Early Transcriptional Activation Research Articles

Suppressing Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene Expression and Replication by RNase P Ribozyme.

Kaposi's sarcoma, an AIDS-defining illness, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic virus. In this study, we engineered ribozymes derived from ribonuclease P (RNase P) catalytic RNA with targeting against the mRNA encoding KSHV immediate early replication and transcription activator (RTA), which is vital for KSHV gene expression. The functional ribozyme F-RTA efficiently sliced the RTA mRNA sequence in vitro. In cells, KSHV production was suppressed with ribozyme F-RTA expression by 250-fold, and RTA expression was suppressed by 92-94%. In contrast, expression of control ribozymes hardly affected RTA expression or viral production. Further studies revealed both overall KSHV early and late gene expression and viral growth decreased because of F-RTA-facilitated suppression of RTA expression. Our results indicate the first instance of RNase P ribozymes having potential for use in anti-KSHV therapy.

Early Transcriptional Activity of Vtr-297 - Results from an Ongoing Phase I Clinical Trial

VTR-297 (Trichostatin A) is a pan-inhibitor of histone deacetylases types 1, 2, 4, 6, and 10. Histone deacetylase inhibitors (HDACi) are well-known small molecules that increase the acetylation of lysine residues by inhibiting the activity of histone deacetylase enzymes. These anticancer agents affect epigenetic and non-epigenetic transcriptional regulation and are already part of effective clinical practice and hematology regimens. As HDACi are known for their relatively wide transcriptional modulation, we have conducted a high throughput screen to elucidate early transcriptional effects related to exposure to Trichostatin A (TSA) on patient-derived, clinical time-series samples. NCT03838926 is an ongoing, open-label, dose-escalation, phase I clinical trial for subjects with relapsed or refractory hematologic malignancies. Enrolled patients were assigned to receive one currently tested dose (1.25 / 2.50 / 5.00 mg; IV infusion) in 28-day cycles, 3-times a week for three weeks with one week off (IV administration days 1,3,5,8,10,12,15,17 and 19) until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint was safety/tolerability and maximum tolerated dose (MTD) of VTR-297 (TSA). Currently, twelve patients enrolled in the study, including Multiple Myeloma (MM, n=5), Non-Hodgkin's Lymphoma (NHL, n=5), and Hodgkin's Lymphoma (HL, n=2) across the first three dose-escalation cohorts that completed (1.25 / 2.50 / 5.00mg). No significant drug-related toxicities were reported, and MTD was not reached (recruitment is currently ongoing in the fourth dose cohort of 10mg). As a part of the exploratory analysis of whole blood (PBMC), patient samples were collected on cycle1 day1 (C1D1; pre-infusion, 15min-post, 30min-post, 1h-post, 2h-post, and 6h-post infusion) to capture any transcriptional changes (RNAseq) related to VTR-297 treatment. In the current analysis, samples were collected and analyzed in respective batches (15min / 30min / 1h / 2h / 6h) and compared with combined baseline samples (pre-infusion). Using Deseq2 to ascertain transcriptionally affected genes (pval<0.05, up/down relative fold-change 1.5x), we detected 136 differentially expressed genes across at least one of the time point analyzed. Among the detected genes, we found novel and previously described genes related to the core pathophysiology of myeloid and lymphoid disease (e.g., iron metabolism, vascular metastasis, and tyrosine kinase activity). In conclusion, these preliminary signatures offer early biomarker information related to a specific transcriptional activity elicited by intravenously delivered VTR-297 in the clinical setting. In an ongoing clinical study, we will further monitor, validate, and expand on these findings. Further observations at higher, safe, and clinically meaningful doses will complement our current, early picture of VTR-297 activity and how it can be used to optimize TSA-based treatments.

