explicit formula for the character of an irreducible minimal support module in type An-1 for c=mn, and an expression of its quasispherical part (i.e., the isotypic part of "hooks") in terms of the HOMFLY polynomial of a torus knot colored by a Young diagram. We use this formula and the work of Calaque, Enriquez and Etingof to give explicit formulas for the characters of the irreducible equivariant D-modules on the nilpotent cone for SLm. Our third result is the construction of the Koszul-BGG complex for the rational Cherednik algebra, which generalizes the construction of the Koszul-BGG resolution from [3] and [21], and the calculation of its homology in type A. We also show in type A that the differentials in the Koszul-BGG complex are uniquely determined by the condition that they are nonzero homomorphisms of modules over the Cherednik algebra. Finally, our fourth result is the symmetry theorem, which identifies the quasispherical components in the representations with minimal support over the rational Cherednik algebras Hmn(Sn) and Hnm(Sm). In fact, we show that the simple quotients of the corresponding quasispherical subalgebras are isomorphic as filtered algebras. This symmetry was essentially established in [8] in the spherical case, and in [24] in the case GCD(m, n)=1, and it has a natural interpretation in terms of invariants of torus knots