SWR-C and INO80 chromatin remodelers recognize nucleosome-free regions near +1 nucleosomes.

SWR-C/SWR1 and INO80 are multisubunit complexes that catalyze the deposition and removal, respectively, of histone variant H2A.Z from the first nucleosome at the start of genes. How they target and engage these +1 nucleosomes is unclear. Using ChIP-exo, we identified the subnucleosomal placement of 20 of their subunits across the yeast genome. The Swc2 subunit of SWR-C bound a narrowly defined region in the adjacent nucleosome-free region (NFR), where it positioned the Swr1 subunit over one of two sites of H2A.Z deposition at +1. The genomic binding maps suggest that many subunits have a rather plastic organization that allows subunits to exchange between the two complexes. One outcome of promoting H2A/H2A.Z exchange was an enhanced turnover of entire nucleosomes, thereby creating dynamic chromatin at the start of genes. Our findings provide unifying concepts on how these two opposing chromatin remodeling complexes function selectively at the +1 nucleosome of nearly all genes.

Size-based expectation maximization for characterizing nucleosome positions and subtypes.

Genome-wide nucleosome profiles are predominantly characterized using MNase-seq, which involves extensive MNase digestion and size selection to enrich for mono-nucleosome-sized fragments. Most available MNase-seq analysis packages assume that nucleosomes uniformly protect 147-bp DNA fragments. However, some nucleosomes with atypical histone or chemical compositions protect shorter lengths of DNA. The rigid assumptions imposed by current nucleosome analysis packages potentially prevent investigators from understanding the regulatory roles played by atypical nucleosomes. To enable the characterization of different nucleosome types from MNase-seq data, we introduce the Size-based Expectation Maximization (SEM) nucleosome-calling package. SEM employs a hierarchical Gaussian mixture model to estimate nucleosome positions and subtypes. Nucleosome subtypes are automatically identified based on the distribution of protected DNA fragments. Benchmark analysis indicates that SEM is on par with existing packages in terms of standard nucleosome-calling accuracy metrics, while uniquely providing the ability to characterize nucleosome subtype identities. Applying SEM to a low-dose MNase-H2B-ChIP-seq dataset from mouse embryonic stem cells, we identified three nucleosome types: short-fragment nucleosomes; canonical nucleosomes; and di-nucleosomes. Short-fragment nucleosomes can be divided further into two subtypes based on their chromatin accessibility. Interestingly, short-fragment nucleosomes in accessible regions exhibit high MNase sensitivity and are enriched at transcription start sites (TSSs) and CTCF peaks, similar to previously reported 'fragile nucleosomes'. These SEM-defined accessible short-fragment nucleosomes are found not just in promoters, but also in distal regulatory regions. Additional analyses reveal their colocalization with the chromatin remodelers Chd6, Chd8, and Ep400. In summary, SEM provides an effective platform for exploration of nonstandard nucleosome subtypes.

Genome-wide nucleosome specificity and directionality of chromatin remodelers.

How chromatin remodelers cooperate to organize nucleosomes around the start and end of genes is not known. We determined the genome-wide binding of remodeler complexes SWI/SNF, RSC, ISW1a, ISW1b, ISW2, and INO80 to individual nucleosomes in Saccharomyces, and determined their functional contributions to nucleosome positioning through deletion analysis. We applied ultra-high-resolution ChIP-exo mapping to Isw2 to determine its subnucleosomal orientation and organization on a genomic scale. Remodelers interacted with selected nucleosome positions relative to the start and end of genes and produced net directionality in moving nucleosomes either away or toward nucleosome-free regions at the 5' and 3' ends of genes. Isw2 possessed a subnucleosomal organization in accord with biochemical and crystallographic-based models that place its linker binding region within promoters and abutted against Reb1-bound locations. Together, these findings reveal a coordinated position-specific approach taken by remodelers to organize genic nucleosomes into arrays.

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6.

The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.

Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells.

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

Structural evidence for Nap1-dependent H2A-H2B deposition and nucleosome assembly.

