H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction.

Polycomb repressive complex 1 (PRC1) is a key transcriptional regulator in development via modulating chromatin structure and catalyzing histone H2A ubiquitination at Lys119 (H2AK119ub1). H2AK119ub1 is one of the most abundant histone modifications in mammalian cells. However, the function of H2AK119ub1 in polycomb-mediated gene silencing remains debated. In this study, we reveal that H2AK119ub1 has two distinct roles in gene expression, through differentially modulating chromatin compaction mediated by canonical PRC1 and the linker histone H1. Interestingly, we find that H2AK119ub1 plays a positive role in transcription through interfering with the binding of canonical PRC1 to nucleosomes and therefore counteracting chromatin condensation. Conversely, we demonstrate that H2AK119ub1 facilitates H1-dependent chromatin condensation and enhances the silencing of developmental genes in mouse embryonic stem cells, suggesting that H1 may be one of several possible pathways for H2AK119ub1 in repressing transcription. These results provide insights and molecular mechanisms by which H2AK119ub1 differentially fine-tunes developmental gene expression.

Histone Modifications Regulate Chromatin Compartmentalization by Contributing to a Phase Separation Mechanism.

Eukaryotic chromosomes contain compartments of various functions, which are marked by and enriched with specific histone modifications. However, the molecular mechanisms by which these histone marks function in chromosome compartmentalization are poorly understood. Constitutive heterochromatin is a largely silent chromosome compartment characterized in part by H3K9me2 and 3. Here, we show that heterochromatin protein 1 (HP1), an H3K9me2 and 3 "reader," interacts with SUV39H1, an H3K9me2 and 3 "writer," and with TRIM28, an abundant HP1 scaffolding protein, to form complexes with increased multivalent engagement of H3K9me2 and 3-modified chromatin. H3K9me2 and 3-marked nucleosomal arrays and associated complexes undergo phase separation to form macromolecule-enriched liquid droplets. The droplets are reminiscent of heterochromatin as they are highly dense chromatin-containing structures that are resistant to DNase and exclude the general transcription factor TFIIB. Our data suggest a general mechanism by which histone marks regulate chromosome compartmentalization by promoting phase separation.

H2A mono-ubiquitination differentiates FACT's functions in nucleosome assembly and disassembly.

The histone chaperone FACT (FAcilitates Chromatin Transcription) plays an essential role in transcription and DNA replication by its dual functions on nucleosome assembly to maintain chromatin integrity and nucleosome disassembly to destabilize nucleosome and facilitate its accessibility simultaneously. Mono-ubiquitination at Lysine 119 of H2A (ubH2A) has been suggested to repress transcription by preventing the recruitment of FACT at early elongation process. However, up to date, how ubH2A directly affects FACT on nucleosome assembly and disassembly remains elusive. In this study, we demonstrated that the dual functions of FACT are differently regulated by ubH2A. The H2A ubiquitination does not affect FACT's chaperone function in nucleosome assembly and FACT can deposit ubH2A-H2B dimer on tetrasome to form intact nucleosome. However, ubH2A greatly restricts FACT binding on nucleosome and inhibits its activity of nucleosome disassembly. Interestingly, deubiquitination of ubH2A rescues the nucleosome disassembly function of FACT to activate gene transcription. Our findings provide mechanistic insights of how H2A ubiquitination affects FACT in breaking nucleosome and maintaining its integrity, which sheds light on the biological function of ubH2A and various FACT's activity under different chromatin states.

Integrated Nursing and Psychological Intervention for Tuberculosis Complicated by Lung Cancer: Clinical Efficacy.