Ciliogenesis requires sphingolipid-dependent membrane and axoneme interaction

Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome footprinting, we performed global translatome profiling during cilia regeneration in Chlamydomonas and uncovered that flagellar genes undergo an early transcriptional activation but late translational repression. This pattern guided our identification of sphingolipid metabolism enzymes, including serine palmitoyltransferase (SPT), as essential regulators for ciliogenesis. Cryo-electron tomography showed that ceramide loss abnormally increased the membrane-axoneme distance and generated bulged cilia. We found that ceramides interact with intraflagellar transport (IFT) particle proteins that IFT motors transport along axoneme microtubules (MTs), suggesting that ceramide-IFT particle-IFT motor-MT interactions connect the ciliary membrane with the axoneme to form rod-shaped cilia. SPT-deficient vertebrate cells were defective in ciliogenesis, and SPT mutations from patients with hereditary sensory neuropathy disrupted cilia, which could be restored by sphingolipid supplementation. These results reveal a conserved role of sphingolipid in cilium formation and link compromised sphingolipid production with ciliopathies.

Time Is of the Essence-Early Activation of the Mevalonate Pathway in Apple Challenged With Gray Mold Correlates With Reduced Susceptibility During Postharvest Storage.

Apple is typically stored under low temperature and controlled atmospheric conditions to ensure a year round supply of high quality fruit for the consumer. During storage, losses in quality and quantity occur due to spoilage by postharvest pathogens. One important postharvest pathogen of apple is Botrytis cinerea. The fungus is a broad host necrotroph with a large arsenal of infection strategies able to infect over 1,400 different plant species. We studied the apple-B. cinerea interaction to get a better understanding of the defense response in apple. We conducted an RNAseq experiment in which the transcriptome of inoculated and non-inoculated (control and mock) apples was analyzed at 0, 1, 12, and 28 h post inoculation. Our results show extensive reprogramming of the apple’s transcriptome with about 28.9% of expressed genes exhibiting significant differential regulation in the inoculated samples. We demonstrate the transcriptional activation of pathogen-triggered immunity and a reprogramming of the fruit’s metabolism. We demonstrate a clear transcriptional activation of secondary metabolism and a correlation between the early transcriptional activation of the mevalonate pathway and reduced susceptibility, expressed as a reduction in resulting lesion diameters. This pathway produces the building blocks for terpenoids, a large class of compounds with diverging functions including defense. 1-MCP and hot water dip treatment are used to further evidence the key role of terpenoids in the defense and demonstrate that ethylene modulates this response.

Genomic signatures of the unjamming transition in compressed human bronchial epithelial cells.

Epithelial tissue can transition from a jammed, solid-like, quiescent phase to an unjammed, fluid-like, migratory phase, but the underlying molecular events of the unjamming transition (UJT) remain largely unexplored. Using primary human bronchial epithelial cells (HBECs) and one well-defined trigger of the UJT, compression mimicking the mechanical effects of bronchoconstriction, here, we combine RNA sequencing data with protein-protein interaction networks to provide the first genome-wide analysis of the UJT. Our results show that compression induces an early transcriptional activation of the membrane and actomyosin network and a delayed activation of the extracellular matrix (ECM) and cell-matrix networks. This response is associated with a signaling cascade that promotes actin polymerization and cellular motility through the coordinated interplay of downstream pathways including ERK, JNK, integrin signaling, and energy metabolism. Moreover, in nonasthmatic versus asthmatic HBECs, common genomic patterns associated with ECM remodeling suggest a molecular connection between airway remodeling, bronchoconstriction, and the UJT.

Role of Myc Proto-Oncogene as a Transcriptional Hub to Regulate the Expression of Regeneration-Associated Genes following Preconditioning Peripheral Nerve Injury.

Preconditioning peripheral nerve injury enhances the intrinsic growth capacity of DRGs sensory axons by inducing transcriptional upregulation of the regeneration-associated genes (RAGs). However, it is still unclear how preconditioning injury leads to the orchestrated induction of many RAGs. The present study identified Myc proto-oncogene as a transcriptional hub gene to regulate the expression of a distinct subset of RAGs in DRGs following the preconditioning injury. We demonstrated that c-MYC bound to the promoters of certain RAGs, such as Jun, Atf3, and Sprr1a, and that Myc upregulation following SNI preceded that of the RAGs bound by c-MYC. Marked DNA methylation of the Myc exon 3 sequences was implicated in the early transcriptional activation and accompanied by open histone marks. Myc deletion led to a decrease in the injury-induced expression of a distinct subset of RAGs, which were highly overlapped with the list of RAGs that were upregulated by Myc overexpression. Following dorsal hemisection spinal cord injury in female rats, Myc overexpression in DRGs significantly prevented the retraction of the sensory axons in a manner dependent on its downstream RAG, June Our results suggest that Myc plays a critical role in axon regeneration via its transcriptional activity to regulate the expression of a spectrum of downstream RAGs and subsequent effector molecules. Identification of more upstream hub transcription factors and the epigenetic mechanisms specific for individual hub transcription factors would advance our understanding of how the preconditioning injury induces orchestrated upregulation of RAGs.