Nap1 is a histone chaperone involved in the nuclear import of H2A-H2B and nucleosome assembly. Here, we report the crystal structure of Nap1 bound to H2A-H2B together with in vitro and in vivo functional studies that elucidate the principles underlying Nap1-mediated H2A-H2B chaperoning and nucleosome assembly. A Nap1 dimer provides an acidic binding surface and asymmetrically engages a single H2A-H2B heterodimer. Oligomerization of the Nap1-H2A-H2B complex results in burial of surfaces required for deposition of H2A-H2B into nucleosomes. Chromatin immunoprecipitation-exonuclease (ChIP-exo) analysis shows that Nap1 is required for H2A-H2B deposition across the genome. Mutants that interfere with Nap1 oligomerization exhibit severe nucleosome assembly defects showing that oligomerization is essential for the chaperone function. These findings establish the molecular basis for Nap1-mediated H2A-H2B deposition and nucleosome assembly.

Codependency of H2B monoubiquitination and nucleosome reassembly on Chd1.

Monoubiquitination of histone H2B on Lys 123 (H2BK123ub) is a transient histone modification considered to be essential for establishing H3K4 and H3K79 trimethylation by Set1/COMPASS and Dot1, respectively. Here, we identified Chd1 as a factor that is required for the maintenance of high levels of H2B monoubiquitination, but not for H3K4 and H3K79 trimethylation. Loss of Chd1 results in a substantial loss of H2BK123ub levels with little to no effect on the genome-wide pattern of H3K4 and H3K79 trimethylation. Our data show that nucleosomal occupancy is reduced in gene bodies in both chd1Δ and, as has been shown, K123A mutant backgrounds. We also demonstrated that Chd1's function in maintaining H2BK123ub levels is conserved from yeast to humans. Our study provides evidence that only small levels of H2BK123ub are necessary for full levels of H3K4 and H3K79 trimethylation in vivo and points to a possible role for Chd1 in positively regulating gene expression through promoting nucleosome reassembly coupled with H2B monoubiquitination.

Genome-wide function of H2B ubiquitylation in promoter and genic regions.

Nucleosomal organization in and around genes may contribute substantially to transcriptional regulation. The contribution of histone modifications to genome-wide nucleosomal organization has not been systematically evaluated. In the present study, we examine the role of H2BK123 ubiquitylation, a key regulator of several histone modifications, on nucleosomal organization at promoter, genic, and transcription termination regions in Saccharomyces cerevisiae. Using high-resolution MNase chromatin immunoprecipitation and sequencing (ChIP-seq), we map nucleosome positioning and occupancy in mutants of the H2BK123 ubiquitylation pathway. We found that H2B ubiquitylation-mediated nucleosome formation and/or stability inhibits the assembly of the transcription machinery at normally quiescent promoters, whereas ubiquitylation within highly active gene bodies promotes transcription elongation. This regulation does not proceed through ubiquitylation-regulated histone marks at H3K4, K36, and K79. Our findings suggest that mechanistically similar functions of H2B ubiquitylation (nucleosome assembly) elicit different functional outcomes on genes depending on its positional context in promoters (repressive) versus transcribed regions (activating).

SEM: sized-based expectation maximization for characterizing nucleosome positions and subtypes.

Genome-wide nucleosome profiles are predominantly characterized using MNase-seq, which involves extensive MNase digestion and size selection to enrich for mono-nucleosome-sized fragments. Most available MNase-seq analysis packages assume that nucleosomes uniformly protect 147bp DNA fragments. However, some nucleosomes with atypical histone or chemical compositions protect shorter lengths of DNA. The rigid assumptions imposed by current nucleosome analysis packages ignore variation in nucleosome lengths, potentially blinding investigators to regulatory roles played by atypical nucleosomes. To enable the characterization of different nucleosome types from MNase-seq data, we introduce the Size-based Expectation Maximization (SEM) nucleosome calling package. SEM employs a hierarchical Gaussian mixture model to estimate the positions and subtype identity of nucleosomes from MNase-seq fragments. Nucleosome subtypes are automatically identified based on the distribution of protected DNA fragment lengths at nucleosome positions. Benchmark analysis indicates that SEM is on par with existing packages in terms of standard nucleosome-calling accuracy metrics, while uniquely providing the ability to characterize nucleosome subtype identities. Using SEM on a low-dose MNase H2B MNase-ChIP-seq dataset from mouse embryonic stem cells, we identified three nucleosome types: short-fragment nucleosomes, canonical nucleosomes, and di-nucleosomes. The short-fragment nucleosomes can be divided further into two subtypes based on their chromatin accessibility. Interestingly, the subset of short-fragment nucleosomes in accessible regions exhibit high MNase sensitivity and display distribution patterns around transcription start sites (TSSs) and CTCF peaks, similar to the previously reported "fragile nucleosomes". These SEM-defined accessible short-fragment nucleosomes are found not just in promoters, but also in enhancers and other regulatory regions. Additional investigations reveal their co-localization with the chromatin remodelers Chd6, Chd8, and Ep400. In summary, SEM provides an effective platform for distinguishing various nucleosome subtypes, paving the way for future exploration of non-standard nucleosomes.

