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Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) is widely used in studying chromatin biology, but a comprehensive review of the analysis tools has not been completed yet. Here, we discuss the major steps in ATAC-seq data analysis, including pre-analysis (quality check and alignment), core analysis (peak calling), and advanced analysis (peak differential analysis and annotation, motif enrichment, footprinting, and nucleosome position analysis). We also review the reconstruction of transcriptional regulatory networks with multiomics data and highlight the current challenges of each step. Finally, we describe the potential of single-cell ATAC-seq and highlight the necessity of developing ATAC-seq specific analysis tools to obtain biologically meaningful insights.

Mammalian gene silencing is associated with both histone and DNA methylation. The PRMT5 arginine histone methyltransferase is now found to affect DNA methylation at the γ-globin locus in mice. This is mediated by an effect on recruitment of the DNA methyltransferase DNMT3A, but through interaction with the product of PRMT5 activity. This suggests that DNMT3A reads the histone methylation, coupling it to nearby DNA methylation. Mammalian gene silencing is established through methylation of histones and DNA, although the order in which these modifications occur remains contentious. Using the human β-globin locus as a model, we demonstrate that symmetric methylation of histone H4 arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation. H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif. Loss of the H4R3me2s mark through short hairpin RNA–mediated knockdown of PRMT5 leads to reduced DNMT3A binding, loss of DNA methylation and gene activation. In primary erythroid progenitors from adult bone marrow, H4R3me2s marks the inactive methylated globin genes coincident with localization of PRMT5. Our findings define DNMT3A as both a reader and a writer of repressive epigenetic marks, thereby directly linking histone and DNA methylation in gene silencing.

Intensive chemotherapy for acute leukemia can usually induce complete remission, but fails in many patients to eradicate the leukemia stem cells responsible for relapse. There is accumulating evidence that these relapse-inducing cells are maintained and protected by signals provided by the microenvironment. Thus, inhibition of niche signals is a proposed strategy to target leukemia stem cells but this requires knowledge of the critical signals and may be subject to compensatory mechanisms. Signals from the niche require receptor-mediated endocytosis, a generic process dependent on the Dynamin family of large GTPases. Here, we show that Dynole 34-2, a potent inhibitor of Dynamin GTPase activity, can block transduction of key signalling pathways and overcome chemoresistance of leukemia stem cells. Our results provide a significant conceptual advance in therapeutic strategies for acute leukemia that may be applicable to other malignancies in which signals from the niche are involved in disease progression and chemoresistance. The tumour microenvironment provides signals to support leukaemic stem cells (LSC) maintenance and chemoresistance. Here, the authors show that disrupting niche-associated signalling by inhibiting receptor-mediated endocytosis with a dynamin GTPase inhibitor overcomes chemoresistance of LSC.

Pre-leukemic stem cells (pre-LSCs) give rise to leukemic stem cells through acquisition of additional gene mutations and are an important source of relapse following chemotherapy. We postulated that cell-cycle kinetics of pre-LSCs may be an important determinant of clonal evolution and therapeutic resistance. Using a doxycycline-inducible H2B-GFP transgene in a mouse model of T-cell acute lymphoblastic leukemia to study cell cycle in vivo, we show that self-renewal, clonal evolution and therapeutic resistance are limited to a rare population of pre-LSCs with restricted cell cycle. We show that proliferative pre-LSCs are unable to return to a cell cycle-restricted state. Cell cycle-restricted pre-LSCs have activation of p53 and its downstream cell-cycle inhibitor p21. Furthermore, absence of p21 leads to proliferation of pre-LSCs, with clonal extinction through loss of asymmetric cell division and terminal differentiation. Thus, inducing proliferation of pre-LSCs represents a promising strategy to increase cure rates for acute leukemia. Cell cycle kinetics of pre-leukemic stem cells (pre-LSCs) may be an important determinant of clonal evolution and therapeutic resistance. Here, the AUs use a transgenic T-ALL mouse model that allows non-dividing cells to be tracked and identify a subset of non-dividing pre-LSCs maintained by p21.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21688-1

