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ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1–RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-d-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.

The role of Fat Mass and Obesity-associated protein （FTO） and its substrate N6-methyladenosine （m6A） in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adi- pogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5＇- and 3＇-splice sites, spatially over- lapping with mRNA splicing regulatory serine/arginine-rich （SR） protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenie regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice. Molecular diversity of proprioceptive neuron types (Ia, Ib and II PNs) is unclear. Here, the authors characterized the functional organization and development of eight subtypes of PNs in mice. Importantly, Ia subtypes are plastic, suggesting a role in adaptive proprioception during motor behavior.

Retinal ganglion cells (RGCs) convey the major output of information collected from the eye to the brain. Thirty subtypes of RGCs have been identified to date. Here, we analyze 6225 RGCs (average of 5000 genes per cell) from right and left eyes by single-cell RNA-seq and classify them into 40 subtypes using clustering algorithms. We identify additional subtypes and markers, as well as transcription factors predicted to cooperate in specifying RGC subtypes. Zic1, a marker of the right eye-enriched subtype, is validated by immunostaining in situ. Runx1 and Fst, the markers of other subtypes, are validated in purified RGCs by fluorescent in situ hybridization (FISH) and immunostaining. We show the extent of gene expression variability needed for subtype segregation, and we show a hierarchy in diversification from a cell-type population to subtypes. Finally, we present a website for comparing the gene expression of RGC subtypes. Retinal ganglion cells (RGCs) are diverse in cellular function and physiology. This study demonstrates additional RGC heterogeneity using single cell transcriptomic analyses to classify 40 classes of RGCs in early postnatal mice before eye opening.

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb , Gfi1 , Runx1 , and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0–8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8–20), RUNX1 + FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20–28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders. The authors reprogram in vitro endothelial cells from adult mice into engraftable haematopoietic stem cells that display single-cell and multilineage properties, are capable of long-term self-renewal and can reconstitute T cell adaptive immune function. Generating functional haematopoietic stem cells The transition pathways of endothelial cells into haematopoietic stem cells remain undefined. Shahin Rafii and colleagues reprogrammed in vitro mouse adult endothelial cells into mouse engraftable haematopoietic stem cells which display the range of functional properties expected from true haematopoietic stem cells. They use a sequential approach to express transcription factors known to participate in the induction of haematopoiesis from the embryonic endothelium, as well as an endothelial cell line that acts as the vascular niche to provide the required signals. The reprogramed cells display single-cell and multilineage properties, long-term self-renewal and reconstitute T cell adaptive immune function. Converting endothelial cells into functional and self-renewing haematopoietic stem cells could open up treatment opportunities for haematological disorders, the authors suggest. Elsewhere in this issue, George Daley and colleagues differentiated human pluripotent stem cells to the haemogenic endothelium by expressing a set of transcription factors that regulate hematopoiesis.

Background Runt-related transcription factor 1 (RUNX1) plays the roles of an oncogene and an anti-oncogene in epithelial tumours, and abnormally elevated RUNX1 has been suggested to contribute to the carcinogenesis of colorectal cancer (CRC). However, the mechanism remains unclear. Methods The expression of RUNX1 in CRC and normal tissues was detected by real-time quantitative PCR and Western blotting. The effect of RUNX1 on CRC migration and invasion was conducted by functional experiments in vitro and in vivo. Chromatin Immunoprecipitation assay verified the direct regulation of RUNX1 on the promoter of the KIT, which leads to the activation of Wnt/β-catenin signaling. Results RUNX1 expression is upregulated in CRC tissues. Upregulated RUNX1 promotes cell metastasis and epithelial to mesenchymal transition (EMT) of CRC both in vitro and in vivo. Furthermore, RUNX1 can activate Wnt/β-catenin signalling in CRC cells by directly interacting with β-catenin and targeting the promoter and enhancer regions of KIT to promote KIT transcription. These observations demonstrate that RUNX1 upregulation is a common event in CRC specimens and is closely correlated with cancer metastasis and that RUNX1 promotes EMT of CRC cells by activating Wnt/β-catenin signalling. Moreover, RUNX1 is regulated by Wnt/β-catenin. Conclusion Our findings first demonstrate that RUNX1 promotes CRC metastasis by activating the Wnt/β-catenin signalling pathway and EMT.