Context: The incidence of tuberculosis (TB) complicated by lung cancer has been increasing yearly worldwide. The overlapping effects of these two diseases leads to difficulties in clinical treatment and care. Single-care modalities fail to meet the clinical-care requirements of these complex diseases for both psychological and physical treatment. Objective: The study intended to evaluate the clinical efficacy of integrated nursing plus a psychological intervention for patients with TB complicated by lung cancer. Design: The research team conducted a randomized controlled study. Setting: The study took place at the Affiliated Hospital of Hebei University in Baoding, Hebei, China. Participants: Participants were 60 patients with pulmonary TB complicated by lung cancer who received treatment at the hospital between January 2022 and December 2022. Interventions: The research team randomly assigned participants to one of two groups, each with 30 participants: (1) the control group, who received integrated nursing and (2) the intervention group who received integrated nursing plus a psychological intervention. Outcome Measures: The research team evaluated: (1) short-term clinical efficacy; (2) quality of life, using the Medical Outcomes Study's (MOS') 36-item Short-form Health Survey (SF-36); (3) levels of anxiety and depression, using the Self-Rating Anxiety Scale (SAS) and Self-Rating Depression Scale (SDS), respectively; and (4) nursing satisfaction. Results: No significant differences existed between the groups in demographic or clinical characteristics at baseline (P > .05). Compared to the control group, the intervention group's; (1) short-term clinical efficacy was significantly higher (P = .035); (2) scores on the SF-36 were significantly higher (all P < .001; (3) scores on the SAS and SDS were significantly lower (both P < .001); and (4) nursing satisfaction was significantly higher (P = .000). Conclusions: Integrated nursing plus psychological intervention can improve the quality of life of patients with TB complicated by lung cancer, alleviate their negative emotions, and enhance nursing satisfaction, thereby promoting patients' recoveries.

Application of magnetic resonance image compilation (MAGiC) in the diagnosis of middle-aged and elderly women with osteoporosis.

OBJECTIVE: To investigate the feasibility of diagnosing osteoporosis (OP) in women through magnetic resonance image compilation (MAGiC). METHODS: A total of 110 patients who underwent lumbar magnetic resonance imaging and dual X-ray absorptiometry examinations were collected and divided into two groups according bone mineral density: osteoporotic group (OP) and non-osteoporotic group (non-OP). The variation trends of T1 (longitudinal relaxation time), T2 (transverse relaxation time) and BMD (bone mineral density) with the increase of age, and the correlation of T1 and T2 with BMD were examined by establishing a clinical mathematical model. RESULTS: With the increase of age, BMD and T1 value decreased gradually, while T2 value increased. T1 and T2 had statistical significance in diagnosing OP (P < 0.001), and there is moderate positive correlation between T1 and BMD values (R = 0.636, P < 0.001), while moderate negative correlation between T2 and BMD values (R=-0.694, P < 0.001). Receiver characteristic curve test showed that T1 and T2 had high accuracy in diagnosing OP (T1 AUC = 0.982, T2 AUC = 0.978), and the critical values of T1 and T2 for evaluating osteoporosis were 0.625s and 0.095s, respectively. Besides, the combined utilization of T1 and T2 had higher diagnostic efficiency (AUC = 0.985). Combined T1 and T2 had higher diagnostic efficiency (AUC = 0.985). Function fitting results of OP group: BMD=-0.0037* age - 0.0015*T1 + 0.0037*T2 + 0.86, sum of squared error (SSE) = 0.0392, and non-OP group: BMD = 0.0024* age - 0.0071*T1 + 0.0007*T2 + 1.41, SSE = 0.1007. CONCLUSION: T1 and T2 value of MAGiC have high efficiency in diagnosing OP by establishing a function fitting formula of BMD with T1, T2 and age.

Probing the Effect of Ubiquitinated Histone on Mononucleosomes by Translocation Dynamics Study through Solid-State Nanopores.

