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• Histone H2B monoubiquitination (H2Bub1) is recognized as a crucial eukaryotic regulatory mechanism that controls a range of cellular processes during both development and adaptation to environmental changes. In Arabidopsis, the E2 conjugated enzymes UBIQUITIN CARRIER PROTEINs (UBCs) -1 and -2 mediate ubiquitination of H2B. Here, we elucidated the functions of UBC1 and -2 in salt-stress responses and revealed their downstream target genes. • Real-time quantitative PCR assays showed that UBC1 and -2 positively regulated the salt-induced expression of MYB42 and Mitogen-Activated Protein Kinase 4 (MPK4). Chromatin immunoprecipitation assays revealed that H2Bub1 was enriched weakly on the chromatin of MYB42 and MPK4 in the ubc1,2 mutant. We further determined that UBC1/2-mediated H2Bub1 enhanced the level of histone H3 tri-methylated on K4 (H3K4me3) in the chromatin of MYB42 and MPK4 under salt-stress conditions. • MPK4 interacted with and phosphorylated MYB42. The MPK4-mediated MYB42 phosphorylation enhanced the MYB42 protein stability and transcriptional activity under salt-stress conditions. We further showed that MYB42 directly bound to the SALT OVERLY SENSITIVE 2 (SOS2) promoter and mediated the rapid induction of its expression after a salt treatment. • Our results indicate that UBC1 and -2 positively regulate salt-stress responses by modulating MYB42-mediated SOS2 expression.

The H2B.8 variant has been diverged from other variants by its extended N-terminal region that possesses a conserved domain. We generated transgenic Arabidopsis plants expressing H2B.9 (class I), H2B.5 (class II) and H2B.8 (class III) fused to GFP under the 35 S promoter and studied their nuclear distribution and function. H2B.8-GFP showed peculiar nuclear localization at chromocenters in all cell types examined, while H2B.5-GFP and H2B.9-GFP displayed various patterns often dependent on cell types. H2B variants faithfully assembled onto nucleosomes showing no effect on nuclear organization; H2B.8-GFP appeared as three distinct isoforms in which one isoform appeared to be SUMOylated. Interestingly, transient expression in protoplasts revealed H2B.8 nuclear localization distinct from transgenic plants as it was restricted to the nuclear periphery generating a distinctive ring-like appearance accompanied by nuclear size reduction. This unique appearance was abolished by deletion of the N-terminal conserved domain or when H2B.8-GFP is transiently expressed in ddm1 protoplasts. GFP-TRAP-coupled proteome analysis uncovered H2B.8-partner proteins including H2A.W.12, which characterizes heterochromatin. Thus, our data highlight H2B.8 as a unique variant evolved in angiosperms to control chromatin compaction/aggregation and uncover cis- and trans-regulatory elements underlying its nuclear distribution and function.

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

Objectives Cell reprogramming has significant impacts on their potential application in regenerative medicine. Chromatin remodelling plays a very important role in cell reprogramming, but its underlying mechanism remains poorly understood. Materials and methods RNA‐seq, quantitative RT‐PCR and western blot analysis were applied to study the role of RNF20 and H2B ubiquitination during mouse somatic cell reprogramming. Chromatin structure and the recruitment of transcription factors were analysed by ChIP‐seq, micrococcal nuclease sensitivity assays and immunofluorescence staining. Results We show that RNF20 is highly expressed at the early stage of reprogramming along with the accumulation of H2B ubiquitination at the same stage, and Rnf20 knockout results in the failure of reprogramming at the initial stage but not the maturation and stabilization stages. RNA‐seq showed that Rnf20 knockout mainly affects the early stage of cell reprogramming by impairing the transcription of MET‐related genes and early pluripotency genes. Importantly, Rnf20 knockout results in a more compacted chromosomes structure in reprogrammable cells, suppressing the recruitment of reprogramming transcription factors to their proper locations on the chromosomes, and finally resulting in the failure of pluripotent gene network establishment. Conclusions Histone H2B ubiquitination mediated chromatin relaxation is essential for the induction of somatic cell reprogramming. RNF20 and H2Bub are highly expressed in the early stages of cell reprogramming, and Rnf20 knockout mainly affects the early stages of cell reprogramming. The deficiency of RNF20 in reprogrammable cells affected chromatin relaxation by reducing H2B ubiquitination, thus preventing the recruitment of SOX2 to the proper positions on the chromosome and repressing the transcription of pluripotency genes and MET‐related genes during the early stages of cell reprogramming.

