The winged helix domain of MORF binds CpG islands and the TAZ2 domain of p300.

Acetylation of histones by lysine acetyltransferases (KATs) provides a fundamental mechanism by which chromatin structure and transcriptional programs are regulated. Here, we describe a dual binding activity of the first winged helix domain of human MORF KAT (MORFWH1) that recognizes the TAZ2 domain of p300 KAT (p300TAZ2) and CpG rich DNA sequences. Structural and biochemical studies identified distinct DNA and p300TAZ2 binding sites, allowing MORFWH1 to independently engage either ligand. Genomic data show that MORF/MOZWH1 colocalizes with H3K18ac, a product of enzymatic activity of p300, on CpG rich promoters of target genes. Our findings suggest a functional cooperation of MORF and p300 KATs in transcriptional regulation.

Guiding the HBO1 complex function through the JADE subunit.

JADE is a core subunit of the HBO1 acetyltransferase complex that regulates developmental and epigenetic programs and promotes gene transcription. Here we describe the mechanism by which JADE facilitates recruitment of the HBO1 complex to chromatin and mediates its enzymatic activity. Structural, genomic and complex assembly in vivo studies show that the PZP (PHD1-zinc-knuckle-PHD2) domain of JADE engages the nucleosome through binding to histone H3 and DNA and is necessary for the association with chromatin targets. Recognition of unmethylated H3K4 by PZP directs enzymatic activity of the complex toward histone H4 acetylation, whereas H3K4 hypermethylation alters histone substrate selectivity. We demonstrate that PZP contributes to leukemogenesis, augmenting transforming activity of the NUP98-JADE2 fusion. Our findings highlight biological consequences and the impact of the intact JADE subunit on genomic recruitment, enzymatic function and pathological activity of the HBO1 complex.

BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.

ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.

Metabolic intervention by low carbohydrate diet suppresses the onset and progression of neuroendocrine tumors.

Insulin signaling often plays a role in the regulation of cancer, including tumor initiation, progression, and response to treatment. In addition, the insulin-regulated PI3K-Akt-mTOR pathway plays an important role in the regulation of islet cell proliferation, and this pathway is hyperactivated in human non-functional pancreatic neuroendocrine tumors (PanNETs). We, therefore, investigated the effect of a very low carbohydrate diet (ketogenic diet) on a mouse model that develops non-functional PanNETs to ask how reduced PI3K-Akt-mTOR signaling might affect the development and progression of non-functional PanNET. We found that this dietary intervention resulted in lower PI3K-Akt-mTOR signaling in islet cells and a significant reduction in PanNET formation and progression. We also found that this treatment had a significant effect on the suppression of pituitary NET development. Furthermore, we found that non-functional PanNET patients with lower blood glucose levels tend to have a better prognosis than patients with higher blood glucose levels. This preclinical study shows that a dietary intervention that results in lower serum insulin levels leads to lower insulin signals within the neuroendocrine cells and has a striking suppressive effect on the development and progression of both pancreatic and pituitary NETs.

MOZ/ENL complex is a recruiting factor of leukemic AF10 fusion proteins.

Changes in the transcriptional machinery cause aberrant self-renewal of non-stem hematopoietic progenitors. AF10 fusions, such as CALM-AF10, are generated via chromosomal translocations, causing malignant leukemia. In this study, we demonstrate that AF10 fusion proteins cause aberrant self-renewal via ENL, which binds to MOZ/MORF lysine acetyltransferases (KATs). The interaction of ENL with MOZ, via its YEATS domain, is critical for CALM-AF10-mediated leukemic transformation. The MOZ/ENL complex recruits DOT1L/AF10 fusion complexes and maintains their chromatin retention via KAT activity. Therefore, inhibitors of MOZ/MORF KATs directly suppress the functions of AF10 fusion proteins, thereby exhibiting strong antitumor effects on AF10 translocation-induced leukemia. Combinatorial inhibition of MOZ/MORF and DOT1L cooperatively induces differentiation of CALM-AF10-leukemia cells. These results reveal roles for the MOZ/ENL complex as an essential recruiting factor of the AF10 fusion/DOT1L complex, providing a rationale for using MOZ/MORF KAT inhibitors in AF10 translocation-induced leukemia.

MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain.

Human acetyltransferases MOZ and MORF are implicated in chromosomal translocations associated with aggressive leukemias. Oncogenic translocations involve the far amino terminus of MOZ/MORF, the function of which remains unclear. Here, we identified and characterized two structured winged helix (WH) domains, WH1 and WH2, in MORF and MOZ. WHs bind DNA in a cooperative manner, with WH1 specifically recognizing unmethylated CpG sequences. Structural and genomic analyses show that the DNA binding function of WHs targets MORF/MOZ to gene promoters, stimulating transcription and H3K23 acetylation, and WH1 recruits oncogenic fusions to HOXA genes that trigger leukemogenesis. Cryo-EM, NMR, mass spectrometry and mutagenesis studies provide mechanistic insight into the DNA-binding mechanism, which includes the association of WH1 with the CpG-containing linker DNA and binding of WH2 to the dyad of the nucleosome. The discovery of WHs in MORF and MOZ and their DNA binding functions could open an avenue in developing therapeutics to treat diseases associated with aberrant MOZ/MORF acetyltransferase activities.

Mathematical models of the solid cancer induced by atomic bomb and the spontaneous cancer in the daily life-Proposal of a new medical treatment for cancers.

BACKGROUND: A mathematical model of the radiation-induced cancer was devised to explain the change of incidence rates pursued by Radiation Effect Research Foundation for 25 years. AIM: The aim of this work is construction of mechanisms of radiation-induced cancer and cancers observed in the daily life. METHODS AND RESULTS: First, we found a way to separate spontaneous cancers from radiation-induced cancers observed among atomic-bomb victims in Hiroshima and Nagasaki districts by using a constructed algorithm. The isolated incidence rates of radiation-induced cancers were reproduced by a two-stage model mechanical collision of impinging radiation with cells and succeeding mutation of the damaged cell to cancer. This model satisfactorily reproduced observed solid cancer incidence rates. We further attempted to construct a mathematical model for the age-dependence of spontaneous cancers appearing in the daily life and concluded that the cancer should be generated at cell division. CONCLUSION: With these findings, we reached to a conclusion that cancers may be suppressed by eliminating damaged cells with mild-dose radiation.

RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4.

Chromosomal translocation generates the MLL-AF4 fusion gene, which causes acute leukemia of multiple lineages. MLL-AF4 is a strong oncogenic driver that induces leukemia without additional mutations and is the most common cause of pediatric leukemia. However, establishment of a murine disease model via retroviral transduction has been difficult owning to a lack of understanding of its regulatory mechanisms. Here, we show that MLL-AF4 protein is post-transcriptionally regulated by RNA-binding proteins, including those of KHDRBS and IGF2BP families. MLL-AF4 translation is inhibited by ribosomal stalling, which occurs at regulatory sites containing AU-rich sequences recognized by KHDRBSs. Synonymous mutations disrupting the association of KHDRBSs result in proper translation of MLL-AF4 and leukemic transformation. Consequently, the synonymous MLL-AF4 mutant induces leukemia in vivo. Our results reveal that post-transcriptional regulation critically controls the oncogenic activity of MLL-AF4; these findings might be valuable in developing novel therapies via modulation of the activity of RNA-binding proteins.

DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.

Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5' splice site (5'SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5'SS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.

Chromatin-remodeling factor BAZ1A/ACF1 targets UV damage sites in an MLL1-dependent manner to facilitate nucleotide excision repair.

