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Although establishment and maintenance of mitochondria are essential for the production of massive amounts of heme in erythroblasts, mitochondria must be degraded upon terminal differentiation to red blood cells (RBCs), thus creating a biphasic regulatory process. Previously, we reported that iron deficiency in mice promotes mitochondrial retention in RBCs, suggesting that a proper amount of iron and/or heme is necessary for the degradation of mitochondria during erythroblast maturation. Because the transcription factor GATA1 regulates autophagy in erythroid cells, which involves mitochondrial clearance (mitophagy), we investigated the relationship between iron or heme and mitophagy by analyzing the expression of genes related to GATA1 and autophagy and the impact of iron or heme restriction on the amount of mitochondria. We found that heme promotes the expression of GATA1-regulated mitophagy-related genes and the induction of mitophagy. GATA1 might induce the expression of the autophagy-related genes Atg4d and Stk11 for mitophagy through a heme-dependent mechanism in murine erythroleukemia (MEL) cells and a genetic rescue system with G1E-ER-GATA1 erythroblast cells derived from Gata1-null murine embryonic stem cells. These results provide evidence for a biphasic mechanism in which mitochondria are essential for heme generation, and the heme generated during differentiation promotes mitophagy and mitochondrial disposal. This mechanism provides a molecular framework for understanding this fundamentally important cell biological process.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1−/− accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1−/− (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

The hematopoietic transcription factor GATA1 induces heme accumulation during erythropoiesis by directly activating genes mediating heme biosynthesis. In addition to its canonical functions as a hemoglobin prosthetic group and enzyme cofactor, heme regulates gene expression in erythroid cells both transcriptionally and post-transcriptionally. Heme binding to the transcriptional repressor BACH1 triggers its proteolytic degradation. In heme-deficient cells, BACH1 accumulates and represses transcription of target genes, including α- and β-like globin genes, preventing the accumulation of cytotoxic free globin chains. A recently described BACH1-independent mechanism of heme-dependent transcriptional regulation is associated with a DNA motif termed heme-regulated motif (HERM), which resides at the majority of loci harboring heme-regulated chromatin accessibility sites. Progress on these problems has led to a paradigm in which cell type-specific transcriptional mechanisms determine the expression of enzymes mediating the synthesis of small molecules, which generate feedback loops, converging upon the transcription factor itself and the genome. This marriage between transcription factors and the small molecules that they control is predicted to be a canonical attribute of regulatory networks governing cell state transitions such as differentiation in the hematopoietic system and more broadly.

The role of BrKAO2 in leafy head formation was confirmed by using two allelic Chinese cabbage mutants. Chinese cabbage yield and quality are determined by leafy head formation. Cloning and characterising the key genes regulating leafy head formation are essential for its varietal improvement. We used an EMS-mutagenised population of the heading type 'FT' Chinese cabbage line and identified two allelic non-heading mutants, i.e. nhm3-1 and nhm3-2. Genetic analysis showed that the mutant trait was controlled by a single recessive gene. MutMap and Kompetitive Allele Specific PCR genotyping revealed that BraA05g012440.3C was the candidate gene, which encodes ent-kaurenoic acid oxidase 2 in gibberellin (GA) biosynthetic pathway. It was named BrKAO2. Two non-synonymous mutations in the second BrKAO2 exon, respectively, accounted for the mutant phenotypes of nhm3-1 and nhm3-2. BrKAO2 was expressed during all leaf development stages, and there were no significant differences between the wild type and mutants in terms of BrKAO2 expression. The mutant phenotypes were restored to the wild type via exogenous GA3 application. RNA-Seq was performed on wild-type 'FT', nhm3-1, and nhm3-1 + GA3 rosette leaves, and several key genes involved in GA biosynthesis, signal transduction, and leafy head development were identified. These findings indicate that BrKAO2 is responsible for the leafy head formation in nhm3 mutants.

