Microsporidian EnP1 alters host cell H2B monoubiquitination and prevents ferroptosis facilitating microsporidia survival.

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1's translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell's resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

Revealing cis- and trans-regulatory elements underlying nuclear distribution and function of the Arabidopsis histone H2B.8 variant.

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.

Usp7 Regulates Glial Lineage Cell-Specific Transcription Factors by Modulating Histone H2B Monoubiquitination.

Histone H2B monoubiquitination (H2Bub1) is a dynamic posttranslational modification which are linked to DNA damage and plays a key role in a wide variety of regulatory transcriptional programs. Cancer cells exhibit a variety of epigenetic changes, particularly any aberrant H2Bub1 has frequently been associated with the development of tumors. Nevertheless, our understanding of the mechanisms governing the histone H2B deubiquitination and their associated functions during stem cell differentiation remain only partially understood. In this study, we wished to investigate the role of deubiquitinating enzymes (DUBs) on H2Bub1 regulation during stem cell differentiation. In a search for potential DUBs for H2B monoubiquitination, we identified Usp7, a ubiquitin-specific protease that acts as a negative regulator of H2B ubiquitination during the neuronal differentiation of mouse embryonic carcinoma cells. Loss of function of the Usp7 gene by a CRISPR/Cas9 system during retinoic acid-mediated cell differentiation contributes to the increase in H2Bub1. Furthermore, knockout of the Usp7 gene particularly elevated the expression of neuronal differentiation related genes including astryocyte-specific markers and oligodendrocyte-specific markers. In particular, glial lineage cell-specific transcription factors including oligodendrocyte transcription factor 2, glial fibrillary acidic protein, and SRY-box transcription factor 10 was significantly upregulated during neuronal differentiation. Thus, our findings suggest a novel role of Usp7 in gliogenesis in mouse embryonic carcinoma cells.

Progress on H2B as a multifunctional protein related to pathogens.

Histone H2B is a member of the core histones, which together with other histones form the nucleosome, the basic structural unit of chromosomes. As scientists delve deeper into histones, researchers gradually realize that histone H2B is not only an important part of nucleosomes, but also plays a momentous role in regulating gene transcription, acting as a receptor and antimicrobial action outside the nucleus. There are a variety of epigenetically modified sites in the H2B tail rich in arginine and lysine, which can occur in ubiquitination, phosphorylation, methylation, acetylation, etc. When stimulated by foreign factors such as bacteria, viruses or parasites, histone H2B can act as a receptor for the recognition of these pathogens, and induce an intrinsic immune response to enhance host defense. In addition, the extrachromosomal histone H2B is also an important anti-microorganism agent, which may be the key to the development of antibiotics in the future. This review aims to summarize the interaction between histone H2B and etiological agents and explore the role of H2B in epigenetic modifications, receptors and antimicrobial activity.

Analysis of Nuclear Dynamics in Nematode-Trapping Fungi Based on Fluorescent Protein Labeling.

Nematophagous fungi constitute a category of fungi that exhibit parasitic behavior by capturing, colonizing, and poisoning nematodes, which are critical factors in controlling nematode populations in nature, and provide important research materials for biological control. Arthrobotrys oligospora serves as a model strain among nematophagous fungi, which begins its life as conidia, and then its hyphae produce traps to capture nematodes, completing its lifestyle switch from saprophytic to parasitic. There have been many descriptions of the morphological characteristics of A. oligospora lifestyle changes, but there have been no reports on the nuclear dynamics in this species. In this work, we constructed A. oligospora strains labeled with histone H2B-EGFP and observed the nuclear dynamics from conidia germination and hyphal extension to trap formation. We conducted real-time imaging observations on live cells of germinating and extending hyphae and found that the nucleus was located near the tip. It is interesting that the migration rate of this type of cell nucleus is very fast, and we speculate that this may be related to the morphological changes involved in the transformation to a predatory lifestyle. We suggest that alterations in nuclear shape and fixation imply the immediate disruption of the interaction with cytoskeletal mechanisms during nuclear migration. In conclusion, these findings suggest that the signal initiating nuclear migration into fungal traps is generated at the onset of nucleus entry into a trap cell. Our work provides a reference for analysis of the dynamics of nucleus distribution and a means to visualize protein localization and interactions in A. oligospora.