Ultraviolet (UV) light irradiation generates pyrimidine dimers on DNA, such as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Such dimers distort the high-order DNA structure and prevent transcription and replication. The nucleotide excision repair (NER) system contributes to resolving this type of DNA lesion. There are two pathways that recognize pyrimidine dimers. One acts on transcribed strands of DNA (transcription-coupled NER), and the other acts on the whole genome (global genome-NER; GG-NER). In the latter case, DNA damage-binding protein 2 (DDB2) senses pyrimidine dimers with several histone modification enzymes. We previously reported that histone acetyltransferase binding to ORC1 (HBO1) interacts with DDB2 and facilitates recruitment of the imitation switch chromatin remodeler at UV-irradiated sites via an unknown methyltransferase. Here, we found that the phosphorylated histone methyltransferase mixed lineage leukemia 1 (MLL1) was maintained at UV-irradiated sites in an HBO1-dependent manner. Furthermore, MLL1 catalyzed histone H3K4 methylation and recruited the chromatin remodeler bromodomain adjacent to zinc finger domain 1A (BAZ1A)/ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1). Depletion of MLL1 suppressed BAZ1A accumulation at UV-irradiated sites and inhibited the removal of CPDs. These data indicate that the DDB2-HBO1-MLL1 axis is essential for the recruitment of BAZ1A to facilitate GG-NER.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Role of the MOZ/MLL-mediated transcriptional activation system for self-renewal in normal hematopoiesis and leukemogenesis.

Homeostasis in the blood system is maintained by the balance between self-renewing stem cells and nonstem cells. To promote self-renewal, transcriptional regulators maintain epigenetic information during multiple rounds of cell division. Mutations in such transcriptional regulators cause aberrant self-renewal, leading to leukemia. MOZ, a histone acetyltransferase, and MLL, a histone methyltransferase, are transcriptional regulators that promote the self-renewal of hematopoietic stem cells. Gene rearrangements of MOZ and MLL generate chimeric genes encoding fusion proteins that function as constitutively active forms. These MOZ and MLL fusion proteins constitutively activate transcription of their target genes and cause aberrant self-renewal in committed hematopoietic progenitors, which normally do not self-renew. Recent progress in the field suggests that MOZ and MLL are part of a transcriptional activation system that activates the transcription of genes with nonmethylated CpG-rich promoters. The nonmethylated state of CpGs is normally maintained during cell divisions from the mother cell to the daughter cells. Thus, the MOZ/MLL-mediated transcriptional activation system replicates the expression profile of mother cells in daughter cells by activating the transcription of genes previously transcribed in the mother cell. This review summarizes the functions of the components of the MOZ/MLL-mediated transcriptional activation system and their roles in the promotion of self-renewal.

[The mechanism of MLL-rearranged leukemogenesis and its targeted therapies].

Leukemia is caused by uncontrolled proliferation of immature hematopoietic progenitors. MLL fusion proteins, generated by chromosomal translocations, activate a broad range of previously transcribed genes to achieve the same expression profile as that of the parent cell in the daughter cells, thereby promoting self-renewal. Normally, replication of the expression profile only occurs in the hematopoietic stem cells (HSCs). A transactivation system comprised of MLL and AF4/ENL/P-TEFb (AEP) complexes promotes it by reactivating CpG-rich promoters. In the normal hematopoietic development, this system is tightly regulated and progressively suppressed during the course of hematopoietic differentiation so that non-HSC hematopoietic cells would not self-renew. Genetic mutations such as fusions of MLL and AEP components generate a constitutively active form of the MLL transcriptional machinery, which aberrantly promotes self-renewal even in non-HSC hematopoietic cells. In this review, I depict a molecular mechanism of MLL fusion-mediated leukemogenesis from a standpoint that leukemogenesis is driven by aberrant self-renewal that is mediated by hyper-active transcriptional machinery, and introduce several molecularly targeted therapies in the making which specifically perturb this transactivation system.

A histone modifier, ASXL1, interacts with NONO and is involved in paraspeckle formation in hematopoietic cells.

