SGOL1-AS1 enhances cell survival in acute myeloid leukemia by maintaining pro-inflammatory signaling.

Epigenetic dysregulation is a key feature of most acute myeloid leukemia (AML). Recently, it has become clear that long noncoding RNAs (lncRNAs) can play a key role in epigenetic regulation, and consequently also dysregulation. Currently, our understanding of the requirements and roles of lncRNAs in AML is still limited. Here, using CRISPRi screening, we identified the lncRNA SGOL1-AS1 as an essential regulator of survival in THP-1 AML cells. We demonstrated that SGOL1-AS1 interacts with chromatin-modifying proteins involved in gene repression and that SGOL1-AS1 knockdown is associated with increased heterochromatin formation. We also observed that loss of SGOLl-AS1 results in increased apoptosis and the downregulation of pro-inflammatory genes. In AML patients, high expression of SGOL1-AS1 correlates with both pro-inflammatory gene expression and poor survival. Altogether, our data reveal that SGOL1-AS1 is an essential regulator of cell survival in AML cell lines and a possible regulator of pro-inflammatory signaling in AML patients.

Murine AGM single-cell profiling identifies a continuum of hemogenic endothelium differentiation marked by ACE.

In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.

Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.

Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.

Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia.

BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.

Contributions of Embryonic HSC-Independent Hematopoiesis to Organogenesis and the Adult Hematopoietic System.

During ontogeny, the establishment of the hematopoietic system takes place in several phases, separated both in time and location. The process is initiated extra-embryonically in the yolk sac (YS) and concludes in the main arteries of the embryo with the formation of hematopoietic stem cells (HSC). Initially, it was thought that HSC-independent hematopoietic YS cells were transient, and only required to bridge the gap to HSC activity. However, in recent years it has become clear that these cells also contribute to embryonic organogenesis, including the emergence of HSCs. Furthermore, some of these early HSC-independent YS cells persist into adulthood as distinct hematopoietic populations. These previously unrecognized abilities of embryonic HSC-independent hematopoietic cells constitute a new field of interest. Here, we aim to provide a succinct overview of the current knowledge regarding the contribution of YS-derived hematopoietic cells to the development of the embryo and the adult hematopoietic system.

CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.

The RUNX1b Isoform Defines Hemogenic Competency in Developing Human Endothelial Cells.

The transcription factor RUNX1 is a master regulator of blood cell specification. During embryogenesis, hematopoietic progenitors are initially generated from hemogenic endothelium through an endothelium-to-hematopoietic transition controlled by RUNX1. Several studies have dissected the expression pattern and role of RUNX1 isoforms at the onset of mouse hematopoiesis, however the precise pattern of RUNX1 isoform expression and biological output of RUNX1-expressing cells at the onset of human hematopoiesis is still not fully understood. Here, we investigated these questions using a RUNX1b:VENUS RUNX1c:TOMATO human embryonic stem cell line which allows multi-parameter single cell resolution via flow cytometry and isolation of RUNX1b-expressing cells for further analysis. Our data reveal the sequential expression of the two RUNX1 isoforms with RUNX1b expressed first in a subset of endothelial cells and during the endothelial to hematopoietic transition while RUNX1c only becomes expressed in fully specified blood cells. Furthermore, our data show that RUNX1b marks endothelial cells endowed with hemogenic potential and that RUNX1b expression level determines hemogenic competency in a dose-dependent manner. Together our data reveal the dynamic of RUNX1 isoforms expression at the onset of human blood specification and establish RUNX1b isoform as the earliest known marker for hemogenic competency.

RUNX1 Dosage in Development and Cancer.

The transcription factor RUNX1 first came to prominence due to its involvement in the t(8;21) translocation in acute myeloid leukemia (AML). Since this discovery, RUNX1 has been shown to play important roles not only in leukemia but also in the ontogeny of the normal hematopoietic system. Although it is currently still challenging to fully assess the different parameters regulating RUNX1 dosage, it has become clear that the dose of RUNX1 can greatly affect both leukemia and normal hematopoietic development. It is also becoming evident that varying levels of RUNX1 expression can be used as markers of tumor progression not only in the hematopoietic system, but also in non-hematopoietic cancers. Here, we provide an overview of the current knowledge of the effects of RUNX1 dosage in normal development of both hematopoietic and epithelial tissues and their associated cancers.