Studies in Saccharomyces cerevisiae and Schizosaccharomyces pombe have enhanced our understanding of the regulation and functions of histone H2B monoubiquitination (H2Bub1), a key epigenetic marker with important roles in transcription and other processes. The detection of H2Bub1 in yeasts using immunoblotting has been greatly facilitated by the commercial availability of antibodies against yeast histone H2B and the cross-reactivity of an antibody raised against monoubiquitinated human H2BK120. These antibodies have obviated the need to express epitope-tagged histone H2B to detect H2Bub1 in yeasts. Here, we provide a step-by-step protocol and best practices for the quantification of H2Bub1 in yeast systems, from cell extract preparation to immunoblotting using the commercially available antibodies. We demonstrate that the commercial antibodies can effectively and accurately detect H2Bub1 in S. cerevisiae and S. pombe. Further, we show that the C-terminal epitope-tagging of histone H2B alters the steady-state levels of H2Bub1 in yeast systems. We report a sectioned blot probing approach combined with the serial dilution of protein lysates and the use of reversibly stained proteins as loading controls that together provide a cost-effective and sensitive method for the quantitative evaluation of H2Bub1 in yeast.

Histones are constitutive components of nucleosomes and key regulators of chromatin structure. We previously observed that an extrachromosomal histone H2B (ecH2B) localizes at the intercellular bridge (ICB) connecting the two daughter cells during cytokinesis independently of DNA and RNA. Here, we show that ecH2B binds and colocalizes with CHMP4B, a key component of the ESCRT-III machinery responsible for abscission, the final step of cell division. Abscission requires the formation of an abscission site at the ICB where the ESCRT-III complex organizes into narrowing cortical helices that drive the physical separation of sibling cells. ecH2B depletion does not prevent membrane cleavage rather results in abscission delay and accumulation of abnormally long and thin ICBs. In the absence of ecH2B, CHMP4B and other components of the fission machinery, such as IST1 and Spastin, are recruited to the ICB and localize at the midbody. However, in the late stage of abscission, these fission factors fail to re-localize at the periphery of the midbody and the abscission site fails to form. These results show that extrachromosomal activity of histone H2B is required in the formation of the abscission site and the proper localization of the fission machinery.

BackgroundThe coprophilous ascomycete Sordaria fimicola usually reproduces sexually. Sexual differentiation in S. fimicola is accompanied by cellular and morphological changes, followed by multicellular tissue development to complete the sexual cycle. Although the morphological features of the sexual reproductive structure in S. fimicola have been well characterized, little is known about the nuclear dynamics and organization during these processes. Therefore, in this study, we successfully developed an Agrobacterium-mediated protoplast transformation protocol and generated histone H2B-mCherry-labeled S. fimicola strains. The life cycle of S. fimicola begins with germination of the ascospore and ends with the formation and discharge of new ascospores from the mature black sexual fruiting bodies, the so-called perithecia. The nuclear dynamics of the fluorescently labeled strains were examined during ascospore germination, hyphal elongation, and hyphal fusion using fluorescent microscopy.ResultsLive imaging revealed that the nuclei in the germlings and fusion hyphae during the pre-contact interaction are located adjacent to the tip.ConclusionsThis is the first report of the application of a fluorescence labeling technique in S. fimicola. This application will help researchers gain a better understanding of nuclear distribution and investigate the protein–protein interaction networks during fruiting body formation for advanced molecular genetic studies in S. fimicola.

Methyltransferases of the mixed-lineage leukaemia (MLL) family—which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B—implement methylation of histone H3 on lysine 4 (H3K4), and have critical and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development 1 – 6 . The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are associated with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities 7 – 9 . Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 methyl marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favours open chromatin structures, but the underlying mechanism remains unknown 10 – 12 . Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules associated with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the association between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification. Cryo-electron microscopy structures of mixed-lineage leukaemia methyltransferases 1 and 3 associated with unmodified or mono-ubiquitinated nucleosome reveal the structural basis for the activity specificity and regulation of these enzymes.

Chromatin features are widely used for genome-scale mapping of enhancers. However, discriminating active enhancers from other cis -regulatory elements, predicting enhancer strength and identifying their target genes is challenging. Here we establish histone H2B N-terminus multisite lysine acetylation (H2BNTac) as a signature of active enhancers. H2BNTac prominently marks candidate active enhancers and a subset of promoters and discriminates them from ubiquitously active promoters. Two mechanisms underlie the distinct H2BNTac specificity: (1) unlike H3K27ac, H2BNTac is specifically catalyzed by CBP/p300; (2) H2A–H2B, but not H3–H4, are rapidly exchanged through transcription-induced nucleosome remodeling. H2BNTac-positive candidate enhancers show a high validation rate in orthogonal enhancer activity assays and a vast majority of endogenously active enhancers are marked by H2BNTac and H3K27ac. Notably, H2BNTac intensity predicts enhancer strength and outperforms current state-of-the-art models in predicting CBP/p300 target genes. These findings have broad implications for generating fine-grained enhancer maps and modeling CBP/p300-dependent gene regulation. Histone H2B N-terminus multisite lysine acetylation (H2BNTac) is identified as a signature of active enhancers. H2BNTac-positive putative enhancers are validated using orthogonal enhancer activity assays.

The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.