Hepatitis delta virus genome RNA synthesis initiates at position 1646 with a non-templated guanosine

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense ssRNA virus that produces three RNAs in infected cells: genome, antigenome and mRNA; the latter encodes hepatitis delta antigen, the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5’ end of the genome, including the synthesis start site and its chemical composition, are not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling detection of the 5’ end of the genome RNA. The 5’ end of this RNA is capped, as expected for a Pol II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA Polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how synthesis of the viral RNA begins, nor even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed hairpin-like structure of the viral RNA. The 5’ end of the RNA is capped, as expected for Pol II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses Pol II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication.

Genome-wide identification of enhancers and transcription factors regulating the myogenic differentiation of bovine satellite cells

Background Satellite cells are the myogenic precursor cells in adult skeletal muscle. The objective of this study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells into myotubes. Results Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to identify genomic regions where lysine 27 of H3 histone is acetylated (H3K27ac), i.e., active enhancers, from bovine satellite cells before and during differentiation into myotubes. A total of 19,027 and 47,669 H3K27ac-marked enhancers were consistently identified from two biological replicates of before- and during-differentiation bovine satellite cells, respectively. Of these enhancers, 5882 were specific to before-differentiation, 35,723 to during-differentiation, and 13,199 common to before- and during-differentiation bovine satellite cells. Whereas most of the before- or during-differentiation-specific H3K27ac-marked enhancers were located distally to the transcription start site, the enhancers common to before- and during-differentiation were located both distally and proximally to the transcription start site. The three sets of H3K27ac-marked enhancers were associated with functionally different genes and enriched with different transcription factor binding sites. Specifically, many of the H3K27ac-marked enhancers specific to during-differentiation bovine satellite cells were associated with genes involved in muscle structure and development, and were enriched with binding sites for the MyoD, AP-1, KLF, TEAD, and MEF2 families of transcription factors. A positive role was validated for Fos and FosB, two AP-1 family transcription factors, in the differentiation of bovine satellite cells into myotubes by siRNA-mediated knockdown. Conclusions Tens of thousands of H3K27ac-marked active enhancers have been identified from bovine satellite cells before or during differentiation. These enhancers contain binding sites not only for transcription factors whose role in satellite cell differentiation is well known but also for transcription factors whose role in satellite cell differentiation is unknown. These enhancers and transcription factors are valuable resources for understanding the complex mechanism that mediates gene expression during satellite cell differentiation. Because satellite cell differentiation is a key step in skeletal muscle growth, the enhancers, the transcription factors, and their target genes identified in this study are also valuable resources for identifying and interpreting skeletal muscle trait-associated DNA variants in cattle.

The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters

The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene expression is not well understood. During Drosophila oogenesis, the enzymatic functions are not equally required, which may indicate that different genes require different enzymatic functions. An analogy for this phenomenon is the handyman principle: while a handyman has many tools, which tool he uses depends on what requires maintenance. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis, which interacts with TBP. We show that depletion of SAGA-specific core subunits blocked egg chamber development at earlier stages than depletion of enzymatic subunits. These results, as well as additional genetic analyses, point to an interaction with TBP and suggest a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments, and the complex was not specifically associated with distinct promoter types in the ovary. The high-resolution genomic binding profiles were congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. Our data illustrate that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present and suggests that the biological context defines which module functions are critical.

Elucidating the Importance and Regulation of Key Enhancers for Human MEIS1 Expression

Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is deregulated in a large subset of acute myeloid leukemia (AML) by yet unknown mechanisms. We previously identified 3 candidate enhancer regions: enhancer region 1 (E1) at -2 kb upstream; enhancer region 2 (E2) at +10.6 kb downstream inside intron 6; and enhancer region 3 (E3) +140 kb downstream of the translation start site. In the current study, we utilized CRISPR-Cas9 genome editing to further characterize these enhancers in a human AML cell line and identify the key transcription factors (TFs) associated with their function. To efficiently track MEIS1 expression levels, a GFP reporter, a P2A self-cleaving peptide tag and a hemagglutinin tag at its translation start site was introduced in a MEIS1 high expressing human AML cell line, U937. Then we introduced random mutations (Indels) along the MEIS1 locus utilizing a CRISPR-Cas9 mediated genome editing vector system in mono-allelic MEIS1-GFP-tagged U937 cells with special focus on the previously identified enhancer regions to find the key sequences important to the function of the MEIS1 enhancer regions. Two targeted regions yielding the highest proportion of GFP - cells corresponded to the E2 enhancer region within intron 6 and were referred to as E2.1 and E2.2. Using chromosome conformation capture (3C) assay, we detected a significantly decreased interaction (p=0.0022) between the promoter and the intron 6 region surrounding the E2 region in E2.2 targeted cells compared to the parental cells. Moreover, our data indicated that the DNA sequence within E2.2 is highly critical to this region's enhancer function which is further influenced by the larger genomic region surrounding the E2.1 gRNA targeted site. To identify TFs binding to the E2 region, we further scrutinized the E2.2 indel region for loss of TF binding sites. We performed TF prediction analysis and performed a protein pull down-mass spectrometry experiment to identify TF candidates. The overlap yielded a list of 7 TFs, each of which we targeted via CRISPR/Cas9. Reduction in GFP levels was only observed for FLI1 locus targeting but not for the other 6 TFs. Concordant reduction in MEIS1 and FLI1 levels were confirmed by immunoblotting. Additionally, chromatin immunoprecipitation (ChIP) followed by quantitative PCR revealed significant FLI1 enrichment at the promoter and at 3 sites surrounding the E2.2 region (p=0.0004) compared to 4 control regions scattered along the MEIS1 locus. Given a previous study indicating MEIS1 upregulation of FLI1 in normal hematopoiesis, we hypothesised that a positive feedback loop may exist between FLI1 and MEIS1 in AML. Since MEIS1 levels are frequently elevated in normal karyotype AML (CN-AML), we used the murine Hoxa9/Meis1 AML model as a surrogate for CN-AML and performed Meis1 ChIP-seq analysis. We detected direct Meis1 binding to the intronic region of the mouse Fli1 gene as well as other ETS factor loci, in Hoxa9/Meis1 cells. To better understand the clinical relevance of FLI1 in AML, we analyzed the Beat AML dataset. High FLI1 transcript levels correlated with adverse overall survival in CN-AML (p=0.044). Additionally, we observed a trend towards worse outcome with high FLI1 in the NPM1-mutated CN-AML subtype (p=0.069). We also observed a similar correlation in CN-AML for another ETS factor, ELF1, which we had previously shown to bind and upregulate MEIS1 expression in AML, suggesting a broader unrecognized role for ETS factors in AML. In summary, we have developed a rapid flow cytometry-based readout for the fine dissection and characterization of the cis-regulatory elements and associated TFs critical for MEIS1 transcription via CRISPR-Cas9 genetic manipulation. Our study revealed FLI1 as the candidate key regulator of MEIS1 expression and a positive correlation between FLI1 mRNA levels and worse overall survival in MEIS1-high AML subgroups. Disclosures No relevant conflicts of interest to declare.