Post-translational modifications (PTMs), such as ubiquitination, are critically important in regulating genetic expressions by adjusting the nucleosome stability. A fast and label-free technology inspecting dynamic nucleosome structures can facilitate the interrogation of PTMs effects. Here we leverage the advantages of mechanically stable solid-state nanopores and detect the effect of a ubiquitinated histone on mononucleosomes at the single-molecule level. By comparing the translocation dynamics of natural and cross-linked mononucleosomes, we verified that the nucleosomal DNA unravelled from histones in natural mononucleosomes. Furthermore, we found that a turning point of voltage corresponds to the onset of nucleosome rupture. More importantly, we reveal that ubH2A stabilizes the nucleosome by shifting the turning point to a larger value and investigated the effect of ubiquitination on different histones (ubH2A and ubH2B). These findings open promising possibilities for developing a miniaturized and portable device for the fast screening of PTMs on nucleosomes.

Curaxin-Induced DNA Topology Alterations Trigger the Distinct Binding Response of CTCF and FACT at the Single-Molecule Level.

The candidate anticancer drug curaxins can insert into DNA base pairs and efficiently inhibit the growth of various cancers. However, how curaxins alter the genomic DNA structure and affect the DNA binding property of key proteins remains to be clarified. Here, we first showed that curaxin CBL0137 strongly stabilizes the interaction between the double strands of DNA and reduces DNA bending and twist rigidity simultaneously, by single-molecule magnetic tweezers. More importantly, we found that CBL0137 greatly impairs the binding of CTCF but facilitates trapping FACT on DNA. We revealed that CBL0137 clamps the DNA double helix that may induce a huge barrier for DNA unzipping during replication and transcription and causes the distinct binding response of CTCF and FACT on DNA. Our work provides a novel mechanical insight into CBL0137's anticancer mechanisms at the nucleic acid level.

Dual-Colored Fluorescence Imaging of Mitochondrial HNO and Golgi-HNO in Mice with DILI.

Drug-induced liver injury (DILI) is the most common reason for the post-marketing withdrawal of drugs. Poor understanding of the mechanisms of DILI presents a large challenge in clinical diagnosis. Previous evidences indicate a potential relationship between reactive nitrogen species (RNS) and DILI. Hence, we developed two specific probes, Golgi-HNO and Mito-HNO, for the multicolored and simultaneous in situ imaging of nitroxyl (HNO) in the Golgi apparatus and mitochondria, respectively. We discovered a significant rise in HNO levels in the livers of mice with DILI, which means that for the first time, we revealed a positive correlation between HNO levels and DILI. Based on changes in the HNO level, we also successfully explored the extent of liver damage induced by an anticarcinogen, bleomycin. In addition, we uncovered catalase was involved in HNO synthesis, which is the unprecedented function of catalase. These findings demonstrate that HNO is an ideal biomarker for DILI diagnosis, and Golgi-HNO and Mito-HNO are ideal fluorescent probes to study in situ HNO changes in various physiological and biochemical processes.

Histone H2A Ubiquitination Reinforces Mechanical Stability and Asymmetry at the Single-Nucleosome Level.

Monoubiquitination at lysine 119 of histone H2A (ubH2A) is a prevalent post-translational modification that is associated with gene repression in the context of chromatin. However, the direct function of ubH2A on nucleosome is poorly understood. Here we identified the effect of ubH2A on nucleosome using single-molecule magnetic tweezers. We revealed that ubH2A stabilizes the nucleosome by blocking the peeling of DNA from the histone octamer. Each ubH2A reinforces one-half of the outer wrap and introduces a robust asymmetry for nucleosome unfolding. Furthermore, a real-time deubiquitination process confirmed that ubH2A-nucleosome is sequentially deubiquitinated and restored to the unmodified nucleosome state. These results provide a novel mechanism to understand the repression of the passage of RNA or DNA polymerases through the ubH2A-nucleosome barrier during gene transcription or replication.

Histone variants H2A.Z and H3.3 coordinately regulate PRC2-dependent H3K27me3 deposition and gene expression regulation in mES cells.