Physiological, biochemical and transcriptional responses of Passiflora edulis Sims f. edulis under progressive drought stress

Water deficit stress is responsible for at least 40 % of crop losses worldwide, positioning itself as a strong threat to food security. Purple passion fruit (Passiflora edulis Sims f. edulis) is one of the most desirable exotic fruits on the world market mainly for its organoleptic properties. Information on the response to water deficit stress for purple passion fruit is scarce and in some cases contradictory. Therefore, the objective of the present work was to evaluate the morphological, physiological, biochemical and transcriptional response of purple passion fruit during water deficit and depict its strategy. The response of purple passion fruit plants to two water deficit treatments (T1: 50 % of irrigation, T2: 0% of irrigation compared to control) was evaluated during 40 days of stress, measuring changes in physiological variables related to growth and water relations, as well as the content of metabolites related to stress and the expression of genes associated with drought stress. Results show that the plant prevents water loss, by stomata closure, modulation of growth and accumulation of proline and sugars, while promoting root growth, features associated with tolerance to water deficit. At the expression level, early transcriptional activation of PeDREB2A and PeRD21A were detected in plants under the most severe stress, while PeGOLS1 and PeAFL1 upregulation were observed in plants under milder stress. These results suggest that purple passion fruit plants display an isohydric behavior and evade water deficit stress as a response strategy, where loss of water is avoided.

Coordination of transcriptional and translational regulations in human epithelial cells infected by Listeria monocytogenes

ABSTRACT The invasion of mammalian cells by intracellular bacterial pathogens reshuffles their gene expression and functions; however, we lack dynamic insight into the distinct control levels that shape the host response. Here, we have addressed the respective contribution of transcriptional and translational regulations during a time-course of infection of human intestinal epithelial cells by an epidemic strain of Listeria monocytogenes, using transcriptome analysis paralleled with ribosome profiling. Upregulations were dominated by early transcriptional activation of pro-inflammatory genes, whereas translation inhibition appeared as the major driver of downregulations. Instead of a widespread but transient shutoff, translation inhibition affected specifically and durably transcripts encoding components of the translation machinery harbouring a 5ʹ-terminal oligopyrimidine motif. Pre-silencing the most repressed target gene (PABPC1) slowed down the intracellular multiplication of Listeria monocytogenes, suggesting that the infected host cell can benefit from the repression of genes involved in protein synthesis and thereby better control infection.

Nucleosome sliding, histone modifications, and the epigenetic regulation of the Simian Virus 40 life cycle

