Time-Resolved Expression Profiling of the Nuclear Receptor Superfamily in Human Adipogenesis

Background: The differentiation of fibroblast-like pre-adipocytes to lipid-loaded adipocytes is regulated by a network of transcription factors, the most prominent one being the nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma. However, many of the other 47 members of the nuclear receptor superfamily have an impact on adipogenesis, which in human cells has not been investigated in detail. Methodology/Principal Findings: We analyzed by quantitative PCR all human nuclear receptors at multiple time points during differentiation of SGBS pre-adipocytes. The earliest effect was the down-regulation of the genes RARG, PPARD, REVERBA, REV-ERBB, VDR and GR followed by the up-regulation of PPARG, LXRA and AR. These observations are supported with data from 3T3-L1 mouse pre-adipocytes and primary human adipocytes. Investigation of the effects of the individual differentiation mix components in short-term treatments and of their omission from the full mix showed that the expression levels of the early-regulated nuclear receptor genes were most affected by the glucocorticoid receptor (GR) ligand cortisol and the phosophodiesterase inhibitor IBMX. Interestingly, the effects of both compounds converged to repress the genes PPARD, REV-ERBA, REV-ERBB, VDR and GR, whereas cortisol and IBMX showed antagonistic interaction for PPARG, LXRA and AR causing a time lag in their up-regulation. We hypothesize that the well-known auto-repression of GR fine-tunes the detected early responses. Consistently, chromatin immunoprecipitation experiments showed that GR association increased on the transcription start sites of the genes RARG, REV-ERBB, VDR and GR. Conclusions/Significance: Adipocyte differentiation is a process, in which many members of the nuclear receptor superfamily change their mRNA expression. The actions of cortisol and IBMX converged to repress several nuclear receptors early in differentiation, while up-regulation of other nuclear receptor genes showed a time lag due to antagonisms of the signals. Our results place GR and its ligand cortisol as central regulatory factors controlling early regulatory events in human adipogenesis that precedes the regulation of the later events by PPARG

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities

Background Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. Methods We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. Results We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. Conclusions Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities

Background: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. Methods: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. Results: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. Conclusions: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.publishedVersionPeer reviewe

In vivo interrogation of regulatory genomes reveals extensive quasi-insufficiency in cancer evolution.

In contrast to mono- or biallelic loss of tumor-suppressor function, effects of discrete gene dysregulations, as caused by non-coding (epi)genome alterations, are poorly understood. Here, by perturbing the regulatory genome in mice, we uncover pervasive roles of subtle gene expression variation in cancer evolution. Genome-wide screens characterizing 1,450 tumors revealed that such quasi-insufficiency is extensive across entities and displays diverse context dependencies, such as distinct cell-of-origin associations in T-ALL subtypes. We compile catalogs of non-coding regions linked to quasi-insufficiency, show their enrichment with human cancer risk variants, and provide functional insights by engineering regulatory alterations in mice. As such, kilo-/megabase deletions in a Bcl11b-linked non-coding region triggered aggressive malignancies, with allele-specific tumor spectra reflecting gradual gene dysregulations through modular and cell-type-specific enhancer activities. Our study constitutes a first survey toward a systems-level understanding of quasi-insufficiency in cancer and gives multifaceted insights into tumor evolution and the tissue-specific effects of non-coding mutations