Cross-species transcriptomics reveals bifurcation point during the arterial-to-hemogenic transition.

Hemogenic endothelium (HE) with hematopoietic stem cell (HSC)-forming potential emerge from specialized arterial endothelial cells (AECs) undergoing the endothelial-to-hematopoietic transition (EHT) in the aorta-gonad-mesonephros (AGM) region. Characterization of this AECs subpopulation and whether this phenomenon is conserved across species remains unclear. Here we introduce HomologySeeker, a cross-species method that leverages refined mouse information to explore under-studied human EHT. Utilizing single-cell transcriptomic ensembles of EHT, HomologySeeker reveals a parallel developmental relationship between these two species, with minimal pre-HSC signals observed in human cells. The pre-HE stage contains a conserved bifurcation point between the two species, where cells progress towards HE or late AECs. By harnessing human spatial transcriptomics, we identify ligand modules that contribute to the bifurcation choice and validate CXCL12 in promoting hemogenic choice using a human in vitro differentiation system. Our findings advance human arterial-to-hemogenic transition understanding and offer valuable insights for manipulating HSC generation using in vitro models.

Corrigendum: RUNX1 Upregulates CENPE to Promote Leukemic Cell Proliferation.

[This corrects the article DOI: 10.3389/10.3389/fmolb.2021.692880.].

Chemical screening reveals Ronidazole is a superior prodrug to Metronidazole for nitroreductase-induced cell ablation system in zebrafish larvae.

The Metronidazole (MTZ)/nitroreductase (NTR)-mediated cell ablation system is the most commonly used chemical-genetic cell ablation method in zebrafish. This system can specifically ablate target cells under spatial and temporal control. The MTZ/NTR system has become a widely used cell ablation system in biological, developmental, and functional studies. However, the inadequate cell-ablation ability of some cell types and the side effects of high concentration MTZ impede extensive applications of the MTZ/NTR system. In the present study, the US drug collection library was searched to extend the NTR system. Six MTZ analogs were found, and the cell-ablation ability of these analogs was tested in zebrafish larvae. The results revealed that two of the NTR substrates, Furazolidone and Ronidazole, ablated target cells more efficiently than MTZ at lower concentrations. Furthermore, the working concentration of Ronidazole, but not Furazolidone and MTZ, did not affect axonal bridge formation during spinal cord regeneration. Our results, taken together, indicate that Ronidazole is a superior prodrug to MTZ for the NTR system, especially for the study of neuron regeneration in zebrafish larvae.

Asxl1 C-terminal mutation perturbs neutrophil differentiation in zebrafish.

ASXL1 is one of the most frequently mutated genes in malignant myeloid diseases. In patients with myeloid malignancies, ASXL1 mutations are usually heterozygous frameshift or nonsense mutations leading to C-terminal truncation. Current disease models have predominantly total loss of ASXL1 or overexpressed C-terminal truncations. These models cannot fully recapitulate leukemogenesis and disease progression. We generated an endogenous C-terminal-truncated Asxl1 mutant in zebrafish that mimics human myeloid malignancies. At the embryonic stage, neutrophil differentiation was explicitly blocked. At 6 months, mutants initially exhibited a myelodysplastic syndrome-like phenotype with neutrophilic dysplasia. At 1 year, about 13% of mutants further acquired the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or increased progenitors, which mimics acute myeloid leukemia. These features are comparable to myeloid malignancy progression in humans. Furthermore, transcriptome analysis, inhibitor treatment, and rescue assays indicated that asxl1-induced neutrophilic dysplasia was associated with reduced expression of bmi1a, a subunit of polycomb repressive complex 1 and a reported myeloid leukemia-associated gene. Our model demonstrated that neutrophilic dysplasia caused by asxl1 mutation is a foundation for the progression of myeloid malignancies, and illustrated a possible effect of the Asxl1-Bmi1a axis on regulating neutrophil development.