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors ( ERG , HOXA5 , HOXA9 , HOXA10 , LCOR , RUNX1 and SPI1 ) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. Haematopoietic stem and progenitor cell conversion of human pluripotent stem cell-derived haemogenic endothelium. Generating functional haematopoietic stem cells Obtaining functional human haematopoietic stem cells (HSCs) from differentiated pluripotent stem cells (PSCs) is proving a challenge for the field. George Daley and colleagues used a morphogen-based approach to differentiate human PSCs to the haemogenic endothelium, where endothelial cells and HSCs commonly originate. They then screened 26 candidate HSC-specifying transcription factors for their ability to confer multi-lineage blood engraftment to the haemogenic endothelial cells when transplanted into mice. They defined a set of seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that were sufficient to allow engraftment of myeloid, B and T cells in primary and secondary murine recipients. The cells obtained could one day enable researchers to model haematopoietic disease in humanized mice. Elsewhere in this issue, Shahin Rafii and colleagues reprogrammed in vitro mouse adult endothelial cells into mouse engraftable haematopoietic stem cells displaying some key functional properties.

While biomarkers have been shown to enhance the prognosis of patients with colorectal cancer (CRC) compared to conventional treatments, there is a pressing need to discover novel biomarkers that can assist in assessing the prognostic impact of immunotherapy and in formulating individualized treatment plans. The RUNX family, consisting of RUNX1, RUNX2, and RUNX3, has been recognized as crucial regulators in developmental processes, with dysregulation of these genes also being implicated in tumorigenesis and cancer progression. In our present study, we demonstrated a crucial regulatory role of RUNX in CD8 + T and CD103 + CD8 + T cell-mediated anti-tumor response within the tumor microenvironment (TME) of human CRC. Specifically, RUNXs were significantly differentially expressed between tumor and normal tissues in CRC. Patients with a greater proportion of infiltrating CD8 + RUNX1 + , CD103 + CD8 + RUNX1 + , CD8 + RUNX2 + , CD103 + CD8 + RUNX2 + , CD8 + RUNX3 + , or CD103 + CD8 + RUNX3 + T cells demonstrated improved outcomes compared to those with lower proportions. Additionally, the proportions of infiltrating CD8 + RUNX1 + T and CD8 + RUNX3 + T cells may serve as valuable prognostic predictors for CRC patients, independent of other clinicopathological factors. Moreover, further bioinformatic analysis conducted utilizing the TISIDB and TIMER platforms demonstrated significant associations between the members of the RUNX family and immune-infiltrating cells, specifically diverse subpopulations of CD8 + TILs. Our study of human colorectal cancer tissue microarray (TMA) also revealed positive and statistically significant correlations between the expressions of RUNX1, RUNX2, and RUNX3 in both CD8 + T cells and CD103 + CD8 + T cells. Our study comprehensively revealed the varied expressions and prognostic importance of the RUNX family in human colorectal cancer tissues. It underscored their potential as vital biomarkers for prognostic evaluation in colorectal cancer patients and as promising targets for immunotherapy in treating this disease.

Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We showed active chromatin remodeling at two transition stages: bone marrow–to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Therefore, our study represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease. Neutrophils demonstrate highly dynamic functional and transcriptional changes depending on their tissue environment. Udalova and colleagues use an inflammation model to examine neutrophils and find that the transcription factors RUNX1 and KLF6 control maturation; RELB, IRF5 and JUNB drive effector responses; and RFX2 and RELB promote survival.

Microglia are the resident macrophages of the central nervous system and are associated with the pathogenesis of many neurodegenerative and brain inflammatory diseases; however, the origin of adult microglia remains controversial. We show that postnatal hematopoietic progenitors do not significantly contribute to microglia homeostasis in the adult brain. In contrast to many macrophage populations, we show that microglia develop in mice that lack colony stimulating factor-1 (CSF-1) but are absent in CSF-1 receptor-deficient mice. In vivo lineage tracing studies established that adult microglia derive from primitive myeloid progenitors that arise before embryonic day 8. These results identify microglia as an ontogenically distinct population in the mononuclear phagocyte system and have implications for the use of embryonically derived microglial progenitors for the treatment of various brain disorders.