We have recently shown that at late times in an SV40 infection a nucleosome containing modified histones is positioned over the enhancer region consistent with repression of early transcription and activation of late transcription. However, this nucleosome appears to slide toward the late region during virion formation consistent with a role in the activation of early transcription and repression of late transcription in a subsequent infection. Because the transcription factor SP1 plays a major role in regulating SV40 early and late transcription and its binding should be sensitive to nucleosome positioning, we measured the ability of HIS‐tagged SP1 to bind to various forms of SV40 chromatin in vitro and compared this to the presence of SP1 in SV40 chromatin derived in vivo. We found that HIS‐tagged SP1 bound well to chromatin from virions and very poorly to other forms of SV40 chromatin in vitro, while cellular SP1 was associated with minichromosomes isolated from infected cells and was not found in chromatin from virions. In order to determine whether the location of nucleosomes and associated histone modifications plays a role in the activation and continuation of early transcription during the initiation of an infection, we have mapped the location of RNAPII and nucleosomes containing a number of histone modifications in chromatin from virions and minichromosomes isolated 30 minutes and 2 hours post‐infection using ChIP‐Seq. During the first two hours of infection we found that RNAPII first associates with the miRNA site in the SV40 genome (at 30 minutes post‐infection) and then shifts to the early regulatory region around the enhancer (2 hours post‐infection). We next compared the location of RNAPII at the two time points to the location of nucleosomes carrying one of nine different specific histone modifications in the chromatin from virions, and minichromosomes isolated at these times. Chromatin from virions was used because it is the substrate for the activation of transcription, while chromatin from the minichromosomes would indicate any changes occurring as a consequence of transcriptional activation or extension. Based upon this rationale, we identified acetylated H3 as the most likely histone modification in SV40 predicting subsequent binding by RNAPII. We also identified acetylated H4 and the shifting of nucleosomes away from the RNAPII binding site as changes which result from activation of transcription. Together these results indicate that positioned nucleosomes carrying certain histone modifications may control accessibility of transcription factors and also serve as marks for either the binding of RNAPII during initiation of transcription or subsequent re‐initiation during active transcription.Support or Funding InformationNIH 1R03AI127969‐01NIH 1R03AI142011‐01

Circulating CD30+CD4+ T Cells Increase Before Human Immunodeficiency Virus Rebound After Analytical Antiretroviral Treatment Interruption.

Identification of nonviral markers of human immunodeficiency virus (HIV) infection that increase before viral rebound during analytical treatment interruption (ATI) may affect HIV persistence research. We previously showed that HIV ribonucleic acid (RNA) is enriched in CD30+CD4+ T cells in many individuals. Here, we studied CD30+CD4+ T-cell dynamics before ATI, during ATI (before detectable plasma RNA), and after HIV rebound. Peripheral blood mononuclear cells from 23 participants collected longitudinally from 5 Adult AIDS Clinical Trials Group studies incorporating ATI were included in this study. Flow cytometric characterization of expression of CD30 and markers of T-cell activation and exhaustion were performed along with HIV-1 RNA and deoxyribonucleic acid quantification and measurement of soluble plasma CD30 and CD30 ligand. The percentage of CD4+ T cells expressing CD30 significantly increased from pre-ATI to postinterruption time points before detectible viremia (1.65 mean relative increase, P = .005). Seventy-seven percent of participants experienced an increase in CD30+ cells before viral rebound. In contrast, there were no significant differences between pre-ATI and postinterruption pre-rebound time points in percentages of lymphocytes expressing CD69, CD38/HLA-DR, or PD-1 until after HIV recrudescence. CD30 may be a surrogate marker of early replication or viral transcriptional activity before detection by routine peripheral blood sampling.

Single-cell DNA replication profiling identifies spatiotemporal developmental dynamics of chromosome organization.

In mammalian cells, chromosomes are partitioned into megabase-sized topologically associating domains (TADs). TADs can be in either A (active) or B (inactive) subnuclear compartments, which exhibit early and late replication timing (RT), respectively. Here, we show that A/B compartments change coordinately with RT changes genome wide during mouse embryonic stem cell (mESC) differentiation. While A to B compartment changes and early to late RT changes were temporally inseparable, B to A changes clearly preceded late to early RT changes and transcriptional activation. Compartments changed primarily by boundary shifting, altering the compartmentalization of TADs facing the A/B compartment interface, which was conserved during reprogramming and confirmed in individual cells by single-cell Repli-seq. Differentiating mESCs altered single-cell Repli-seq profiles gradually but uniformly, transiently resembling RT profiles of epiblast-derived stem cells (EpiSCs), suggesting that A/B compartments might also change gradually but uniformly toward a primed pluripotent state. These results provide insights into how megabase-scale chromosome organization changes in individual cells during differentiation.

A clinically relevant murine model unmasks a "two-hit" mechanism for reactivation and dissemination of cytomegalovirus after kidney transplant.