The Role of Histone H2B Acetylation Modification in Aluminum-Induced Cognitive Dysfunction.

Aluminum (Al) is a low toxic trace element that can accumulate in the nervous system and induce cognitive disorders characterized by reduced learning and memory ability. Neuroepigenetic effects are structural changes in cellular function by the brain in response to environmental stimuli by altering the expression of specific genes and repressing normal cellular transcription, leading to abnormalities in a variety of biological processes within the nervous system and affecting neurobehavioral responses. One of the most important mechanisms of epigenetic control on chromatin shape is histone modification. In the present study, we established an offspring rat model of Al intoxication to investigate the changes in spatial learning and memory retention abilities and the relationship with histone H2B acetylation modification in rats exposed to different doses of Al over a long period of time. The results demonstrated that long-term AlCl3 staining resulted in decreased CBP gene and protein expression, increased HDAC3 gene and protein levels, as well as decreased histone H2B and acH2BK20 protein expression levels in the hippocampus of rats. In conclusion, long-term exposure to Al may vary the expression of histone H2B and acH2BK20 through the regulation of enzymes that specifically regulate histone acetylation, hence hastening the deterioration of the nervous system that impairs cognitive function.

Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A.

Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.

Multiple intra-articular injections with adipose-derived stem cells for knee osteoarthritis cause severe arthritis with anti-histone H2B antibody production.

Introduction: Osteoarthritis (OA) is the most common form of arthritis. OA results from the breakdown of cartilage, which leads to deterioration of the entire joint and the connective tissue that holds the joint together, and gradually and irreversibly worsens over time. Adipose-derived stem/stromal cells (ADSCs) have been used in the treatment of knee OA. However, the safety and efficacy of ADSC treatment of OA remain unclear. In this study, we investigated the pathophysiology of severe knee arthritis that occurred after ADSC treatment by screening for autoantibodies in synovial fluid from patients who received ADSC treatment. Methods: Adult Japanese patients with OA who received ADSC treatment at Saitama Cooperative Hospital between June 2018 and October 2021 were enrolled. Antibodies (Abs) were screened using immunoprecipitation (IPP) with [35S]-methionine-labeled HeLa cell extracts. The detected protein was identified by liquid chromatography coupled with time-of-flight mass spectrometry (MS) and ion trap MS, and the corresponding proteins were confirmed as autoantigens using immunoblotting. Ab titers were measured using an enzyme-linked immunosorbent assay. Results: A total of 113 patients received ADSC treatment, and 75% (85/113) received ADSC injection at least twice with a 6-month interval between. No obvious abnormalities were observed in any patient after their first treatment; by contrast, 53% (45/85) of patients who received their second or third ADSC injection showed severe knee arthritis. IPP detected a common anti-15 kDa Ab in synovial fluid of 62% (8/13) of the samples analyzed from patients who showed severe arthritis. This Ab was not detected in synovial fluid obtained from the same joints before treatment. The corresponding autoantigen was identified as histone H2B. All available synovial samples from patients who tested positive for anti-histone H2B Ab were newly positive after the treatment; that is, none had been positive for anti-histone H2B Ab before treatment. Conclusions: Multiple ADSC injections for OA induced severe arthritis in a high percentage of patients, particularly after the second injection. Synovial fluid from some patients with knee arthritis contained Ab to histone H2B that appeared only after ADSC treatment. These findings provide new insights into the pathogenesis of ADSC treatment-induced severe arthritis.

Two-photon lifetime-based photoconversion of EGFP for 3D-photostimulation in FLIM.

Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and biomedical applications. Surprisingly, some interesting photochemical properties of EGFP have remained unexplored. Here we report on two-photon-induced photoconversion of EGFP, which can be permanently converted by intense IR irradiation to a form with a short fluorescence lifetime and spectrally conserved emission. Photoconverted EGFP thus can be distinguished from the unconverted tag by the time-resolved detection. Nonlinear dependence of the two-photon photoconversion efficiency on the light intensity allows for an accurate 3D localization of the photoconverted volume within cellular structures, which is especially useful for kinetic FLIM applications. For illustration, we used the two photon photoconversion of EGFP for measurements of redistribution kinetics of nucleophosmin and histone H2B in nuclei of live cells. Measurements revealed high mobility of fluorescently tagged histone H2B in the nucleoplasm and their redistribution between spatially separated nucleoli.

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6.

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6-HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.

BMAL1-TTK-H2Bub1 loop deficiency contributes to impaired BM-MSC-mediated bone formation in senile osteoporosis.

During the aging process, the reduced osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) results in decreased bone formation, which contributes to senile osteoporosis. Previous studies have confirmed that interrupted circadian rhythm plays an indispensable role in age-related disease. However, the mechanism underlying the impaired osteogenic differentiation of BM-MSCs during aging and its relationship with interrupted circadian rhythm remains unclear. In this study, we confirmed that the circadian rhythm was interrupted in aging mouse skeletal systems. The level of the core rhythm component BMAL1 but not that of CLOCK in the osteoblast lineage was decreased in senile osteoporotic specimens from both human and mouse. BMAL1 targeted TTK as a circadian-controlled gene to phosphorylate MDM2 and regulate H2Bub1 level, while H2Bub1 in turn regulated the expression of BMAL1. The osteogenic capacity of BM-MSCs was maintained by a positive loop formed by BMAL1-TTK-MDM2-H2Bub1. Furthermore, we demonstrated that using bone-targeting recombinant adeno-associated virus 9 (rAAV9) to enhance Bmal1 or Ttk might have a therapeutic effect on senile osteoporosis and delays bone repair in aging mice. In summary, our study indicated that targeting the BMAL1-TTK-MDM2-H2Bub1 axis via bone-targeting rAAV9 might be a promising strategy for the treatment of senile osteoporosis.

The key role of a basic domain of histone H2B N-terminal tail in the action of 5-bromodeoxyuridine to induce cellular senescence.

5-Bromodeoxyuridine (BrdU), a thymidine analogue, is an interesting reagent that modulates various biological phenomena. BrdU, upon incorporation into DNA, causes destabilized nucleosome positioning which leads to changes in heterochromatin organization and gene expression in cells. We have previously shown that BrdU effectively induces cellular senescence, a phenomenon of irreversible growth arrest in mammalian cells. Identification of the mechanism of action of BrdU would provide a novel insight into the molecular mechanisms of cellular senescence. Here, we showed that a basic domain in the histone H2B N-terminal tail, termed the HBR (histone H2B repression) domain, is involved in the action of BrdU. Notably, deletion of the HBR domain causes destabilized nucleosome positioning and derepression of gene expression, as does BrdU. We also showed that the genes up-regulated by BrdU significantly overlapped with those by deletion of the HBR domain, the result of which suggested that BrdU and deletion of the HBR domain act in a similar way. Furthermore, we showed that decreased HBR domain function induced cellular senescence or facilitated the induction of cellular senescence. These findings indicated that the HBR domain is crucially involved in the action of BrdU, and also suggested that disordered nucleosome organization may be involved in the induction of cellular senescence.

Polylactide Nanoparticles as a Biodegradable Vaccine Adjuvant: A Study on Safety, Protective Immunity and Efficacy against Human Leishmaniasis Caused by Leishmania Major.