Comprehensive understanding of Tn5 insertion preference improves transcription regulatory element identification.

Tn5 transposase, which can efficiently tagment the genome, has been widely adopted as a molecular tool in next-generation sequencing, from short-read sequencing to more complex methods such as assay for transposase-accessible chromatin using sequencing (ATAC-seq). Here, we systematically map Tn5 insertion characteristics across several model organisms, finding critical parameters that affect its insertion. On naked genomic DNA, we found that Tn5 insertion is not uniformly distributed or random. To uncover drivers of these biases, we used a machine learning framework, which revealed that DNA shape cooperatively works with DNA motif to affect Tn5 insertion preference. These intrinsic insertion preferences can be modeled using nucleotide dependence information from DNA sequences, and we developed a computational pipeline to correct for these biases in ATAC-seq data. Using our pipeline, we show that bias correction improves the overall performance of ATAC-seq peak detection, recovering many potential false-negative peaks. Furthermore, we found that these peaks are bound by transcription factors, underscoring the biological relevance of capturing this additional information. These findings highlight the benefits of an improved understanding and precise correction of Tn5 insertion preference.

Prognostic Prediction of Cytogenetically Normal Acute Myeloid Leukemia Based on a Gene Expression Model.

Acute myeloid leukemia (AML) refers to a heterogeneous group of hematopoietic malignancies. The well-known European Leukemia Network (ELN) stratifies AML patients into three risk groups, based primarily on the detection of cytogenetic abnormalities. However, the prognosis of cytogenetically normal AML (CN-AML), which is the largest AML subset, can be hard to define. Moreover, the clinical outcomes associated with this subgroup are diverse. In this study, using transcriptome profiles collected from CN-AML patients in the BeatAML cohort, we constructed a robust prognostic Cox model named NEST (Nine-gEne SignaTure). The validity of NEST was confirmed in four external independent cohorts. Moreover, the risk score predicted by the NEST model remained an independent prognostic factor in multivariate analyses. Further analysis revealed that the NEST model was suitable for bone marrow mononuclear cell (BMMC) samples but not peripheral blood mononuclear cell (PBMC) samples, which indirectly indicated subtle differences between BMMCs and PBMCs. Our data demonstrated the robustness and accuracy of the NEST model and implied the importance of the immune dysfunction in the leukemogenesis that occurs in CN-AML, which shed new light on the further exploration of molecular mechanisms and treatment guidance for CN-AML.

RUNX1 Upregulates CENPE to Promote Leukemic Cell Proliferation.

RUNX1 is a Runt family transcription factor that plays a critical role in normal hematopoiesis, including the differentiation and proliferation of hematopoietic cells. RUNX1 mutations, including chromosomal translocations, cause abnormal cell differentiation, but the mutation alone is not sufficient to cause leukemia. In MLL-fusion-induced leukemia, dysregulated wild-type RUNX1 can promote leukemia survival. Nevertheless, the underlying mechanisms of dysregulated wild-type RUNX1 in leukemia development have not been fully elucidated. This study overexpressed and knocked down RUNX1 expression in THP-1 human leukemia cells and CD34+ hematopoietic stem/progenitor cells to investigate the biological functions affected by dysregulated RUNX1. Our data indicated RUNX1 facilitated proliferation to promote leukemia cell growth. Furthermore, we demonstrated that RUNX1 knockdown in leukemia cells drastically diminished colony-forming ability. Finally, the RUNX1-knocked down cell depletion phenotype could be rescued by overexpression of CENPE, a cell proliferation gene and a RUNX1 direct target gene. Our results indicate a possible mechanism involving the RUNX1-CENPE axis on promoting leukemic cell growth.

Functional Verification of Novel ELMO1 Variants by Live Imaging in Zebrafish.

