A Data-Driven Approach to Sugarcane Breeding Programs with Agronomic Characteristics and Amino Acid Constituent Profiling.

Sugarcane (Saccharum spp. hybrids) and its processed products have supported local industries such as those in the Nansei Islands, Japan. To improve the sugarcane quality and productivity, breeders select better clones by evaluating agronomic characteristics, such as commercially recoverable sugar and cane yield. However, other constituents in sugarcane remain largely unutilized in sugarcane breeding programs. This study aims to establish a data-driven approach to analyze agronomic characteristics from breeding programs. This approach also determines a correlation between agronomic characteristics and free amino acid composition to make breeding programs more efficient. Sugarcane was sampled in clones in the later stage of breeding selection and cultivars from experimental fields on Tanegashima Island. Principal component analysis and hierarchical cluster analysis using agronomic characteristics revealed the diversity and variability of each sample, and the data-driven approach classified cultivars and clones into three groups based on yield type. A comparison of free amino acid constituents between these groups revealed significant differences in amino acids such as asparagine and glutamine. This approach dealing with a large volume of data on agronomic characteristics will be useful for assessing the characteristics of potential clones under selection and accelerating breeding programs.

Canonical BAF complex regulates the oncogenic program in human T-cell acute lymphoblastic leukemia.

ABSTRACT: Acute leukemia cells require bone marrow microenvironments, known as niches, which provide leukemic cells with niche factors that are essential for leukemic cell survival and/or proliferation. However, it remains unclear how the dynamics of the leukemic cell-niche interaction are regulated. Using a genome-wide CRISPR screen, we discovered that canonical BRG1/BRM-associated factor (cBAF), a variant of the switch/sucrose nonfermenting chromatin remodeling complex, regulates the migratory response of human T-cell acute lymphoblastic leukemia (T-ALL) cells to a niche factor CXCL12. Mechanistically, cBAF maintains chromatin accessibility and allows RUNX1 to bind to CXCR4 enhancer regions. cBAF inhibition evicts RUNX1 from the genome, resulting in CXCR4 downregulation and impaired migration activity. In addition, cBAF maintains chromatin accessibility preferentially at RUNX1 binding sites, ensuring RUNX1 binding at these sites, and is required for expression of RUNX1-regulated genes, such as CDK6; therefore, cBAF inhibition negatively impacts cell proliferation and profoundly induces apoptosis. This anticancer effect was also confirmed using T-ALL xenograft models, suggesting cBAF as a promising therapeutic target. Thus, we provide novel evidence that cBAF regulates the RUNX1-driven leukemic program and governs migration activity toward CXCL12 and cell-autonomous growth in human T-ALL.

RENGE infers gene regulatory networks using time-series single-cell RNA-seq data with CRISPR perturbations.

Single-cell RNA-seq analysis coupled with CRISPR-based perturbation has enabled the inference of gene regulatory networks with causal relationships. However, a snapshot of single-cell CRISPR data may not lead to an accurate inference, since a gene knockout can influence multi-layered downstream over time. Here, we developed RENGE, a computational method that infers gene regulatory networks using a time-series single-cell CRISPR dataset. RENGE models the propagation process of the effects elicited by a gene knockout on its regulatory network. It can distinguish between direct and indirect regulations, which allows for the inference of regulations by genes that are not knocked out. RENGE therefore outperforms current methods in the accuracy of inferring gene regulatory networks. When used on a dataset we derived from human-induced pluripotent stem cells, RENGE yielded a network consistent with multiple databases and literature. Accurate inference of gene regulatory networks by RENGE would enable the identification of key factors for various biological systems.

Sugarcane Metabolome Compositional Stability in Pretreatment Processes for NMR Measurements.

Sugarcane is essential for global sugar production and its compressed juice is a key raw material for industrial products. Sugarcane juice includes various metabolites with abundances and compositional balances influencing product qualities and functionalities. Therefore, understanding the characteristic features of the sugarcane metabolome is important. However, sugarcane compositional variability and stability, even in pretreatment processes for nuclear magnetic resonance (NMR)-based metabolomic studies, remains elusive. The objective of this study is to evaluate sugarcane juice metabolomic variability affected by centrifugation, filtration, and thermal pretreatments, as well as the time-course changes for determining optimal conditions for NMR-based metabolomic approach. The pretreatment processes left the metabolomic compositions unchanged, indicating that these pretreatments are compatible with one another and the studied metabolomes are comparable. The thermal processing provided stability to the metabolome for more than 32 h at room temperature. Based on the determined analytical conditions, we conducted an NMR-based metabolomic study to discriminate the differences in the harvest period and allowed for successfully identifying the characteristic metabolome. Our findings denote that NMR-based sugarcane metabolomics enable us to provide an opportunity to collect a massive amount of data upon collaboration between multiple researchers, resulting in the rapid construction of useful databases for both research purposes and industrial use.