The membrane associated accessory protein is an adeno-associated viral egress factor

Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

The chromatin remodeler Ino80 mediates RNAPII pausing site determination

Background Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive. Results Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells. Conclusions Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

The Contribution of the Predicted Sorting Platform Component HrcQ to Type III Secretion in Xanthomonas campestris pv. vesicatoria Depends on an Internal Translation Start Site

Pathogenicity of the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. T3S systems are conserved in plant- and animal-pathogenic bacteria and consist of at least nine structural core components, which are designated Sct (secretion and cellular translocation) in animal-pathogenic bacteria. Sct proteins are involved in the assembly of the membrane-spanning secretion apparatus which is associated with an extracellular needle structure and a cytoplasmic sorting platform. Components of the sorting platform include the ATPase SctN, its regulator SctL, and pod-like structures at the periphery of the sorting platform consisting of SctQ proteins. Members of the SctQ family form a complex with the C-terminal protein domain, SctQC, which is translated as separate protein and likely acts either as a structural component of the sorting platform or as a chaperone for SctQ. The sorting platform has been intensively studied in animal-pathogenic bacteria but has not yet been visualized in plant pathogens. We previously showed that the SctQ homolog HrcQ from X. campestris pv. vesicatoria assembles into complexes which associate with the T3S system and interact with components of the ATPase complex. Here, we report the presence of an internal alternative translation start site in hrcQ leading to the separate synthesis of the C-terminal protein region (HrcQC). The analysis of genomic hrcQ mutants showed that HrcQC is essential for pathogenicity and T3S. Increased expression levels of hrcQ or the T3S genes, however, compensated the lack of HrcQC. Interaction studies and protein analyses suggest that HrcQC forms a complex with HrcQ and promotes HrcQ stability. Furthermore, HrcQC colocalizes with HrcQ as was shown by fluorescence microscopy, suggesting that it is part of the predicted cytoplasmic sorting platform. In agreement with this finding, HrcQC interacts with the inner membrane ring protein HrcD and the SctK-like linker protein HrpB4 which contributes to the docking of the HrcQ complex to the membrane-spanning T3S apparatus. Taken together, our data suggest that HrcQC acts as a chaperone for HrcQ and as a structural component of the predicted sorting platform.

Expression Analysis of Lanosterol synthase Gene in Dynamic Accumulation of Triterpenoids in Sanghuangporus baumii

Background: Sanghuangporus baumii is a traditional Chinese medicine with anti-cancer, anti-tumor, and anti-inflammatory effects. Triterpenoids are one of the main medicinal ingredients found in S. baumii. However, the dynamic changes of triterpenoids content and its molecular regulation mechanism are still unclear. Objective: Some studies have shown that Lanosterol synthase (LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing S. baumii triterpenoids. Therefore, the study of LS gene and expression characteristics can provide clues for the further study of triterpenoids synthesis. Methods: The PCR, RACE PCR, RT-PCR, seamless cloning and prokaryotic expression technology were used to research the gene characteristic and dynamic changes of LS transcription level. Results: The S. baumii LS sequence included a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased. Results: The S. baumii LS sequence included a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased. Conclusion: The S. baumii LS was cloned and characterized to help to understand the mechanism of triterpenoids synthesis. In addition, we studied the relationship between LS transcription level and triterpenoid dynamic accumulation, and we found that they had a certain correlation.

The MuvB Complex Binds and Stabilizes Nucleosomes Downstream of the Transcription Start Site of Cell-Cycle Dependent Genes

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene-expression.