ELMO1 (Engulfment and Cell Motility1) is a gene involved in regulating cell motility through the ELMO1-DOCK2-RAC complex. Contrary to DOCK2 (Dedicator of Cytokinesis 2) deficiency, which has been reported to be associated with immunodeficiency diseases, variants of ELMO1 have been associated with autoimmune diseases, such as diabetes and rheumatoid arthritis (RA). To explore the function of ELMO1 in immune cells and to verify the functions of novel ELMO1 variants in vivo, we established a zebrafish elmo1 mutant model. Live imaging revealed that, similar to mammals, the motility of neutrophils and T-cells was largely attenuated in zebrafish mutants. Consequently, the response of neutrophils to injury or bacterial infection was significantly reduced in the mutants. Furthermore, the reduced mobility of neutrophils could be rescued by the expression of constitutively activated Rac proteins, suggesting that zebrafish elmo1 mutant functions via a conserved mechanism. With this mutant, three novel human ELMO1 variants were transiently and specifically expressed in zebrafish neutrophils. Two variants, p.E90K (c.268G>A) and p.D194G (c.581A>G), could efficiently recover the motility defect of neutrophils in the elmo1 mutant; however, the p.R354X (c.1060C>T) variant failed to rescue the mutant. Based on those results, we identified that zebrafish elmo1 plays conserved roles in cell motility, similar to higher vertebrates. Using the transient-expression assay, zebrafish elmo1 mutants could serve as an effective model for human variant verification in vivo.

Snf1-independent, glucose-resistant transcription of Adr1-dependent genes in a mediator mutant of Saccharomyces cerevisiae.

Glucose represses transcription of a network of co-regulated genes in Saccharomyces cerevisiae, ensuring that it is utilized before poorer carbon sources are metabolized. Adr1 is a glucose-regulated transcription factor whose promoter binding and activity require Snf1, the yeast homologue of the AMP-activated protein kinase in higher eukaryotes. In this study we found that a temperature-sensitive allele of MED14, a Mediator middle subunit that tethers the tail to the body, allowed a low level of Adr1-independent ADH2 expression that can be enhanced by Adr1 in a dose-dependent manner. A low level of TATA-independent ADH2 expression was observed in the med14-truncated strain and transcription of ADH2 and other Adr1-dependent genes occurred in the absence of Snf1 and chromatin remodeling coactivators. Loss of ADH2 promoter nucleosomes had occurred in the med14 strain in repressing conditions and did not require ADR1. A global analysis of transcription revealed that loss of Med14 function was associated with both up- and down- regulation of several groups of co-regulated genes, with ADR1-dependent genes being the most highly represented in the upregulated class. Expression of most genes was not significantly affected by the loss of Med14 function.

Ectopically Expressed IL-34 Can Efficiently Induce Macrophage Migration to the Liver in Zebrafish.

Interleukin-34 (IL-34) is a known cytokine that plays an important role in the survival, proliferation, and differentiation of macrophages. In previous studies, IL-34 can induce macrophage migration through syndecan-1 or focal adhesion kinase and extracellular signal-related kinase 1 and 2 pathway. These studies mainly focused on in vitro experiments, but the effect of IL-34 on macrophage migration in vivo is less understood. In our study, we artificially induced macrophage, but not neutrophil, enrichment in the skin or liver by overexpressing IL-34 in epidermal cells or hepatocytes in zebrafish. Live imaging showed that the enrichment of macrophages in the liver is due to the direct attraction of macrophages by IL-34. Our results demonstrated that ectopically expressed IL-34 can induce macrophage migration to liver in vivo.

Targeting of apoptosis gene loci by reprogramming factors leads to selective eradication of leukemia cells.

Applying somatic cell reprogramming strategies in cancer cell biology is a powerful approach to analyze mechanisms of malignancy and develop new therapeutics. Here, we test whether leukemia cells can be reprogrammed in vivo using the canonical reprogramming transcription factors-Oct4, Sox2, Klf4, and c-Myc (termed as OSKM). Unexpectedly, we discover that OSKM can eradicate leukemia cells and dramatically improve survival of leukemia-bearing mice. By contrast, OSKM minimally impact normal hematopoietic cells. Using ATAC-seq, we find OSKM induce chromatin accessibility near genes encoding apoptotic regulators in leukemia cells. Moreover, this selective effect also involves downregulation of H3K9me3 as an early event. Dissection of the functional effects of OSKM shows that Klf4 and Sox2 play dominant roles compared to c-Myc and Oct4 in elimination of leukemia cells. These results reveal an intriguing paradigm by which OSKM-initiated reprogramming induction can be leveraged and diverged to develop novel anti-cancer strategies.