Detection of a major QTL related to smut disease resistance inherited from a Japanese wild sugarcane using GRAS-Di technology.

Smut disease of sugarcane causes considerable yield losses and the use of resistant varieties is the best control practice. Our group identified a Japanese wild sugarcane with highly smut disease resistance named 'Iriomote8'. In this study, we conducted QTL analysis for smut disease resistance using a mapping population derived from a resistant variety 'Yaenoushie', in which resistance is inherited from 'Iriomote8'. We identified 4813 non-redundant markers using GRAS-Di technology and developed a linkage map of mapping parents. We evaluated smut disease resistance of the mapping population by the inoculation test. Consequently, a large number of clones did not show the disease symptoms and the distribution of smut disease incidence tended to be "L shaped". Composite interval mapping detected an identical QTL for indices of smut disease incidence with a markedly high LOD score (26.6~45.6) at the end of linkage group 8 of 'Yaenoushie'. This QTL explained approximately 50% of the cases of smut disease incidence. In the mapping population, there were no correlations between the indices of smut disease incidence and other agronomic traits. In conclusion, this QTL could be used for marker-assisted selection to significantly improve smut disease resistance without negative effects on other agronomic traits.

Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia.

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2, and SATB1. They are organized convergently in genetically diverse subtypes of AML and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.

KAT7 is a genetic vulnerability of acute myeloid leukemias driven by MLL rearrangements.

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype.

Salt-inducible kinase inhibition suppresses acute myeloid leukemia progression in vivo.

Lineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a salt-inducible kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF myocyte enhancer factor (MEF2C). In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. In this study, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells under in vitro and in vivo conditions. Similar phenotypes were obtained when cells were exposed to YKL-05-099, which caused cell-cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele of SIK3, we found that the antiproliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated 2 different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progression in vivo and extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-addicted AML and provide a rationale for developing druglike inhibitors of SIK3 for definitive preclinical investigation and for studies in human patients.

Telomere-binding proteins Taz1 and Rap1 regulate DSB repair and suppress gross chromosomal rearrangements in fission yeast.

Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs.

Tankyrase inhibition sensitizes cells to CDK4 blockade.

Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the ß-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.

A Transcription Factor Addiction in Leukemia Imposed by the MLL Promoter Sequence.

The Mixed Lineage Leukemia gene (MLL) is altered in leukemia by chromosomal translocations to produce oncoproteins composed of the MLL N-terminus fused to the C-terminus of a partner protein. Here, we used domain-focused CRISPR screening to identify ZFP64 as an essential transcription factor in MLL-rearranged leukemia. We show that the critical function of ZFP64 in leukemia is to maintain MLL expression via binding to the MLL promoter, which is the most enriched location of ZFP64 occupancy in the human genome. The specificity of ZFP64 for MLL is accounted for by an exceptional density of ZFP64 motifs embedded within the MLL promoter. These findings demonstrate how a sequence anomaly of an oncogene promoter can impose a transcriptional addiction in cancer.

LKB1, Salt-Inducible Kinases, and MEF2C Are Linked Dependencies in Acute Myeloid Leukemia.

The lineage-specific transcription factor (TF) MEF2C is often deregulated in leukemia. However, strategies to target this TF have yet to be identified. Here, we used a domain-focused CRISPR screen to reveal an essential role for LKB1 and its Salt-Inducible Kinase effectors (SIK3, in a partially redundant manner with SIK2) to maintain MEF2C function in acute myeloid leukemia (AML). A key phosphorylation substrate of SIK3 in this context is HDAC4, a repressive cofactor of MEF2C. Consequently, targeting of LKB1 or SIK3 diminishes histone acetylation at MEF2C-bound enhancers and deprives leukemia cells of the output of this essential TF. We also found that MEF2C-dependent leukemias are sensitive to on-target chemical inhibition of SIK activity. This study reveals a chemical strategy to block MEF2C function in AML, highlighting how an oncogenic TF can be disabled by targeting of upstream kinases.

A TFIID-SAGA Perturbation that Targets MYB and Suppresses Acute Myeloid Leukemia.

Targeting of general coactivators is an emerging strategy to interfere with oncogenic transcription factors (TFs). However, coactivator perturbations often lead to pleiotropic effects by influencing numerous TFs. Here we identify TAF12, a subunit of TFIID and SAGA coactivator complexes, as a selective requirement for acute myeloid leukemia (AML) progression. We trace this dependency to a direct interaction between the TAF12/TAF4 histone-fold heterodimer and the transactivation domain of MYB, a TF with established roles in leukemogenesis. Ectopic expression of the TAF4 histone-fold fragment can efficiently squelch TAF12 in cells, suppress MYB, and regress AML in mice. Our study reveals a strategy for potent MYB inhibition in AML and highlights how an oncogenic TF can be selectively neutralized by targeting a general coactivator complex.