Paraspeckles are membraneless organelles formed through liquid-liquid phase separation and consist of multiple proteins and RNAs, including NONO, SFPQ, and NEAT1. The role of paraspeckles and the component NONO in hematopoiesis remains unknown. In this study, we show histone modifier ASXL1 is involved in paraspeckle formation. ASXL1 forms phase-separated droplets, upregulates NEAT1 expression, and increases NONO-NEAT1 interactions through the C-terminal intrinsically disordered region (IDR). In contrast, a pathogenic ASXL mutant (ASXL1-MT) lacking IDR does not support the interaction of paraspeckle components. Furthermore, paraspeckles are disrupted and Nono localization is abnormal in the cytoplasm of hematopoietic stem and progenitor cells (HSPCs) derived from ASXL1-MT knockin mice. Nono depletion and the forced expression of cytoplasmic NONO impair the repopulating potential of HSPCs, as does ASXL1-MT. Our study indicates a link between ASXL1 and paraspeckle components in the maintenance of normal hematopoiesis.

Cross-talk among MEN1, p53 and Notch regulates the proliferation of pancreatic neuroendocrine tumor cells by modulating INSM1 expression and subcellular localization.

Genomic analysis of Pancreatic Neuroendocrine Tumors (PanNETs) has revealed that these tumors often lack mutations in typical cancer-related genes such as the tumor suppressor gene p53. Instead, PanNET tumorigenesis usually involves mutations in specific PanNET-related genes, such as tumor suppressor gene MEN1. Using a PanNET mouse model, human tissues and human cell lines, we studied the cross-talk among MEN1, p53 and Notch signaling pathways and their role in PanNETs. Here, we show that reactivation of the early developmental program of islet cells underlies PanNET tumorigenesis by restoring the proliferative capacity of PanNET cells. We investigated the role of INSM1, a transcriptional regulator of islet cells' development, and revealed that its expression and subcellular localization is regulated by MEN1 and p53. Both human and mouse data show that loss of MEN1 in a p53 wild-type genetic background results in increased nuclear INSM1 expression and cell proliferation. Additionally, inhibition of Notch signaling in a p53 wild-type background reduces the proliferation of PanNET cells, due to repression of INSM1 transcription and nuclear localization. Our study elucidates the molecular mechanisms governing the interactions of INSM1 with MEN1, p53 and Notch and their roles in PanNET tumorigenesis, suggesting INSM1 as a key transcriptional regulator of PanNET cell proliferation.

HBO1-MLL interaction promotes AF4/ENL/P-TEFb-mediated leukemogenesis.

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development.

Leukemogenesis via aberrant self-renewal by the MLL/AEP-mediated transcriptional activation system.

Homeostasis of the hematopoietic system is achieved in a hierarchy, with hematopoietic stem cells at the pinnacle. Because only hematopoietic stem cells (HSCs) can self-renew, the size of the hematopoietic system is strictly controlled. In hematopoietic reconstitution experiments, 1 HSC can reconstitute the entire hematopoietic system, whereas 50 multipotent progenitors cannot. This indicates that only HSCs self-renew, whereas non-HSC hematopoietic progenitors are programmed to differentiate or senesce. Oncogenic mutations of the mixed lineage leukemia gene (MLL) overcome this "programmed differentiation" by conferring the self-renewing ability to non-HSC hematopoietic progenitors. In leukemia, mutated MLL proteins constitutively activate a broad range of previously transcribed CpG-rich promoters by an MLL-mediated transcriptional activation system. This system promotes self-renewal by replicating an expression profile similar to that of the mother cell in its daughter cells. In this transcriptional activation system, MLL binds to unmethylated CpG-rich promoters and recruits RNA polymerase II. MLL recruits p300/CBP through its transcriptional activation domain, which acetylates histone H3 at lysines 9, 18, and 27. The AF4 family/ENL family/P-TEFb complex (AEP) binds to acetylated H3K9/18/27 to activate transcription. Gene rearrangements of MLL with AEP- or CBP/p300-complex components generate constitutively active transcriptional machinery of this transcriptional activation system, which causes aberrant self-renewal of leukemia stem cells. Inhibitors of the components of this system effectively decrease their leukemogenic potential.

