A COMPASS histone H3K4 trimethyltransferase pentamer transactivates drought tolerance and growth/biomass production in Populus trichocarpa.

Histone H3 lysine-4 trimethylation (H3K4me3) activating drought-responsive genes in plants for drought adaptation has long been established, but the underlying regulatory mechanisms are unknown. Here, using yeast two-hybrid, bimolecular fluorescence complementation, biochemical analyses, transient and CRISPR-mediated transgenesis in Populus trichocarpa, we unveiled in this adaptation a regulatory interplay between chromatin regulation and gene transactivation mediated by an epigenetic determinant, a PtrSDG2-1-PtrCOMPASS (complex proteins associated with Set1)-like H3K4me3 complex, PtrSDG2-1-PtrWDR5a-1-PtrRbBP5-1-PtrAsh2-2 (PtrSWRA). Under drought conditions, a transcription factor PtrAREB1-2 interacts with PtrSWRA, forming a PtrSWRA-PtrAREB1-2 pentamer, to recruit PtrSWRA to specific promoter elements of drought-tolerant genes, such as PtrHox2, PtrHox46, and PtrHox52, for depositing H3K4me3 to promote and maintain activated state of such genes for tolerance. CRISPR-edited defects in the pentamer impaired drought tolerance and elevated expression of PtrHox2, PtrHox46, or PtrHox52 improved the tolerance as well as growth in P. trichocarpa. Our findings revealed the identity of the underlying H3K4 trimethyltransferase and its interactive arrangement with the COMPASS for catalysis specificity and efficiency. Furthermore, our study uncovered how the H3K4 trimethyltransferase-COMPASS complex is recruited to the effector genes for elevating H3K4me3 marks for improved drought tolerance and growth/biomass production in plants.

Two fragments of HBV DNA integrated into chrX: 11009033 and its genetic regulation in HepG2.2.15.

Hepatitis B virus (HBV) integration into human genome causes hepatocellular carcinoma (HCC). The present study used inverse nested PCR; the full sequence of HBV DNA fragments of the chrX: 111009033 integration site was detected (987 bp), containing two fragments of double­stranded linear DNA with the same orientation (1,744­1,094 and 1,565­1,228 nt). By reverse transcription­quantitative PCR, HBV­cell fusion transcript was observed in HepG2.2.15 cells. The mean copy number of this site in cells with H2O2 treatment (8.73x10­2±1.65x10­2 copies/cell) was significantly higher than that in the cells without H2O2 treatment (3.02x10­2±2.33x10­2 copies/cell; P<0.0001). The mean levels of P21­activated kinase 3 (PAK3) were 15.67±5.65 copies/cell in HepG2.2.15 cells with H2O2 treatment, significantly higher than in the cells without H2O2 treatment (11.34±4.58 copies/cell, P=0.0076) and in HepG2 cells (5.92±1.54 copies/cell, P<0.0001). Significant difference of PAK3 levels was also found between HepG2.2.15 cells without H2O2 treatment and HepG2 cells (11.34±4.58 vs. 5.92±1.54 copies/cell, P<0.0001). The average copy numbers of the integration site chrX: 111009033 were positively correlated with the average levels of PAK3 (P=0.0013). The overall trend of PAK3 expression was significantly increased in HepG2.2.15 cells with H2O2 treatment compared with that in HepG2.2.15 cells without H2O2 treatment (37.63±8.16 and 31.38±7.94, P=0.008) and HepG2 cells (21.67±7.88, P<0.0001). In summary, the chrX: 11009033 integration site may originate from primary human hepatocytes, occurrence and clonal expansion of which may upregulate PAK3 expression, which may contribute to hepatocarcinogenesis.

PtrVCS2 Regulates Drought Resistance by Changing Vessel Morphology and Stomatal Closure in Populus trichocarpa.