Reactivation of latent cytomegalovirus remains an important complication after transplant. Although immunosuppression (IS) has been implicated as a primary cause, we have previously shown that the implantation response of a kidney allograft can lead to early transcriptional activation of latent murine cytomegalovirus (MCMV) genes in an immune-competent host and to MCMV reactivation and dissemination to other organs in a genetically immune-deficient recipient. We now describe a model that allows us to separately analyze the impact of the implantation effect vs that of a clinically relevant IS regimen. Treatment with IS of latently infected mice alone does not induce viral reactivation, but transplant of latently infected allogeneic kidneys combined with IS facilitates MCMV reactivation in the graft and dissemination to other organs. The IS regimen effectively dampens allo-immune inflammatory pathways and depletes recipient anti-MCMV but does not affect ischemia-reperfusion injury pathways. MCMV reactivation similar to that seen in allogeneic transplants combined with also occurs after syngeneic transplants. Thus, our data strongly suggest that while ischemia-reperfusion injury of the implanted graft is sufficient and necessary to initiate transcriptional reactivation of latent MCMV ("first hit"), IS is permissive to the first hit and facilitates dissemination to other organs ("second hit").

KSHV lytic proteins K-RTA and K8 bind to cellular and viral chromatin to modulate gene expression.

The oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) has two distinct life cycles with lifelong latent/non-productive and a sporadic lytic-reactivating/productive phases in the infected immune compromised human hosts. The virus reactivates from latency in response to various chemical or environmental stimuli, which triggers the lytic cascade and leads to the expression of immediate early gene, i.e. Replication and Transcription Activator (K-RTA). K-RTA, the latent-to-lytic switch protein, activates the expression of early (E) and late (L) lytic genes by transactivating multiple viral promoters. Expression of K-RTA is shown to be sufficient and essential to switch the latent virus to enter into the lytic phase of infection. Similarly, the virus-encoded bZIP family of protein, K8 also plays an important role in viral lytic DNA replication. Although, both K-RTA and K8 are found to be the ori-Lyt binding proteins and are required for lytic DNA replication, the detailed DNA-binding profile of these proteins in the KSHV and host genomes remains uncharacterized. In this study, using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) assay, we performed a comprehensive analysis of K-RTA and K8 binding sites in the KSHV and human genomes in order to identify specific DNA binding sequences/motifs. We identified two novel K-RTA binding motifs, (i.e. AGAGAGAGGA/motif RB and AGAAAAATTC/motif RV) and one K8 binding motif (i.e. AAAATGAAAA/motif KB), respectively. The binding of K-RTA/K8 proteins with these motifs and resulting transcriptional modulation of downstream genes was further confirmed by DNA electrophoretic gel mobility shift assay (EMSA), reporter promoter assay, Chromatin Immunoprecipitation (ChIP) assay and mRNA quantitation assay. Our data conclusively shows that K-RTA/K8 proteins specifically bind to these motifs on the host/viral genomes to modulate transcription of host/viral genes during KSHV lytic reactivation.

Global translation inhibition yields condition-dependent de-repression of ribosome biogenesis mRNAs.

Ribosome biogenesis (RiBi) is an extremely energy intensive process that is critical for gene expression. It is thus highly regulated, including through the tightly coordinated expression of over 200 RiBi genes by positive and negative transcriptional regulators. We investigated RiBi regulation as cells initiated meiosis in budding yeast and noted early transcriptional activation of RiBi genes, followed by their apparent translational repression 1 hour (h) after stimulation to enter meiosis. Surprisingly, in the representative genes examined, measured translational repression depended on their promoters rather than mRNA regions. Further investigation revealed that the signature of this regulation in our data depended on pre-treating cells with the translation inhibitor, cycloheximide (CHX). This treatment, at 1 h in meiosis, but not earlier, rapidly resulted in accumulation of RiBi mRNAs that were not translated. This effect was also seen in with CHX pre-treatment of cells grown in media lacking amino acids. For NSR1, this effect depended on the –150 to –101 region of the promoter, as well as the RiBi transcriptional repressors Dot6 and Tod6. Condition-specific RiBi mRNA accumulation was also seen with translation inhibitors that are dissimilar from CHX, suggesting that this phenomenon might represent a feedback response to global translation inhibition.