BACKGROUND: The hierarchical organization of eukaryotic chromatin plays a central role in gene regulation, by controlling the extent to which the transcription machinery can access DNA. The histone variants H3.3 and H2A.Z have recently been identified as key regulatory players in this process, but the underlying molecular mechanisms by which they permit or restrict gene expression remain unclear. Here, we investigated the regulatory function of H3.3 and H2A.Z on chromatin dynamics and Polycomb-mediated gene silencing. RESULTS: Our ChIP-seq analysis reveals that in mouse embryonic stem (mES) cells, H3K27me3 enrichment correlates strongly with H2A.Z. We further demonstrate that H2A.Z promotes PRC2 activity on H3K27 methylation through facilitating chromatin compaction both in vitro and in mES cells. In contrast, PRC2 activity is counteracted by H3.3 through impairing chromatin compaction. However, a subset of H3.3 may positively regulate PRC2-dependent H3K27 methylation via coordinating depositions of H2A.Z to developmental and signaling genes in mES cells. Using all-trans retinoic acid (tRA)-induced gene as a model, we show that the dynamic deposition of H2A.Z and H3.3 coordinately regulates the PRC2-dependent H3K27 methylation by modulating local chromatin structure at the promoter region during the process of turning genes off. CONCLUSIONS: Our study provides key insights into the mechanism of how histone variants H3.3 and H2A.Z function coordinately to finely tune the PRC2 enzymatic activity during gene silencing, through promoting or impairing chromosome compaction respectively.

MicroRNA expression data analysis to identify key miRNAs associated with Alzheimer's disease.

BACKGROUND: MicroRNAs (miRNAs) have become increasingly prevalent as a result of the association of their deregulation with neurodegenerative disorders, especially Alzheimer's disease (AD). However, the association between miRNAs and AD remains unclear. METHODS: In the present study, Nine representative miRNA datasets were selected for the identification of the critical miRNAs by analyzing the overlapping relationships among them. TargetScan software (http://www.targetscan.org) was used to predict the target genes of these miRNAs. In addition, the Database for Annotation Visualization and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov) and TfactS (http://www.tfacts.org) datasets were used for combined analysis of functional enrichment and transcription factor (TF) analysis. RESULTS: Thirteen key miRNAs were identified, of which four were significantly up-regulated (hsa-miR-101,hsa-miR-155, has-miR-34a, has-miR-9) and eight were found to be significantly down-regulated (hsa-let-7d-5p, hsa-let-7 g-5p, hsa-miR-15b, has-miR-191-5p, hsa-miR-125b, has-miR-26b-5p, hsa-miR-29b, hsa-miR-342-3p). The functional enrichment analysis indicated that up-regulated signature miRNA targets were associated with transcription from the RNA polymerase II promoter process and the chemical synaptic transmission process. Down-regulated signature miRNA targets were mostly enriched with respect to positive regulation of transcription from the RNA polymerase II promoter process, p53 signaling, and microRNAs in cancer pathways. TF analysis showed that 87 TFs were influenced by the up-regulated miRNAs, and 134 TFs were influenced by the down-regulated miRNAs. In total, 70 (45.5%) TFs were affected by both up-regulated and down-regulated miRNAs. CONCLUSIONS: In summary, 13 key miRNAs were found to have a vital function in the pathological progress of AD, as well as the target genes and TFs of these miRNAs. The potential functions of these miRNAs as diagnostic and therapeutic targets of the AD are revealed by the present study.

Quantitative analysis of chromatin accessibility in mouse embryonic fibroblasts.

Genomic DNA of eukaryotic cells is hierarchically packaged into chromatin by histones. The dynamic organization of chromatin fibers plays a critical role in the regulation of gene transcription and other DNA-associated biological processes. Recently, numerous approaches have been developed to map the chromatin organization by characterizing chromatin accessibilities in genome-wide. However, reliable methods to quantitatively map chromatin accessibility are not well-established, especially not on a genome-wide scale. Here, we developed a modified MNase-seq for mouse embryonic fibroblasts, wherein chromatin was partially digested at multiple digestion times using micrococcal nuclease (MNase), allowing quantitative analysis of local yet genome-wide chromatin compaction. Our results provide strong evidence that the chromatin accessibility at promoter regions are positively correlated with gene activity. In conclusion, our assay is an ideal tool for the quantitative study of gene regulation in the perspective of chromatin accessibility.