HOXA9 promotes MYC-mediated leukemogenesis by maintaining gene expression for multiple anti-apoptotic pathways.

HOXA9 is often highly expressed in leukemias. However, its precise roles in leukemogenesis remain elusive. Here, we show that HOXA9 maintains gene expression for multiple anti-apoptotic pathways to promote leukemogenesis. In MLL fusion-mediated leukemia, MLL fusion directly activates the expression of MYC and HOXA9. Combined expression of MYC and HOXA9 induced leukemia, whereas single gene transduction of either did not, indicating a synergy between MYC and HOXA9. HOXA9 sustained expression of the genes implicated in the hematopoietic precursor identity when expressed in hematopoietic precursors, but did not reactivate it once silenced. Among the HOXA9 target genes, BCL2 and SOX4 synergistically induced leukemia with MYC. Not only BCL2, but also SOX4 suppressed apoptosis, indicating that multiple anti-apoptotic pathways underlie cooperative leukemogenesis by HOXA9 and MYC. These results demonstrate that HOXA9 is a crucial transcriptional maintenance factor that promotes MYC-mediated leukemogenesis, potentially explaining why HOXA9 is highly expressed in many leukemias.

Protocol for fractionation-assisted native ChIP (fanChIP) to capture protein-protein/DNA interactions on chromatin.

Conventional chromatin immunoprecipitation (ChIP) includes many steps that need to be optimized. Here, we have described a protocol of fractionation-assisted native ChIP (fanChIP) which combines subfractionation and native ChIP to purify protein/chromatin complexes applicable for analyses of both protein-protein and protein-DNA interactions within a short period of time. fanChIP is advantageous as subcellular fractionation removes chromatin-unbound materials before immunoprecipitation, and the chromatin fragmentation by micrococcal nuclease (MNase) in a mild condition enables one-step purification of intact protein/chromatin complexes. For complete details on the use and execution of this protocol, please refer to Miyamoto et al. (2020).

Multiorgan failure with abnormal receptor metabolism in mice mimicking Samd9/9L syndromes.

Autosomal dominant sterile α motif domain containing 9 (Samd9) and Samd9L (Samd9/9L) syndromes are a large subgroup of currently established inherited bone marrow failure syndromes that includes myelodysplasia, infection, growth restriction, adrenal hypoplasia, genital phenotypes, and enteropathy (MIRAGE), ataxia pancytopenia, and familial monosomy 7 syndromes. Samd9/9L genes are located in tandem on chromosome 7 and have been known to be the genes responsible for myeloid malignancies associated with monosomy 7. Additionally, as IFN-inducible genes, Samd9/9L are crucial for protection against viruses. Samd9/9L syndromes are caused by gain-of-function mutations and develop into infantile myelodysplastic syndromes associated with monosomy 7 (MDS/-7) at extraordinarily high frequencies. We generated mice expressing Samd9LD764N, which mimic MIRAGE syndrome, presenting with growth retardation, a short life, bone marrow failure, and multiorgan degeneration. In hematopoietic cells, Samd9LD764N downregulates the endocytosis of transferrin and c-Kit, resulting in a rare cause of anemia and a low bone marrow reconstitutive potential that ultimately causes MDS/-7. In contrast, in nonhematopoietic cells we tested, Samd9LD764N upregulated the endocytosis of EGFR by Ship2 phosphatase translocation to the cytomembrane and activated lysosomes, resulting in the reduced expression of surface receptors and signaling. Thus, Samd9/9L is a downstream regulator of IFN that controls receptor metabolism, with constitutive activation leading to multiorgan dysfunction.