Summary: Protein ensembles control genome function by establishing, maintaining, and deconstructing cell-type-specific chromosomal landscapes. A plethora of small molecules orchestrate cellular functions and therefore may link physiological processes with genome biology. The metabolic enzyme and hemoglobin cofactor heme induces proteolysis of a transcriptional repressor, Bach1, and regulates gene expression post-transcriptionally. However, whether heme controls genome function broadly or through prescriptive actions is unclear. Using assay for transposase-accessible chromatin sequencing (ATAC-seq), we establish a heme-dependent chromatin atlas in wild-type and mutant erythroblasts lacking enhancers that confer normal heme synthesis. Amalgamating chromatin landscapes and transcriptomes in cells with sub-physiological heme and post-heme rescue reveals parallel Bach1-dependent and Bach1-independent mechanisms that target heme-sensing chromosomal hotspots. The hotspots harbor a DNA motif demarcating heme-regulated chromatin and genes encoding proteins not known to be heme regulated, including metabolic enzymes. The heme-omics analysis establishes how an essential biochemical cofactor controls genome function and cellular physiology.

Developmental-regulatory networks often include large gene families encoding mechanistically-related proteins like G-protein-coupled receptors, zinc finger transcription factors and solute carrier (SLC) transporters. In principle, a common mechanism may confer expression of multiple members integral to a developmental process, or diverse mechanisms may be deployed. Using genetic complementation and enhancer-mutant systems, we analyzed the 456 member SLC family that establishes the small molecule constitution of cells. This analysis identified SLC gene cohorts regulated by GATA1 and/or GATA2 during erythroid differentiation. As >50 SLC genes shared GATA factor regulation, a common mechanism established multiple members of this family. These genes included Slc29a1 encoding an equilibrative nucleoside transporter (Slc29a1/ENT1) that utilizes adenosine as a preferred substrate. Slc29a1 promoted erythroblast survival and differentiation ex vivo. Targeted ablation of murine Slc29a1 in erythroblasts attenuated erythropoiesis and erythrocyte regeneration in response to acute anemia. Our results reveal a GATA factor-regulated SLC ensemble, with a nucleoside transporter component that promotes erythropoiesis and prevents anemia, and establish a mechanistic link between GATA factor and adenosine mechanisms. We propose that integration of the GATA factor-adenosine circuit with other components of the GATA factor-regulated SLC ensemble establishes the small molecule repertoire required for progenitor cells to efficiently generate erythrocytes., Author summary GATA transcription factors endow blood stem and progenitor cells with activities to produce progeny that transport oxygen to protect cells and tissues, evade pathogens and control physiological processes. GATA factors regulate hundreds of genes, and the actions of these genes mediate important biological functions. While the genes have been documented, many questions remain regarding how the “network” components mediate biological functions. The networks include members of large gene families, and the relationships between the regulation and function of individual family members is not well understood. Analyzing datasets from genetic complementation and enhancer mutant systems revealed that GATA factors regulate an ensemble of membrane transporters termed solute carrier proteins (SLCs), which dictate the small molecule composition of cells. Genetic analyses with Slc29a1, which transports adenosine, revealed its function to promote erythrocyte development, and Slc29a1 attenuated anemia in a mouse model. This study revealed the importance of SLC transporters in GATA factor networks. We propose that the GATA factor-adenosine circuit integrates with other SLCs to establish/maintain the small molecule constitution of progenitor cells as a new mechanism to control blood cell development.

By functioning as an enzyme cofactor, hemoglobin component, and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we merged GATA-1/heme-regulated sectors of the proteome and transcriptome. This multi-omic analysis revealed a GATA-1/heme circuit involving hemoglobin subunits, ubiquitination components, and proteins not implicated in erythrocyte biology, including the zinc exporter Slc30a1. Though GATA-1 induced expression of Slc30a1 and the zinc importer Slc39a8, Slc39a8 dominantly increased intracellular zinc, which conferred erythroblast survival. Subsequently, a zinc transporter switch, involving decreased importer and sustained exporter expression, reduced intracellular zinc during terminal differentiation. Downregulating Slc30a1 increased intracellular zinc and, strikingly, accelerated differentiation. This analysis established a conserved paradigm in which a GATA-1/heme circuit controls trace metal transport machinery and trace metal levels as a mechanism governing cellular differentiation.