We have performed a genome-wide ENU mutagenesis screen in mice to identify novel genes or alleles that regulate erythropoiesis. Here we describe a recessive mouse strain, called RBC19, harbouring a point mutation within the housekeeping gene, Tpi1, which encodes for the glycolysis enzyme, triosephosphate isomerase (TPI). A serine in place of a phenylalanine at amino acid 57 severely diminishes enzyme activity in red cells and other tissues, resulting in a macrocytic haemolytic phenotype in homozygous mice that closely resembles human TPI deficiency. A rescue study was performed using bone marrow transplantation of wildtype donor cells, which restored all haematological parameters and increased red cell enzyme function to wildtype levels after 7 weeks. This is the first study performed in a mammalian model of TPI deficiency demonstrating that the haematological phenotype can be rescued.

Among patients undergoing stem-cell transplantation from matched related donors, cyclophosphamide plus cyclosporin led to significantly longer GVHD-free, relapse-free survival than standard prophylaxis.

Key Points All members of the human LIM-domain-only (LMO) family of proteins, LMO1–4, are implicated in the onset or the progression of cancers. In particular, LMO1 and LMO2 are linked to the onset of T cell leukaemia; overexpression of LMO4 is a marker of poor prognosis in breast cancer; and LMO1 and LMO3 are associated with neuroblastoma. The overexpression of LMO2 in T cells is caused by chromosomal translocations (in which T cell receptor genes are inserted upstream of LMO2), chromosomal deletions, insertional mutagenesis during gene therapy trials for SCID-X1, and the upregulation of promoters though transcription factors such as FLI1, ERG and LMO2 itself. LMO2-induced T cell leukaemias have a long latency period. The overexpression of LMO2 results in the upregulation of haematopoietic stem cell genes and the downregulation of T cell differentiation genes, which causes developing thymocytes to stall at the DN3 stage and to undergo self-renewal. The self-renewing cells can accumulate additional mutations (such as activating mutations of NOTCH1) that trigger overt leukaemia. LMO2 seems to function primarily through forming transcription factor complexes with TAL1 or LYL1, GATA proteins, LDB1, and E12 or E47 to regulate gene expression. Other LMO proteins may function in the same way, but apart from LMO1 (which can take the place of LMO2 in haematopoietic transcriptional complexes) their protein partners are less well characterized. LMO4 seems to regulate progression through the cell cycle in breast cancer cell lines, and can act at several different stages of the cell cycle, probably either by affecting transcriptional programmes of proteins that directly control cell cycle progression, or through interaction with such proteins. The overexpression of LMO proteins has been identified in many different types of cancers. In some cases this overexpression can have a protective effect, in other cases the LMO proteins actively promote cancer, but in many other cases a causative or protective role has yet to be determined. In cancers in which LMO proteins are oncogenic, such as LMO2-induced T cell leukaemias, inhibitors of the LMO protein may be of therapeutic value. LIM-domain-only (LMO) proteins are a subset of the LIM-domain protein family and function primarily as transcriptional regulators. They are associated with various cancers, including T cell acute lymphoblastic leukaemia (T-ALL) that resulted from unintended activation of LMO2 by insertional mutagenesis in human gene therapy trials. This Review discusses the roles and potential mechanisms of LMO proteins in cancer and the potential for therapeutic targeting. LIM-domain proteins are a large family of proteins that are emerging as key molecules in a wide variety of human cancers. In particular, all members of the human LIM-domain-only (LMO) proteins, LMO1–4, which are required for many developmental processes, are implicated in the onset or the progression of several cancers, including T cell leukaemia, breast cancer and neuroblastoma. These small proteins contain two protein-interacting LIM domains but little additional sequence, and they seem to function by nucleating the formation of new transcriptional complexes and/or by disrupting existing transcriptional complexes to modulate gene expression programmes. Through these activities, the LMO proteins have important cellular roles in processes that are relevant to cancer such as self-renewal, cell cycle regulation and metastasis. These functions highlight the therapeutic potential of targeting these proteins in cancer.

A genome-wide ENU mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX). A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared to that of wildtype littermates, particularly coproporphyrinogen III, while males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5–MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5–MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.