Leishmaniasis is the 3rd most challenging vector-borne disease after malaria and lymphatic filariasis. Currently, no vaccine candidate is approved or marketed against leishmaniasis due to difficulties in eliciting broad immune responses when using sub-unit vaccines. The aim of this work was the design of a particulate sub-unit vaccine for vaccination against leishmaniasis. The poly (D,L-lactide) nanoparticles (PLA-NPs) were developed in order to efficiently adsorb a recombinant L. major histone H2B (L. major H2B) and to boost its immunogenicity. Firstly, a study was focused on the production of well-formed nanoparticles by the nanoprecipitation method without using a surfactant and on the antigen adsorption process under mild conditions. The set-up preparation method permitted to obtain H2B-adsorbed nanoparticles H2B/PLA (adsorption capacity of about 2.8% (w/w)) with a narrow size distribution (287 nm) and a positive zeta potential (30.9 mV). Secondly, an in vitro release assay performed at 37 °C, pH 7.4, showed a continuous release of the adsorbed H2B for almost 21 days (30%) from day 7. The immune response of H2B/PLA was investigated and compared to H2B + CpG7909 as a standard adjuvant. The humoral response intensity (IgG) was substantially similar between both formulations. Interestingly, when challenged with the standard parasite strain (GLC94) isolated from a human lesion of cutaneous leishmaniasis, mice showed a significant reduction in footpad swelling compared to unvaccinated ones, and no deaths occurred until week 17th. Taken together, these results demonstrate that PLA-NPs represent a stable, cost-effective delivery system adjuvant for use in vaccination against leishmaniasis.

Construction of nucleus-directed fluorescent reporter systems and its application to verification of heterokaryon formation in Morchella importuna.

Introduction: Morchella importuna (M. importuna) is a rare fungus with high nutrition value and distinct flavor. Despite the successful artificial cultivation, its genetic characteristics and biological processes such as life cycle, reproductive system, and trophic mode remain poorly understood. Methods: Considering this, we constructed pEH2B and pMH2B vectors by fusing M. importuna endogenous histone protein H2B with fluorescent proteins eGFP or mCherry, respectively. Based on the constructed pEH2B and pMH2B vectors, nuclear fluorescence localization was performed via Agrobacterium tumefaciens-mediated transformation (ATMT). These two vectors were both driven by two endogenous promoters glyceraldehyde 3-phosphate dehydrogenase (GPD) and ubiquitin (UBI). The vector-based reporter systems were tested by the paired culture of two genetically modified strains pEH2B-labeled M04M24 (24e, MAT1-1-1) and pMH2B-abeled M04M26 (26m, MAT1-2-1). Results: The fluorescence observation and molecular identification results indicated the successful hyphal fusion and heterokaryon formation. We found that the expression of the reporter genes was stable, and it did not interfere with the growth of the fungus. Discussion: Our constructed nucleus-directed fluorescent systems in M. importuna can be used for monitoring the dynamic development and reproductive processes in living cells and also for monitoring the interaction between morels and plant roots. Therefore, morels exhibit the potential to be a candidate organism used for the research on basic biology and genetics of ascomycetes.

Insight into the physiological and pathological roles of USP44, a potential tumor target (Review).

Ubiquitin-specific peptidase 44 (USP44) is a member of the ubiquitin-specific proteases (USPs) family and its functions in various biological processes have been gradually elucidated in recent years. USP44 targets multiple downstream factors and regulates multiple mechanisms through its deubiquitination activity. Ubiquitination is, in essence, a process in which a single ubiquitin molecule or a multiubiquitin chain binds to a substrate protein to form an isopeptide bond. Deubiquitination is the catalyzing of the isopeptide bonds between ubiquitin and substrate proteins through deubiquitylating enzymes. These two processes serve an important role in the regulation of the expression, conformation, localization and function of substrate proteins by regulating their binding to ubiquitin. Based on existing research, this paper summarized the current state of knowledge about USP44. The physiological roles of USP44 in various cellular events and its pathophysiological roles in different cancer types are evaluated and the therapeutic potential of USP44 for cancer treatment is evaluated.

Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting.

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.

Reversal of histone H2B mono-ubiquitination is required for replication stress recovery.