Drought has severe effects on plant growth, forest productivity, and survival throughout the world. Understanding the molecular regulation of drought resistance in forest trees can enable effective strategic engineering of novel drought-resistant genotypes of tree species. In this study, we identified a gene, PtrVCS2, encoding a zinc finger (ZF) protein of the ZF-homeodomain transcription factor in Populus trichocarpa (Black Cottonwood) Torr. & A. Gray. ex Hook. Overexpression of PtrVCS2 (OE-PtrVCS2) in P. trichocarpa resulted in reduced growth, a higher proportion of smaller stem vessels, and strong drought-resistance phenotypes. Stomatal movement experiments revealed that the OE-PtrVCS2 transgenics showed lower stomata apertures than wild-type plants under drought conditions. RNA-seq analysis of the OE-PtrVCS2 transgenics showed that PtrVCS2 regulates the expression of multiple genes involved in regulation of stomatal opening and closing, particularly the PtrSULTR3;1-1 gene, and several genes related to cell wall biosynthesis, such as PtrFLA11-12 and PtrPR3-3. Moreover, we found that the water use efficiency of the OE-PtrVCS2 transgenic plants was consistently higher than that of wild type plants when subjected to chronic drought stress. Taken together, our results suggest that PtrVCS2 plays a positive role in improving drought adaptability and resistance in P. trichocarpa.

Cell-type-specific PtrWOX4a and PtrVCS2 form a regulatory nexus with a histone modification system for stem cambium development in Populus trichocarpa.

Stem vascular cambium cells in forest trees produce wood for materials and energy. WOX4 affects the proliferation of such cells in Populus. Here we show that PtrWOX4a is the most highly expressed stem vascular-cambium-specific (VCS) gene in P. trichocarpa, and its expression is controlled by the product of the second most highly expressed VCS gene, PtrVCS2, encoding a zinc finger protein. PtrVCS2 binds to the PtrWOX4a promoter as part of a PtrWOX13a-PtrVCS2-PtrGCN5-1-PtrADA2b-3 protein tetramer. PtrVCS2 prevented the interaction between PtrGCN5-1 and PtrADA2b-3, resulting in H3K9, H3K14 and H3K27 hypoacetylation at the PtrWOX4a promoter, which led to fewer cambium cell layers. These effects on cambium cell proliferation were consistent across more than 20 sets of transgenic lines overexpressing individual genes, gene-edited mutants and RNA interference lines in P. trichocarpa. We propose that the tetramer-PtrWOX4a system may coordinate genetic and epigenetic regulation to maintain normal vascular cambium development for wood formation.

PSDX: A Comprehensive Multi-Omics Association Database of Populus trichocarpa With a Focus on the Secondary Growth in Response to Stresses.

Populus trichocarpa (P. trichocarpa) is a model tree for the investigation of wood formation. In recent years, researchers have generated a large number of high-throughput sequencing data in P. trichocarpa. However, no comprehensive database that provides multi-omics associations for the investigation of secondary growth in response to diverse stresses has been reported. Therefore, we developed a public repository that presents comprehensive measurements of gene expression and post-transcriptional regulation by integrating 144 RNA-Seq, 33 ChIP-seq, and six single-molecule real-time (SMRT) isoform sequencing (Iso-seq) libraries prepared from tissues subjected to different stresses. All the samples from different studies were analyzed to obtain gene expression, co-expression network, and differentially expressed genes (DEG) using unified parameters, which allowed comparison of results from different studies and treatments. In addition to gene expression, we also identified and deposited pre-processed data about alternative splicing (AS), alternative polyadenylation (APA) and alternative transcription initiation (ATI). The post-transcriptional regulation, differential expression, and co-expression network datasets were integrated into a new P. trichocarpa Stem Differentiating Xylem (PSDX) database (http://forestry.fafu.edu.cn/db/SDX), which further highlights gene families of RNA-binding proteins and stress-related genes. The PSDX also provides tools for data query, visualization, a genome browser, and the BLAST option for sequence-based query. Much of the data is also available for bulk download. The availability of PSDX contributes to the research related to the secondary growth in response to stresses in P. trichocarpa, which will provide new insights that can be useful for the improvement of stress tolerance in woody plants.

Integration of hepatitis B virus DNA into p21-activated kinase 3 (PAK3) gene in HepG2.2.15 cells.

Integration of HBV DNA into host chromosomes was found in most of the patients with chronic hepatitis B (CHB). In this study, using inverse nested PCR (invPCR), we found the integration site chrX: 111,009,033, which inserted into the p21-activated kinase 3 (PAK3) gene in HepG2.2.15 cells. The viral-human chimeric transcripts were also observed and, significant differences of the copy numbers of integration site chrX: 111,009,033 (P = 0.012) and intra-cell HBV DNA levels (P = 0.027) were found between the cells with and without H2O2 treatment, respectively. This study may provide a novel insight into the elucidation of etiology of HBV integration.

