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N 6 -methyladenosine (m 6 A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m 6 A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m 6 A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC ) in an m 6 A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m 6 A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m 6 A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m 6 A readers in post-transcriptional gene regulation and cancer biology. Huang et al. identify IGF2BPs as an additional class of N 6 -methyladenosine (m 6 A) reader proteins. They find that IGF2BPs selectively bind to m 6 A-containing mRNAs and promote their stability.

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4 + intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (Sil ThPOK ). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the Sil ThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (T reg ) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITR lo PD-1 lo CD25 lo (Triple lo ) T reg cells and conversion to CD4 + IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance. The transcription factor ThPOK is critical for homeostasis and differentiation of mature helper T cells. Here, Kappes and colleagues describe a ThPOK-mediated positive autoregulatory loop that is crucial for tissue-specific T reg cell differentiation, maintenance of intestinal T reg cell integrity and conversion of these cells into CD4 + intraepithelial lymphocytes.

Effective therapy of acute myeloid leukemia (AML) remains an unmet need. DNA methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Through a series of data analysis and drug screening, we identified two compounds (i.e., NSC-311068 and NSC-370284) that selectively suppress TET1 transcription and 5-hydroxymethylcytosine (5hmC) modification, and effectively inhibit cell viability in AML with high expression of TET1 (i.e., TET1 -high AML), including AML carrying t(11q23)/MLL-rearrangements and t(8;21) AML. NSC-311068 and especially NSC-370284 significantly repressed TET1 -high AML progression in vivo. UC-514321, a structural analog of NSC-370284, exhibited a more potent therapeutic effect and prolonged the median survival of TET1 -high AML mice over three fold. NSC-370284 and UC-514321 both directly target STAT3/5, transcriptional activators of TET1 , and thus repress TET1 expression. They also exhibit strong synergistic effects with standard chemotherapy. Our results highlight the therapeutic potential of targeting the STAT/TET1 axis by selective inhibitors in AML treatment. Ten-eleven translocation 1 (TET1) is a critical oncoprotein in AML. Here, the authors identify 2 compounds that target the binding of STAT3/5 specifically to the TET1 promoter, inhibiting its expression and AML cell viability.

MLL -rearranged acute myeloid leukemia (AML) remains a fatal disease with a high rate of relapse and therapeutic failure due to chemotherapy resistance. In analysis of our Affymetrix microarray profiling and chromatin immunoprecipitation (ChIP) assays, we found that ALOX5 is especially down-regulated in MLL -rearranged AML, via transcription repression mediated by Polycomb repressive complex 2 (PRC2). Colony forming/replating and bone marrow transplantation (BMT) assays showed that Alox5 exhibited a moderate anti-tumor effect both in vitro and in vivo . Strikingly, leukemic cells with Alox5 overexpression showed a significantly higher sensitivity to the standard chemotherapeutic agents, i.e., doxorubicin (DOX) and cytarabine (Ara-C). The drug-sensitizing role of Alox5 was further confirmed in human and murine MLL -rearranged AML cell models in vitro , as well as in the in vivo MLL -rearranged AML BMT model coupled with treatment of “5 + 3” (i.e. DOX plus Ara-C) regimen. Stat and K-Ras signaling pathways were negatively correlated with Alox5 overexpression in MLL-AF9 -leukemic blast cells; inhibition of the above signaling pathways mimicked the drug-sensitizing effect of ALOX5 in AML cells. Collectively, our work shows that ALOX5 plays a moderate anti-tumor role and functions as a drug sensitizer, with a therapeutic potential, in MLL -rearranged AML.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

N -methyladenosine (m A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m A readers in post-transcriptional gene regulation and cancer biology.

The original version of this Article contained an error in the spelling of the author James C. Mulloy, which was incorrectly given as James Mulloy. This has now been corrected in both the PDF and HTML versions of the Article.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease. In this Resource article, the authors integrate genomic, bioinformatic and flow cytometric data from human bone marrow to provide an atlas of hematopoietic progenitor cell states in health and disease.

Hematopoietic stem and progenitor cells (HSPCs) form a complicated system that must fulfill the needs of immune, platelet, and red blood cell development throughout life. It is thought that hematopoietic stem cells (HSCs) become active and generate multilineage progenitors which differentiate to make bipotent followed by lineage committed progenitors. This process of differentiation depends on a complex interplay of intrinsic and extrinsic cellular factors including cytokines and growth factors, epigenetic regulation, signaling pathways, and gene regulatory networks. Cell states were previously identified using small sets of surface markers and functional assays. These approaches were limited in their ability to fully characterize the molecular and functional heterogeneity within the hematopoietic system, as they relied on bulk cell populations rather than individual cells. The advent of single-cell multi-omics technologies has revolutionized our ability to characterize cell states, allowing for the simultaneous capture of multiple molecular features at the single-cell level. These approaches have recently become higher throughput with advancements in microfluidics and sequence barcoding techniques. At the same time, methods in flow cytometry, such as new fluorescent dyes, multi-laser systems, increased detection channels, and spectral unmixing algorithms have made it possible to measure more features at a single-cell level. With the advancement of measuring molecular features at the single cell level, analysis pipelines continue to optimize in throughput and efficiency.In this dissertation, I present my work focusing on the identification and isolation of hematopoietic progenitor cells. Singe-cell omics techniques have made it possible to identify rare and previously unknown cell types. Through the application of high throughput analysis pipelines, we can reconstruct the global structure of hematopoiesis and begin to associate cell states with different stages of developmental trajectories. We built an interactive viewer for the human cell atlas bone marrow tissue project, spanning 100,000 hematopoietic cells across 8 donors. Next, we built a software package for the construction of high dimensional flow cytometry datasets using Infinity Flow. Next, we used cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to generate a comprehensive cell atlas of murine hematopoietic stem and progenitor cells in the bone marrow. Finally, we integrated CITE-seq with Infinity Flow to streamline the development of fluorescence-activated cell sorting (FACS) panels for the isolation of transcriptionally discrete cell populations. These findings have important implications for understanding disease states, as identifying rare cell types that are dysregulated in disease can provide insights into underlying mechanisms and potential therapeutic targets.

The transcription factor ThPOK (encoded by Zbtb7b ) is well known for its role as a master regulator of CD4 lineage commitment in the thymus. Here, we report an unexpected and critical role of ThPOK as a multifaceted regulator of myeloid lineage commitment, differentiation and maturation. Using reporter and knockout mouse models combined with single-cell RNA-sequencing, progenitor transfer and colony assays, we show that ThPOK controls monocyte-dendritic cell versus granulocyte lineage production during homeostatic differentiation, and serves as a brake for neutrophil maturation in granulocyte lineage-specified cells through transcriptional regulation of lineage-specific transcription factors and RNA via altered messenger RNA splicing to reprogram intron retention. Basu et al. find that the transcription factor ThPOK is not restricted to T cells, as it also is expressed in myeloid cell progenitors and contributes to the lineage choice of monocyte-dendritic cells as opposed to neutrophils.