Replication-coupled inheritance of chromatin states.

During the development of eukaryote, faithful inheritance of chromatin states is central to the maintenance of cell fate. DNA replication poses a significant challenge for chromatin state inheritance because every nucleosome in the genome is disrupted as the replication fork passes. It has been found that many factors including DNA polymerases, histone chaperones, as well as, RNA Pol II and histone modifying enzymes coordinate spatially and temporally to maintain the epigenome during this progress. In this review, we provide a summary of the detailed mechanisms of replication-coupled nucleosome assembly and post-replication chromatin maturation, highlight the inheritance of chromatin states and epigenome during these processes, and discuss the future directions and challenges in this field.

Pluripotency state transition of embryonic stem cells requires the turnover of histone chaperone FACT on chromatin.

The differentiation of embryonic stem cells (ESCs) begins with the transition from the naive to the primed state. The formative state was recently established as a critical intermediate between the two states. Here, we demonstrate the role of the histone chaperone FACT in regulating the naive-to-formative transition. We found that the Q265K mutation in the FACT subunit SSRP1 increased the binding of FACT to histone H3-H4, impaired nucleosome disassembly in vitro, and reduced the turnover of FACT on chromatin in vivo. Strikingly, mouse ESCs harboring this mutation showed elevated naive-to-formative transition. Mechanistically, the SSRP1-Q265K mutation enriched FACT at the enhancers of formative-specific genes to increase targeted gene expression. Together, these findings suggest that the turnover of FACT on chromatin is crucial for regulating the enhancers of formative-specific genes, thereby mediating the naive-to-formative transition. This study highlights the significance of FACT in fine-tuning cell fate transition during early development.

Histone H1 facilitates restoration of H3K27me3 during DNA replication by chromatin compaction.

During cell renewal, epigenetic information needs to be precisely restored to maintain cell identity and genome integrity following DNA replication. The histone mark H3K27me3 is essential for the formation of facultative heterochromatin and the repression of developmental genes in embryonic stem cells. However, how the restoration of H3K27me3 is precisely achieved following DNA replication is still poorly understood. Here we employ ChOR-seq (Chromatin Occupancy after Replication) to monitor the dynamic re-establishment of H3K27me3 on nascent DNA during DNA replication. We find that the restoration rate of H3K27me3 is highly correlated with dense chromatin states. In addition, we reveal that the linker histone H1 facilitates the rapid post-replication restoration of H3K27me3 on repressed genes and the restoration rate of H3K27me3 on nascent DNA is greatly compromised after partial depletion of H1. Finally, our in vitro biochemical experiments demonstrate that H1 facilitates the propagation of H3K27me3 by PRC2 through compacting chromatin. Collectively, our results indicate that H1-mediated chromatin compaction facilitates the propagation and restoration of H3K27me3 after DNA replication.

H2B ubiquitination recruits FACT to maintain a stable altered nucleosome state for transcriptional activation.

Histone H2B mono-ubiquitination at lysine 120 (ubH2B) has been found to regulate transcriptional elongation by collaborating with the histone chaperone FACT (Facilitates Chromatin Transcription) and plays essential roles in chromatin-based transcriptional processes. However, the mechanism of how ubH2B directly collaborates with FACT at the nucleosome level still remains elusive. In this study, we demonstrate that ubH2B impairs the mechanical stability of the nucleosome and helps to recruit FACT by enhancing the binding of FACT on the nucleosome. FACT prefers to bind and deposit H2A-ubH2B dimers to form an intact nucleosome. Strikingly, the preferable binding of FACT on ubH2B-nucleosome greatly enhances nucleosome stability and maintains its integrity. The stable altered nucleosome state obtained by ubH2B and FACT provides a key platform for gene transcription, as revealed by genome-wide and time-course ChIP-qPCR analyses. Our findings provide mechanistic insights of how ubH2B directly collaborates with FACT to regulate nucleosome dynamics for gene transcription.