Mono-ubiquitination of histone H2B (H2B-Ub1) is a conserved modification that plays central role in regulating numerous biological processes including the DNA damage response, gene transcription, and DNA replication. Previous studies have revealed that H2B-Ub1 promotes recovery from replication stress by mediating Rad53 phosphorylation (Rad53-P), and activation of the intra-S replication checkpoint, in order to limit fork progression, and associated DNA damage. Since such mono-ubiquitination is a reversible process, we examined the role of H2B-Ub1 deubiquitination during replication stress. Using an experimental system in yeast which mimics H2B-Ub1 accumulation, we show that cells become sensitive to the replication stress induced by HU. This stress response was accompanied by Rad53-P accumulation, and delayed recovery from intra-S checkpoint arrest. Furthermore, we show that similar effects were recapitulated by the accumulation of endogenous H2B-Ub1, induced by the co-inactivation of the deubiquitinating enzyme, Ubp10, and Spt16, a FACT histone chaperone family member. While it has been well established that H2B mono-ubiquitination plays an essential role in recovering from replication stress, our data reveal that H2B-Ub1 deubiquitination is also essential for this process.

SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription.

Dynamic regulation of phosphorylation and dephosphorylation of histones is essential for eukaryotic transcription, but the enzymes engaged in histone dephosphorylation are not fully explored. Here, we show that the tyrosine phosphatase SHP-1 dephosphorylates histone H2B and plays a critical role during transition from the initiation to the elongation stage of transcription. Nuclear-localized SHP-1 is associated with the Paf1 complex at chromatin and dephosphorylates H2B at tyrosine 121. Moreover, knockout of SHP-1, or expression of a mutant mimicking constitutive phosphorylation of H2B Y121, leads to a reduction in genome-wide H2B ubiquitination, which subsequently causes defects in RNA polymerase II-dependent transcription. Mechanistically, we demonstrate that Y121 phosphorylation precludes H2B's interaction with the E2 enzyme, indicating that SHP-1-mediated dephosphorylation of this residue may be a prerequisite for efficient H2B ubiquitination. Functionally, we find that SHP-1-mediated H2B dephosphorylation contributes to maintaining basal autophagic flux in cells through the efficient transcription of autophagy and lysosomal genes. Collectively, our study reveals an important modification of histone H2B regulated by SHP-1 that has a role during eukaryotic transcription.

The roles of epigenetic modifications in the regulation of auxin biosynthesis.

Auxin is one of the most important plant growth regulators of plant morphogenesis and response to environmental stimuli. Although the biosynthesis pathway of auxin has been elucidated, the mechanisms regulating auxin biosynthesis remain poorly understood. The transcription of auxin biosynthetic genes is precisely regulated by complex signaling pathways. When the genes are expressed, epigenetic modifications guide mRNA synthesis and therefore determine protein production. Recent studies have shown that different epigenetic factors affect the transcription of auxin biosynthetic genes. In this review, we focus our attention on the molecular mechanisms through which epigenetic modifications regulate auxin biosynthesis.

N-Terminal Tails of Histones H2A and H2B Differentially Affect Transcription by RNA Polymerase II In Vitro.

Histone N-terminal tails and their post-translational modifications affect various biological processes, often in a context-specific manner; the underlying mechanisms are poorly studied. Here, the role of individual N-terminal tails of histones H2A/H2B during transcription through chromatin was analyzed in vitro. spFRET data suggest that the tail of histone H2B (but not of histone H2A) affects nucleosome stability. Accordingly, deletion of the H2B tail (amino acids 1-31, but not 1-26) causes a partial relief of the nucleosomal barrier to transcribing RNA polymerase II (Pol II), likely facilitating uncoiling of DNA from the histone octamer during transcription. Taken together, the data suggest that residues 27-31 of histone H2B stabilize DNA-histone interactions at the DNA region localized ~25 bp in the nucleosome and thus interfere with Pol II progression through the region localized 11-15 bp in the nucleosome. This function of histone H2B requires the presence of the histone H2A N-tail that mediates formation of nucleosome-nucleosome dimers; however, nucleosome dimerization per se plays only a minimal role during transcription. Histone chaperone FACT facilitates transcription through all analyzed nucleosome variants, suggesting that H2A/H2B tails minimally interact with FACT during transcription; therefore, an alternative FACT-interacting domain(s) is likely involved in this process.