Identification of gene specific cis-regulatory elements during differentiation of mouse embryonic stem cells: An integrative approach using high-throughput datasets.

Gene expression governs cell fate, and is regulated via a complex interplay of transcription factors and molecules that change chromatin structure. Advances in sequencing-based assays have enabled investigation of these processes genome-wide, leading to large datasets that combine information on the dynamics of gene expression, transcription factor binding and chromatin structure as cells differentiate. While numerous studies focus on the effects of these features on broader gene regulation, less work has been done on the mechanisms of gene-specific transcriptional control. In this study, we have focussed on the latter by integrating gene expression data for the in vitro differentiation of murine ES cells to macrophages and cardiomyocytes, with dynamic data on chromatin structure, epigenetics and transcription factor binding. Combining a novel strategy to identify communities of related control elements with a penalized regression approach, we developed individual models to identify the potential control elements predictive of the expression of each gene. Our models were compared to an existing method and evaluated using the existing literature and new experimental data from embryonic stem cell differentiation reporter assays. Our method is able to identify transcriptional control elements in a gene specific manner that reflect known regulatory relationships and to generate useful hypotheses for further testing.

RUNX transcription factors: orchestrators of development.

RUNX transcription factors orchestrate many different aspects of biology, including basic cellular and developmental processes, stem cell biology and tumorigenesis. In this Primer, we introduce the molecular hallmarks of the three mammalian RUNX genes, RUNX1, RUNX2 and RUNX3, and discuss the regulation of their activities and their mechanisms of action. We then review their crucial roles in the specification and maintenance of a wide array of tissues during embryonic development and adult homeostasis.

Regulation of RUNX1 dosage is crucial for efficient blood formation from hemogenic endothelium.

During ontogeny, hematopoietic stem and progenitor cells arise from hemogenic endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in hemogenic endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects hemogenic endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFß allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in hemogenic endothelium maturation and the establishment of the hematopoietic system.

Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate.

The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.

Interplay between SOX7 and RUNX1 regulates hemogenic endothelial fate in the yolk sac.

Endothelial to hematopoietic transition (EHT) is a dynamic process involving the shutting down of endothelial gene expression and switching on of hematopoietic gene transcription. Although the factors regulating EHT in hemogenic endothelium (HE) of the dorsal aorta have been relatively well studied, the molecular regulation of yolk sac HE remains poorly understood. Here, we show that SOX7 inhibits the expression of RUNX1 target genes in HE, while having no effect on RUNX1 expression itself. We establish that SOX7 directly interacts with RUNX1 and inhibits its transcriptional activity. Through this interaction we demonstrate that SOX7 hinders RUNX1 DNA binding as well as the interaction between RUNX1 and its co-factor CBFß. Finally, we show by single-cell expression profiling and immunofluorescence that SOX7 is broadly expressed across the RUNX1+ yolk sac HE population compared with SOX17. Collectively, these data demonstrate for the first time how direct protein-protein interactions between endothelial and hematopoietic transcription factors regulate contrasting transcriptional programs during HE differentiation and EHT.

New insights into the regulation by RUNX1 and GFI1(s) proteins of the endothelial to hematopoietic transition generating primordial hematopoietic cells.

The first hematopoietic cells are generated very early in ontogeny to support the growth of the embryo and to provide the foundation to the adult hematopoietic system. There is a considerable therapeutic interest in understanding how these first blood cells are generated in order to try to reproduce this process in vitro. This would allow generating blood products, or hematopoietic cell populations from embryonic stem (ES) cells, induced pluripotent stem cells or through directed reprogramming. Recent studies have clearly established that the first hematopoietic cells originate from a hemogenic endothelium (HE) through an endothelial to hematopoietic transition (EHT). The molecular mechanisms underlining this transition remain largely unknown with the exception that the transcription factor RUNX1 is critical for this process. In this Extra Views report, we discuss our recent studies demonstrating that the transcriptional repressors GFI1 and GFI1B have a critical role in the EHT. We established that these RUNX1 transcriptional targets are actively implicated in the downregulation of the endothelial program and the loss of endothelial identity during the formation of the first blood cells. In addition, our results suggest that GFI1 expression provides an ideal novel marker to identify, isolate and study the HE cell population.

Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation.

Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.

Expression of the MOZ-TIF2 oncoprotein in mice represses senescence.

The MOZ-TIF2 translocation, which fuses monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase (HAT) with the nuclear co-activator TIF2, is associated with the development of acute myeloid leukemia. We recently found that in the absence of MOZ HAT activity, p16(INK4a) transcriptional levels are significantly increased, triggering an early entrance into replicative senescence. Because oncogenic fusion proteins must bypass cellular safeguard mechanisms, such as senescence and apoptosis, to induce leukemia, we hypothesized that this repressive activity of MOZ over p16(INK4a) transcription could be preserved, or even reinforced, in MOZ leukemogenic fusion proteins, such as MOZ-TIF2. We describe here that, indeed, MOZ-TIF2 silences expression of the CDKN2A locus (p16(INK4a) and p19(ARF)), inhibits the triggering of senescence and enhances proliferation, providing conditions favorable to the development of leukemia. Furthermore, we describe that abolishing the MOZ HAT activity of the fusion protein leads to a significant increase in expression of the CDKN2A locus and the number of hematopoietic progenitors undergoing senescence. Finally, we report that inhibition of senescence by MOZ-TIF2 is associated with increased apoptosis, suggesting a role for the fusion protein in p53 apoptosis-versus-senescence balance. Our results underscore the importance of the HAT activity of MOZ, preserved in the fusion protein, for repression of the CDKN2A locus transcription and the subsequent block of senescence, a necessary step for the survival of leukemic cells.

RUNX1 positively regulates a cell adhesion and migration program in murine hemogenic endothelium prior to blood emergence.

During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called hemogenic endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic program and downregulation of the endothelial program. However, due to the rare and transient nature of the HE, little is known about the initial role of RUNX1 within this population. We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE. This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development.

Smooth muscle cells largely develop independently of functional hemogenic endothelium.

Vascular smooth muscle cells represent a major component of the cardiovascular system. In vitro studies have shown that FLK1(+) cells derived from embryonic stem (ES) cells can differentiate into both endothelial and smooth muscle cells. These FLK1(+) cells also contain a mesodermal precursor, the hemangioblast, able to produce endothelial, blood and smooth muscle cells. The generation of blood precursors from the hemangioblast was recently shown to occur through a transient cell population of specialised endothelium, a hemogenic endothelium. To date, the lineage relationship between this cell population and smooth muscle cell progenitors has not been investigated. In this study, we generated a reporter ES cell line in which expression of the fluorescent protein H2B-VENUS is driven by the α-smooth muscle actin (α-SMA) regulatory sequences. We demonstrated that this reporter cell line efficiently trace smooth muscle development during ES cell differentiation. Although some smooth muscle cells are associated with broad endothelial development, we established that smooth muscle cells are mostly generated independently from a specialised functional hemogenic endothelium. This study provides new and important insights into hematopoietic and vascular development, which may help in driving further progress towards the development of bioengineered vascular grafts for regenerative medicine.

MOZ-mediated repression of p16(INK) (4) (a) is critical for the self-renewal of neural and hematopoietic stem cells.

Although inhibition of p16(INK4a) expression is critical to preserve the proliferative capacity of stem cells, the molecular mechanisms responsible for silencing p16(INK4a) expression remain poorly characterized. Here, we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16(INK4a) . In the absence of the HAT activity of MOZ, expression of p16(INK4a) is upregulated in progenitor and stem cells, inducing an early entrance into replicative senescence. Genetic deletion of p16(INK4a) reverses the proliferative defect in both Moz(HAT) (-) (/) (-) hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16(INK4a) expression and to protect stem cells from early entrance into replicative senescence.

The MYSTerious MOZ, a histone acetyltransferase with a key role in haematopoiesis.

The MOnocytic leukaemia Zing finger (MOZ; MYST3 or KAT6A(1)) gene is frequently found translocated in acute myeloid leukaemia. MOZ encodes a large multidomain protein that contains, besides others, a histone acetyl transferase catalytic domain. Several studies have now established the critical function of MOZ in haematopoiesis. In this review we summarize the recent findings that underscore the relevance of the different biological activities of MOZ in the regulation of haematopoiesis.