Background Structural variation among histone H1 variants confers distinct modes of chromatin binding that are important for differential regulation of chromatin condensation, gene expression and other processes. Changes in the expression and genomic distributions of H1 variants during cell differentiation appear to contribute to phenotypic differences between cell types, but few details are known about the roles of individual H1 variants and the significance of their disparate capacities for phosphorylation. In this study, we investigated the dynamics of interphase phosphorylation at specific sites in individual H1 variants during the differentiation of pluripotent NT2 and mouse embryonic stem cells and characterized the kinases involved in regulating specific H1 variant phosphorylations in NT2 and HeLa cells. Results Here, we show that the global levels of phosphorylation at H1.5-Ser18 (pS18-H1.5), H1.2/H1.5-Ser173 (pS173-H1.2/5) and H1.4-Ser187 (pS187-H1.4) are regulated differentially during pluripotent cell differentiation. Enrichment of pS187-H1.4 near the transcription start site of pluripotency factor genes in pluripotent cells is markedly reduced upon differentiation, whereas pS187-H1.4 levels at housekeeping genes are largely unaltered. Selective inhibition of CDK7 or CDK9 rapidly diminishes pS187-H1.4 levels globally and its enrichment at housekeeping genes, and similar responses were observed following depletion of CDK9. These data suggest that H1.4-S187 is a bona fide substrate for CDK9, a notion that is further supported by the significant colocalization of CDK9 and pS187-H1.4 to gene promoters in reciprocal re-ChIP analyses. Moreover, treating cells with actinomycin D to inhibit transcription and trigger the release of active CDK9/P-TEFb from 7SK snRNA complexes induces the accumulation of pS187-H1.4 at promoters and gene bodies. Notably, the levels of pS187-H1.4 enrichment after actinomycin D treatment or cell differentiation reflect the extent of CDK9 recruitment at the same loci. Remarkably, the global levels of H1.5-S18 and H1.2/H1.5-S173 phosphorylation are not affected by these transcription inhibitor treatments, and selective inhibition of CDK2 does not affect the global levels of phosphorylation at H1.4-S187 or H1.5-S18. Conclusions Our data provide strong evidence that H1 variant interphase phosphorylation is dynamically regulated in a site-specific and gene-specific fashion during pluripotent cell differentiation, and that enrichment of pS187-H1.4 at genes is positively related to their transcription. H1.4-S187 is likely to be a direct target of CDK9 during interphase, suggesting the possibility that this particular phosphorylation may contribute to the release of paused RNA pol II. In contrast, the other H1 variant phosphorylations we investigated appear to be mediated by distinct kinases and further analyses are needed to determine their functional significance. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0135-3) contains supplementary material, which is available to authorized users.

The rapid development of high-throughput technologies enables researchers to sequence the whole metagenome of a microbial community sampled directly from the environment. The assignment of these sequence reads into different species or taxonomical classes is a crucial step for metagenomic analysis, which is referred to as binning of metagenomic data. Most traditional binning methods rely on known reference genomes for accurate assignment of the sequence reads, therefore cannot classify reads from unknown species without the help of close references. To overcome this drawback, unsupervised learning based approaches have been proposed, which need not any known species’ reference genome for help. In this paper, we introduce a novel unsupervised method called MCluster for binning metagenomic sequences. This method uses N-grams to extract sequence features and utilizes automatic feature weighting to improve the performance of the basic K-means clustering algorithm. We evaluate MCluster on a variety of simulated data sets and a real data set, and compare it with three latest binning methods: AbundanceBin, MetaCluster 3.0, and MetaCluster 5.0. Experimental results show that MCluster achieves obviously better overall performance ( $F$ -measure) than AbundanceBin and MetaCluster 3.0 on long metagenomic reads ( $\ge$ 800 bp); while compared with MetaCluster 5.0, MCluster obtains a larger sensitivity, and a comparable yet more stable $F$ -measure on short metagenomic reads ( ${ bp). This suggests that MCluster can serve as a promising tool for effectively binning metagenomic sequences.