The AREB1 Transcription Factor Influences Histone Acetylation to Regulate Drought Responses and Tolerance in Populus trichocarpa.

Plants develop tolerance to drought by activating genes with altered levels of epigenetic modifications. Specific transcription factors are involved in this activation, but the molecular connections within the regulatory system are unclear. Here, we analyzed genome-wide acetylated lysine residue 9 of histone H3 (H3K9ac) enrichment and examined its association with transcriptomes in Populus trichocarpa under drought stress. We revealed that abscisic acid-Responsive Element (ABRE) motifs in promoters of the drought-responsive genes PtrNAC006, PtrNAC007, and PtrNAC120 are involved in H3K9ac enhancement and activation of these genes. Overexpressing these PtrNAC genes in P trichocarpa resulted in strong drought-tolerance phenotypes. We showed that the ABRE binding protein PtrAREB1-2 binds to ABRE motifs associated with these PtrNAC genes and recruits the histone acetyltransferase unit ADA2b-GCN5, forming AREB1-ADA2b-GCN5 ternary protein complexes. Moreover, this recruitment enables GCN5-mediated histone acetylation to enhance H3K9ac and enrich RNA polymerase II specifically at these PtrNAC genes for the development of drought tolerance. CRISPR editing or RNA interference-mediated downregulation of any of the ternary members results in highly drought-sensitive P trichocarpa Thus, the combinatorial function of the ternary proteins establishes a coordinated histone acetylation and transcription factor-mediated gene activation for drought response and tolerance in Populus species.

Different types of viral­host junction found in HBV integration breakpoints in HBV­infected patients.

The present study surveyed the characteristics of hepatitis B virus (HBV) integration in the liver genomes of patients with acute hepatitis B (AHB), carriers of inactive hepatitis B surface antigen (HBsAg), and patients with chronic hepatitis B (CHB) receiving antiviral treatment. 'Short­read' whole genome sequencing (WGS) with an average of 4,879x coverage for HBV integration was performed in three patients with AHB, two carriers of inactive HBsAg, and 13 patients with CHB receiving antiviral treatment. Conventional polymerase chain reaction and Sanger sequencing were used to verify integration breakpoints supported by at least two paired­end reads, and viral­host chimeric transcripts were surveyed simultaneously. HBV integration breakpoints were 100% identified with an average of 138.2±379.9 breakpoints per sample. The numbers of HBV integration breakpoints were positively associated with the sequencing depth coverage numbers and levels of intrahepatic covalently closed circular DNA, respectively (P<0.0001 and P<0.0001). Four types of viral­host junction in 14 HBV integration breakpoints were detected (two viral junctions mapped in the HBs gene, one in the Precore gene, and others within the HBx gene): Forward simple junction, reverse simple junction, forward and reverse complicated junction, and microhomology were found in many of the junctions. Expression of viral­human chimeric transcripts was observed in several breakpoints, including the HBs gene. As a result, HBV can integrate into the host gene in the same manner as non­homologous end joining and microhomology­mediated end joining with numerous sites, and a close association may exist between HBV integration and patient prognosis. HBx integration may be indispensable for viral­host chimeric transcription and HBsAg may be produced from integrated DNA.

Cimicifuga foetida L. plus adefovir effectively inhibits the replication of hepatitis B virus in patients with chronic hepatitis B.

The aim of the present study was to assess the anti-hepatitis B virus (HBV) effect of Cimicifuga foetida L. (C. foetida) in the patients with chronic hepatitis B (CHB). A total of 60 randomly selected patients with CHB were recruited and divided into groups I and II. The patients in group I received a monotherapy of adefovir (ADV), and the patients in group II received a combination therapy of ADV and C. foetida for >48 weeks. Intrahepatic (IH) HBV covalently closed circular DNA (cccDNA), serum HBV DNA, hepatitis B surface antigen (HBsAg), alanine aminotransferase levels and serum interferon-γ (IFN-γ) and transforming growth factor-ß (TGF-ß) levels were quantified during the test. Following the treatment, a significant reduction of the median IH cccDNA level was identified in group II (P=0.017), but not in group I (P=0.05, and P=0.01 between the 2 groups), and a significant reduction of log10 HBsAg was identified in groups I (P=0.012) and II (P<0.0001, and P=0.20 between the 2 groups). A significant increase of the median serum IFN-γ level was found in group II (P=0.0005), but not in group I (P=0.06, and P=0.004 between the 2 groups), and a significant reduction of the median TGF-ß level was identified in groups I (P<0.0001) and II (P<0.0001, and P=0.002 between the 2 groups). A total of 24 patients in group I, and 27 patients in group II achieved a sustained virological response (P=0.0386), and 20 patients in group I and 24 in group II achieved hepatitis B e antigen seroclearance (P=0.0442). In conclusion, C. foetida can effectively inhibit HBV transcription and replication in the patients by stimulating the release of the inflammatory cytokines, such as IFN-γ.