NFIB facilitates replication licensing by acting as a genome organizer.

The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of Nuclear Factor I (NFI) family that was initially purified in host cells to promote adenoviral DNA replication but has since mainly been investigated in transcription regulation, is physically associated with the pre-replication complex (pre-RC) in mammalian cells. Genomic analyses reveal that NFIB facilitates the assembly of the pre-RC by increasing chromatin accessibility. Nucleosome binding and single-molecule magnetic tweezers shows that NFIB binds to and opens up nucleosomes. Transmission electron microscopy indicates that NFIB promotes nucleosome eviction on parental chromatin. NFIB deficiency leads to alterations of chromosome contacts/compartments in both G1 and S phase and affects the firing of a subset of origins at early-replication domains. Significantly, cancer-associated NFIB overexpression provokes gene duplication and genomic alterations recapitulating the genetic aberrance in clinical breast cancer and empowering cancer cells to dynamically evolve growth advantage and drug resistance. Together, these results point a role for NFIB in facilitating replication licensing by acting as a genome organizer, shedding new lights on the biological function of NFIB and on the replication origin selection in eukaryotes.

Preparation and Characterization of Chromatin Templates for Histone Methylation Assays.

In eukaryotic cells, chromatin plays an important role in gene regulation by controlling the access of the transcription machinery to DNA. In this chapter, we will describe methods for generating different chromatin templates to investigate the impact of histone variants and chromatin structure on histone methyltransferase activities. For this purpose, we take Polycomb Repressive Complex 2 (PRC2) as an example and investigate how its activity on H3K27me3 is regulated by the histone variants H3.3 and H2A.Z and higher-order chromatin structure.

Simultaneous fluorescence imaging of Golgi O2•- and Golgi H2O2 in mice with hypertension.

Hypertensive cardiovascular disease is a persistent threat to public health. Elucidating the pathogenesis of hypertension is expected to provide more highly targeted therapies for patients. To date, reactive oxygen species (ROS) induced endothelial nitric oxide synthase (eNOS) uncoupling are generally considered to be common phenomena in hypertension. However, the critical factor contribute to persistent eNOS uncoupling remains poorly understood. Herein, we established a fluorescence probe, GolROS, for the multicolored and simultaneous detection of Golgi O2•- and H2O2 in situ. We successfully detected increases in Golgi ROS levels in hypertensive mice and evaluated the pharmaceutical effects of various antihypertensive drugs. More importantly, we identified the ROS post-transcriptional modification sites on dihydrofolate reductase (DHFR). Altogether, we propose a novel therapeutic target for hypertension, which will promote the development of new antihypertensive drugs, and also developed an ideal fluorescence probe to study in situ Golgi O2•- and H2O2 changes in various biochemical processes.

Acetylation-dependent SAGA complex dimerization promotes nucleosome acetylation and gene transcription.

Cells reprogram their transcriptomes to adapt to external conditions. The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a highly conserved transcriptional coactivator that plays essential roles in cell growth and development, in part by acetylating histones. Here, we uncover an autoregulatory mechanism of the Saccharomyces cerevisiae SAGA complex in response to environmental changes. Specifically, the SAGA complex acetylates its Ada3 subunit at three sites (lysines 8, 14 and 182) that are dynamically deacetylated by Rpd3. The acetylated Ada3 lysine residues are bound by bromodomains within SAGA subunits Gcn5 and Spt7 that synergistically facilitate formation of SAGA homo-dimers. Ada3-mediated dimerization is enhanced when cells are grown under sucrose or under phosphate-starvation conditions. Once dimerized, SAGA efficiently acetylates nucleosomes, promotes gene transcription and enhances cell resistance to stress. Collectively, our work reveals a mechanism for regulation of SAGA structure and activity and provides insights into how cells adapt to environmental conditions.