Additional file 3: Figure S2. H1 variant expression and phosphorylation in differentiating NT2 cells. Crude H1 in acid extracts of nuclei isolated from NT2 cells induced to differentiate with 10 μM retinoic acid for 0, 1, 3 or 7 days was fractionated by hydrophobic interaction chromatography (HIC). Eluate absorbance at 214 nm (Y axis) is plotted relative to time (X axis) for equivalent portions of each separation. The relative elution positions of H1.2, H1.3, H1.4 and H1.5 and the phosphorylation stoichiometry of their major interphase forms, as characterized previously [23], are indicated above the 0 day trace. H1.0 coelutes as a broad peak that overlaps with both phosphorylated and non-phosphorylated H1.5 (data not shown).

Additional file 4: Figure S3. pS187-H1.4 is preferentially enriched in active chromatin. A, HeLa cells were treated with DMSO or 500 nM ActD for 1 h and the levels of CDK9 and pS187-H1.4 at the promoters of ACTG1, GAPDH or MYOD1 and one intergenic region were assessed by ChIP-qPCR. B, HeLa cells were treated with DMSO or 1 µM FLVP for 1 h and the levels of CDK9 and pS187-H1.4 at the promoters of ACTG1, GAPDH or MYOD1 and one intergenic region were assessed by ChIP-qPCR. Negative control ChIP assays employed non-immune rabbit IgG (rIg) in place of primary antisera for the first ChIP. The data are expressed as percent relative to input DNA (mean ± s.e.m., *: p

By functioning as an enzyme cofactor, hemoglobin component and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we used CRISPR/Cas9 to delete intron 1 and 8 GATA motifs in Alas2, encoding the rate-limiting enzyme in heme biosynthesis, in erythroid precursor G1E-ER-GATA-1 cells (Tanimura et al., 2016, EMBO Rep.). These cells stably express a b-estradiol-activated allele encoding the estrogen receptor hormone binding domain fused to GATA-1. The GATA motif mutations abrogated GATA-1-mediated activation of Alas2 and decreased heme ~30 fold. We merged GATA-1/heme-regulated sectors of the proteome and transcriptome. This analysis revealed a GATA-1/heme circuit involving hemoglobin subunits, ubiquitination components, and proteins not implicated in erythrocyte biology, including the zinc exporter Slc30a1. Slc30a1 and the zinc importer Slc39a8 were the most highly expressed zinc transporters in G1E-ER-GATA-1 cells. Both were GATA-1-activated, and Slc30a1, but not Slc39a8, expression declined in heme-deficient Alas2-intronic mutant cells. We tested whether the zinc transporter genes Slc30a1 and Slc39a8 that share GATA-1 regulation and differ in heme regulation are controlled similarly in primary cells. Lineage-depleted hematopoietic precursors from murine fetal livers were cultured for 72 hours, and flow cytometry was used to isolate cells based on erythroid surface markers. Whereas Slc30a1 expression increased 5.7-fold upon differentiation, Slc39a8 increased 4.6-fold during the initial phase, but was downregulated 8.7-fold thereafter. To quantify intracellular zinc levels, we used the fluorescent zinc indicator FluoZin-3. The FluoZin-3 signal increased during early maturation and decreased thereafter, as predicted by the zinc transporter switch, in which expression of the zinc exporter and importer was sustained and decreased, respectively, during terminal differentiation. We demonstrated that the zinc transporter switch occurs in a primary human erythroblast culture system. Intracellular zinc rises during initial erythroid maturation, followed by a steep decline during terminal differentiation. The heme biosynthesis inhibitor succinylacetone reduced SLC30A1 expression 2.8-fold (p < 0.01), as well as other heme-dependent globin genes HBB and HBA1. These primary mouse and human cell studies revealed an evolutionarily conserved mechanism to differentially regulate zinc transporter genes during erythroid differentiation, culminating in a switch from importer and exporter expression to solely exporter expression. To test whether reconfiguring the mechanism governing intracellular zinc impacts differentiation and to elucidate Slc30a1 and Slc39a8 function, we developed multiple shRNAs and used them in loss-of-function studies with lineage-negative hematopoietic precursors from murine fetal livers (E14.5). Slc39a8 downregulation decreased intracellular zinc levels in immature erythroblasts. We also used the zinc chelator TPEN to reduce zinc in immature erythroblasts. TPEN reduced intracellular zinc and considerably decreased the live cell population (p < 0.001). Downregulating Slc30a1 increased intracellular zinc 2.2-fold and, strikingly, accelerated differentiation (p < 0.001). This analysis established a conserved paradigm in which a GATA-1/heme circuit controls trace metal transport machinery and trace metal levels as a mechanism governing cellular differentiation. Ongoing studies involve the development of innovative strategies to leverage this mechanism to enhance or suppress erythrocyte development with primary mouse and human systems. We are conducting multi-disciplinary metallomic analyses to test the hypothesis that the dynamic changes in zinc selectively commission and decommission zinc-binding proteins in erythroblasts. We anticipate that this approach can be readily extended to other sectors of the hematopoietic system and more broadly. Disclosures No relevant conflicts of interest to declare.