Significance of the quantitative measurement of the chr16: 51320015 integration site in hepatocytes of patients with chronic hepatitis B.

The present study reported the presence of a hepatitis B virus (HBV) major integration site (MIS) chr16: 51320015 and discussed the significance of quantitative measurement of this site. A total of 30 hepatitis B e antigen (HBeAg) positive (+) and 30 HBeAg negative (­) patients with chronic hepatitis B (CHB) were enrolled in the present study, and the levels of intrahepatic (IH) covalently closed circular DNA (cccDNA), serum HBV DNA and hepatitis B surface antigen (HBsAg) were detected. Conventional reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and Sanger sequencing were designed to verify the chr16: 51320015 integration site, and the copy numbers of this site were measured using molecular clone and SYBR Green I RT­qPCR. This site was found to be present in the hepatocytes of all the enrolled patients, and the average number of copies was 1.46x10­2 ± 4.94x10­2 copies/cell (3.48x10­5­0.212 copies/cell). No significant difference in the copy numbers of this site were observed between the HBeAg (+) (1.43 ± 9.79x10­1 copies/cell) and HBeAg (­) patients (6.58x10­2 ± 2.47x10­2 copies/cell; P>0.05), which were positively correlated with the levels of serum HBsAg (P=0.0038), but were not correlated with the levels of IH cccDNA (P=0.7785). In conclusion, the chr16:51320015 integration site may be a novel site, which persists in a several patients with HBV infection, and may accumulate in the hepatocytes due to clonal expansion. The diagnostic and therapeutic values of this site require further investigation.

Predictive value of intrahepatic hepatitis B virus covalently closed circular DNA and total DNA in patients with acute hepatitis B and patients with chronic hepatitis B receiving anti­viral treatment.

This study aimed to investigate the persistence and predictive values of intrahepatic (IH) hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and total DNA (tDNA) in patients with acute hepatitis B (AHB) and patients with chronic hepatitis B (CHB) receiving anti­viral treatment. The levels of IH cccDNA and tDNA, serum HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and alanine aminotransferase (ALT) were detected in 11 patients with AHB and 46 patients with CHB who were receiving anti­viral treatment, among whom 21 had primary treatment failure, 11 achieved virological response (VR) and 15 achieved VR and HBsAg seroclearance. The median IH cccDNA and tDNA levels in the patients with AHB (0.002 copies/cell and 0.04 copies/cell, respectively) were significantly lower than those in the patients with CHB. In the patients with CHB, the median IH cccDNA level among individuals who achieved VR and HBsAg seroclearance (0.012 copies/cell) was significantly lower than that in those who had failed primary treatment (4.18 copies/cell, P<0.0001) but not that in those who achieved solely VR (0.039 copies/cell, P=0.169). The median IH tDNA level in patients with CHB who achevied VR and HBsAg seroclearance (0.096 copies/cell) was significantly lower than that in those who failed primary treatment (371 copies/cell, P<0.0001) and those who achieved solely VR (1.62 copies/cell, P=0.001). No significant difference was observed in the area under the receiver operating characteristic (ROC) curve, which was used to predict the likelihood of achieving VR and HBsAg seroclearance, between IH tDNA and IH cccDNA levels (0.96 and 0.88, respectively; P>0.10). IH cccDNA levels were shown to be positively correlated with serum ALT (P=0.024), HBeAg (P=0.001) and IH tDNA levels (P<0.0001), but not with serum HBV DNA (P=0.12) and HBsAg levels in either HBeAg­positive (P=0.84) or in HBeAg­negative (P=0.146) patients. In conclusion, IH cccDNA may persist in patients with AHB and patients with CHB who acheive VR and HBsAg seroclearance following anti­viral treatment. Furthermore, IH cccDNA and tDNA may have potential in predicting successful therapeutic response in patients with CHB who receive anti­viral treatment.