Isoform-Dependent Changes in Cytochrome P450-Mediated Drug Metabolism after Portal Vein Ligation in the Rat

Background: Surgical removal of complicated liver tumors may be realized in two stages via selective portal vein ligation, inducing the atrophy of portally ligated lobes and the compensatory hypertrophy of nonligated liver lobes. Unlike morphological changes, functional aspects such as hepatic cytochrome P450 (CYP)-mediated drug metabolism remain vaguely understood, despite its critical role in both drug biotransformation and hepatic functional analysis. Our goal was the multilevel characterization of hepatic CYP-mediated drug metabolism after portal vein ligation in the rat. Methods: Male Wistar rats (n = 24, 210–230 g) were analyzed either untreated (controls; n = 4) or 24/48/72/168/336 h (n = 4 each) following portal vein ligation affecting approximately 80% of the liver parenchyma. Besides the weights of ligated and nonligated lobes, pentobarbital (30 mg/kg)-induced sleeping time, CYP1A(2), CYP 2B(1/2), CYP2C(6/11/13), CYP3A(1) enzyme activities, and corresponding isoform mRNA expressions, as well as CYP3A1 protein expression were determined by in vivo sleeping test, CYP isoform-selective assays, polymerase chain reaction, and immunohistochemistry, respectively. Results: Portal vein ligation triggered atrophy in ligated lobes and hypertrophy nonligated lobes. Sleeping time was transiently elevated (p = 0.0451). After an initial rise, CYP1A, CYP2B, and CYP3A enzyme activities dropped until 72 h, followed by a potent increase only in the nonligated lobes, paralleled by an early (24–48 h) transcriptional activation only in nonligated lobes. CYP2C enzyme activities and mRNA levels were bilaterally rapidly decreased, showing a late reconvergence only in nonligated lobes. CYP3A1 immunohistochemistry indicated substantial differences in positivity in the early period. Conclusions: Beyond the atrophy-hypertrophy complex, portal vein ligation generated a transient suppression of global and regional drug metabolism, re-established by an adaptive, CYP isoform-dependent transcriptional response of the nonligated lobes.

Semen Exosomes Promote Transcriptional Silencing of HIV-1 by Disrupting NF-κB/Sp1/Tat Circuitry.

Exosomes play various roles in host responses to cancer and infective agents, and semen exosomes (SE) inhibit HIV-1 infection and transmission, although the mechanism(s) by which this occurs is unclear. Here, we show that SE block HIV-1 proviral transcription at multiple transcriptional checkpoints, including transcription factor recruitment to the long terminal repeat (LTR), transcription initiation, and elongation. Biochemical and functional studies show that SE inhibit HIV-1 LTR-driven viral gene expression and virus replication. Through partitioning of the HIV-1 RNA, we found that SE reduced the optimal expression of various viral RNA species. Chromatin immunoprecipitation-real-time quantitative PCR (ChIP-RT-qPCR) and electrophoretic mobility shift assay (EMSA) analysis of infected cells identified the human transcription factors NF-κB and Sp1, as well as RNA polymerase (Pol) II and the viral protein transcriptional activator (Tat), as targets of SE. Of interest, SE inhibited HIV-1 LTR activation mediated by HIV-1 or Tat, but not by the mitogen phorbol myristate acetate (PMA) or tumor necrosis factor alpha (TNF-α). SE inhibited the DNA binding activities of NF-κB and Sp1 and blocked the recruitment of these transcription factors and Pol II to the HIV-1 LTR promoter. Importantly, SE directly blocked NF-κB, Sp1, and Pol II binding to the LTR and inhibited the interactions of Tat/NF-κB and Tat/Sp1, suggesting that SE-mediated inhibition of the functional quadripartite complex NF-κB-Sp1-Pol II-Tat may be a novel mechanism of proviral transcription repression. These data provide a novel molecular basis for SE-mediated inhibition of HIV-1 and identify Tat as a potential target of SE.IMPORTANCE HIV is most commonly transmitted sexually, and semen is the primary vector. Despite progress in studies of HIV pathogenesis and the success of combination antiretroviral therapy in controlling viral replication, current therapy cannot completely control sexual transmission. Thus, there is a need to identify effective methods of controlling HIV replication and transmission. Recently, it was shown that human semen contains exosomes that protect against HIV infection in vitro In this study, we identified a mechanism by which semen exosomes inhibited HIV-1 RNA expression. We found that semen exosomes inhibit recruitment of transcription factors NF-κB and Sp1, as well as RNA Pol II, to the promoter region in the 5' long terminal repeat (LTR) of HIV-1. The HIV-1 early protein transcriptional activator (Tat) was a target of semen exosomes, and semen exosomes inhibited the binding and recruitment of Tat to the HIV-1 LTR.

Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection

The location of nucleosomes in SV40 virions and minichromosomes isolated during infection were determined by next generation sequencing (NGS). The patterns of reads within the regulatory region of chromatin from wild-type virions indicated that micrococcal nuclease-resistant nucleosomes were specifically positioned at nt 5223 and nt 363, while in minichromosomes isolated 48 h post-infection we observed nuclease-resistant nucleosomes at nt 5119 and nt 212. The nucleosomes at nt 5223 and nt 363 in virion chromatin would be expected to repress early and late transcription, respectively. In virions from the mutant cs1085, which does not repress early transcription, we found that these two nucleosomes were significantly reduced compared to wild-type virions confirming a repressive role for them. In chromatin from cells infected for only 30min with wild-type virus, we observed a significant reduction in the nucleosomes at nt 5223 and nt 363 indicating that the potential repression by these nucleosomes appeared to be relieved very early in infection.

Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13

SUMMARYThe combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13−/−; Hoxd13−/−limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.

Mitotic binding of Esrrb marks key regulatory regions of the pluripotency network.

Pluripotent mouse embryonic stem cells maintain their identity throughout virtually infinite cell divisions. This phenomenon, referred to as self-renewal, depends on a network of sequence-specific transcription factors (TFs) and requires daughter cells to accurately reproduce the gene expression pattern of the mother. However, dramatic chromosomal changes take place in mitosis, generally leading to the eviction of TFs from chromatin. Here, we report that Esrrb, a major pluripotency TF, remains bound to key regulatory regions during mitosis. We show that mitotic Esrrb binding is highly dynamic, driven by specific recognition of its DNA-binding motif and is associated with early transcriptional activation of target genes after completion of mitosis. These results indicate that Esrrb may act as a mitotic bookmarking factor, opening another perspective to molecularly understand the role of sequence-specific TFs in the epigenetic control of self-renewal, pluripotency and genome reprogramming.

Caffeic acid attenuated acetaminophen-induced hepatotoxicity by inhibiting ERK1/2-mediated early growth response-1 transcriptional activation

Caffeic acid (CA) is a natural compound abundant in fruits, coffee and plants. This study aims to investigate the involved mechanism of the therapeutic detoxification of CA against acetaminophen (APAP)-induced hepatotoxicity. CA (10, 30 mg/kg) was orally given to mice at 1 h after mice were pre-administrated with APAP (300 mg/kg). The therapeutic detoxification of CA against APAP-induced hepatotoxicity was observed by detecting serum aminotransferases, liver malondialdehyde (MDA) amount and liver histological evaluation in vivo. CA reduced APAP-induced increase in the mRNA expression of early growth response 1 (Egr1) in hepatocytes, and inhibited APAP-induced Egr1 transcriptional activation in vitro and in vivo. CA reduced the increased expression of growth arrest and DNA-damage-inducible protein (Gadd45)α induced by APAP in hepatocytes. Moreover, Egr1 siRNA reduced Gadd45α expression and reversed APAP-induced cytotoxicity in hepatocytes. Further results showed that CA blocked APAP-induced activation of extracellular-regulated protein kinase (ERK1/2) signaling cascade in vivo and in vitro. In addition, the application of ERK1/2 inhibitors (PD98059 and U0126) abrogated the nuclear translocation of Egr1 induced by APAP in hepatocytes. In conclusion, this study demonstrated the therapeutic detoxification of CA against APAP-induced liver injury, and the inhibition of CA on ERK1/2-mediated Egr1 transcriptional activation was involved in this process.