Many metazoan cell types differentially express multiple non-allelic amino acid sequence variants of histone H1. Although early work revealed that H1 variants, collectively, are phosphorylated during interphase and mitosis, differences between individual H1 variants in the sites they possess for mitotic and interphase phosphorylation have been elucidated only relatively recently. Here, we review current knowledge on the regulation and function of interphase H1 phosphorylation, with a particular emphasis on how differences in interphase phosphorylation among the H1 variants of mammalian cells may enable them to have differential effects on transcription and other chromatin processes.

In the past decades, with the rapid development of high-throughput technologies, biology research has generated an unprecedented amount of data. In order to store and process such a great amount of data, cloud computing and MapReduce were applied to many fields of bioinformatics. In this paper, we first introduce the basic concepts of cloud computing and MapReduce, and their applications in bioinformatics. We then highlight some problems challenging the applications of cloud computing and MapReduce to bioinformatics. Finally, we give a brief guideline for using cloud computing in biology research.

Background Plant microRNAs (miRNAs) have been revealed to play important roles in developmental control, hormone secretion, cell differentiation and proliferation, and response to environmental stresses. However, our knowledge about the regulatory mechanisms and functions of miRNAs remains very limited. The main difficulties lie in two aspects. On one hand, the number of experimentally validated miRNA targets is very limited and the predicted targets often include many false positives, which constrains us to reveal the functions of miRNAs. On the other hand, the regulation of miRNAs is known to be spatio-temporally specific, which increases the difficulty for us to understand the regulatory mechanisms of miRNAs. Description In this paper we present miRFANs, an online database for Arabidopsis thaliana miRNA function annotations. We integrated various type of datasets, including miRNA-target interactions, transcription factor (TF) and their targets, expression profiles, genomic annotations and pathways, into a comprehensive database, and developed various statistical and mining tools, together with a user-friendly web interface. For each miRNA target predicted by psRNATarget, TargetAlign and UEA target-finder, or recorded in TarBase and miRTarBase, the effect of its up-regulated or down-regulated miRNA on the expression level of the target gene is evaluated by carrying out differential expression analysis of both miRNA and targets expression profiles acquired under the same (or similar) experimental condition and in the same tissue. Moreover, each miRNA target is associated with gene ontology and pathway terms, together with the target site information and regulating miRNAs predicted by different computational methods. These associated terms may provide valuable insight for the functions of each miRNA. Conclusion First, a comprehensive collection of miRNA targets for Arabidopsis thaliana provides valuable information about the functions of plant miRNAs. Second, a highly informative miRNA-mediated genetic regulatory network is extracted from our integrative database. Third, a set of statistical and mining tools is equipped for analyzing and mining the database. And fourth, a user-friendly web interface is developed to facilitate the browsing and analysis of the collected data.