Fission yeast Stn1 is crucial for semi-conservative replication at telomeres and subtelomeres.

The CST complex is a phylogenetically conserved protein complex consisting of CTC1/Cdc13, Stn1 and Ten1 that protects telomeres on linear chromosomes. Deletion of the fission yeast homologs stn1 and ten1 results in complete telomere loss; however, the precise function of Stn1 is still largely unknown. Here, we have isolated a high-temperature sensitive stn1 allele (termed stn1-1). stn1-1 cells abruptly lost telomeric sequence almost completely at the restrictive temperature. The loss of chromosomal DNA happened without gradual telomere shortening, and extended to 30 kb from the ends of chromosomes. We found transient and modest single-stranded G-strand exposure, but did not find any evidence of checkpoint activation in stn1-1 at the restrictive temperature. When we probed neutral-neutral 2D gels for subtelomere regions, we found no Y-arc-shaped replication intermediates in cycling cells. We conclude that the loss of telomere and subtelomere DNAs in stn1-1 cells at the restrictive temperature is caused by very frequent replication fork collapses specifically in subtelomere regions. Furthermore, we identified two independent suppressor mutants of the high-temperature sensitivity of stn1-1: a multi-copy form of pmt3 and a deletion of rif1. Collectively, we propose that fission yeast Stn1 primarily safeguards the semi-conservative DNA replication at telomeres and subtelomeres.

Receptor for activated C-kinase (RACK1) homolog Cpc2 facilitates the general amino acid control response through Gcn2 kinase in fission yeast.

General amino acid control (GAAC) is crucial for sensing and adaptation to nutrient availability. Amino acid starvation activates protein kinase Gcn2, which plays a central role in the GAAC response by phosphorylating the α-subunit of eukaryotic initiation factor 2 (eIF2α), leading to the translational switch to stimulate selective expression of stress-responsive genes. We report here that in fission yeast Schizosaccharomyces pombe, Cpc2, a homolog of mammalian receptor for activated C-kinase (RACK1), is important for the GAAC response. Deletion of S. pombe cpc2 impairs the amino acid starvation-induced phosphorylation of eIF2α and the expression of amino acid biosynthesis genes, thereby rendering cells severely sensitive to amino acid limitation. Unlike the Saccharomyces cerevisiae Cpc2 ortholog, which normally suppresses the GAAC response, our findings suggest that S. pombe Cpc2 promotes the GAAC response. We also found that S. pombe Cpc2 is required for starvation-induced Gcn2 autophosphorylation, which is essential for Gcn2 function. These results indicate that S. pombe Cpc2 facilitates the GAAC response through the regulation of Gcn2 activation and provide a novel insight for the regulatory function of RACK1 on Gcn2-mediated GAAC response.

HIRA, a conserved histone chaperone, plays an essential role in low-dose stress response via transcriptional stimulation in fission yeast.

Cells that have been pre-exposed to mild stress (priming stress) acquire transient resistance to subsequent severe stress even under different combinations of stresses. This phenomenon is called cross-tolerance. Although it has been reported that cross-tolerance occurs in many organisms, the molecular basis is not clear yet. Here, we identified slm9(+) as a responsible gene for the cross-tolerance in the fission yeast Schizosaccharomyces pombe. Slm9 is a homolog of mammalian HIRA histone chaperone. HIRA forms a conserved complex and gene disruption of other HIRA complex components, Hip1, Hip3, and Hip4, also yielded a cross-tolerance-defective phenotype, indicating that the fission yeast HIRA is involved in the cross-tolerance as a complex. We also revealed that Slm9 was recruited to the stress-responsive gene loci upon stress treatment in an Atf1-dependent manner. The expression of stress-responsive genes under stress conditions was compromised in HIRA disruptants. Consistent with this, Pol II recruitment and nucleosome eviction at these gene loci were impaired in slm9Δ cells. Furthermore, we found that the priming stress enhanced the expression of stress-responsive genes in wild-type cells that were exposed to the severe stress. These observations suggest that HIRA functions in stress response through transcriptional regulation.

Tel1(ATM) and Rad3(ATR) phosphorylate the telomere protein Ccq1 to recruit telomerase and elongate telomeres in fission yeast.

In fission yeast, the DNA damage sensor kinases Tel1(ATM) and Rad3(ATR) exist at telomeres and are required for telomere maintenance, but the biological role they play at telomeres is not known. Here we show that the telomere protein Ccq1 is phosphorylated at Thr 93 (threonine residue at amino acid 93) by Tel1(ATM) and Rad3(ATR) both in vitro and in vivo. A ccq1 mutant in which alanine was substituted for Thr 93 failed to recruit telomerase to telomeres and showed gradual shortening of telomeres. These results indicate that the direct phosphorylation of Ccq1 Thr 93 by Tel1 and Rad3 is involved in the recruitment of telomerase to elongate telomeres.