Unique Regulatory Network Research Articles

Genome-wide chromatin accessibility reveals transcriptional regulation of heterosis in inter-subspecific hybrid rice.

The utilization of rice heterosis is essential for ensuring global food security; however, its molecular mechanism remains unclear. In this study, comprehensive analyses of accessible chromatin regions (ACRs), DNA methylation, and gene expression in inter-subspecific hybrid and its parents were performed to determine the potential role of chromatin accessibility in rice heterosis. The hybrid exhibited abundant ACRs, in which the gene ACRs and proximal ACRs were directly related to transcriptional activation rather than the distal ACRs. Regarding the dynamic accessibility contribution of the parents, paternal ZHF1015 transmitted a greater number of ACRs to the hybrid. Accessible genotype-specific target genes were enriched with overrepresented transcription factors, indicating a unique regulatory network of genes in the hybrid. Compared with its parents, the differentially accessible chromatin regions with upregulated chromatin accessibility were much greater than those with downregulated chromatin accessibility, reflecting a stronger regulation in the hybrid. Furthermore, DNA methylation levels were negatively correlated with ACR intensity, and genes were strongly affected by CHH methylation in the hybrid. Chromatin accessibility positively regulated the overall expression level of each genotype. ACR-related genes with maternal Z04A-bias allele-specific expression tended to be enriched during carotenoid biosynthesis, whereas paternal ZHF1015-bias genes were more active in carbohydrate metabolism. Our findings provide a new perspective on the mechanism of heterosis based on chromatin accessibility in inter-subspecific hybrid rice.

Unique regulatory network of dragon fruit simultaneously mitigates the effect of vanadium pollutant and environmental factors.

Under changing climatic conditions, plants are simultaneously facing conflicting stresses in nature. Plants can sense different stresses, induce systematic ROS signals, and regulate transcriptomic, hormonal, and stomatal responses. We performed transcriptome analysis to reveal the integrative stress response regulatory mechanism underlying heavy metal stress alone or in combination with heat and drought conditions in pitaya (dragon fruit). A total of 70 genes were identified from 31,130 transcripts with conserved differential expression. Furthermore, weighted gene co-expression network analysis (WGCNA) identified trait-associated modules. By integrating information from three modules and protein-protein interaction (PPI) networks, we identified 10 interconnected genes associated with the multifaceted defense mechanism employed by pitaya against co-occurring stresses. To further confirm the reliability of the results, we performed a comparative analysis of 350 genes identified by three trait modules and 70 conserved genes exhibiting their dynamic expression under all treatments. Differential expression pattern of genes and comparative analysis, have proven instrumental in identifying ten putative structural genes. These ten genes were annotated as PLAT/LH2, CAT, MLP, HSP, PB1, PLA, NAC, HMA, and CER1 transcription factors involved in antioxidant activity, defense response, MAPK signaling, detoxification of metals and regulating the crosstalk between the complex pathways. Predictive analysis of putative candidate genes, potentially governing single, double, and multifactorial stress response, by several signaling systems and molecular patterns. These findings represent a valuable resource for pitaya breeding programs, offering the potential to develop resilient "super pitaya" plants.

Diurnal Rhythms in the Red Seaweed Gracilariopsis chorda are Characterized by Unique Regulatory Networks of Carbon Metabolism.

Cellular and physiological cycles are driven by endogenous pacemakers, the diurnal and circadian rhythms. Key functions such as cell cycle progression and cellular metabolism are under rhythmic regulation, thereby maintaining physiological homeostasis. The photoreceptors phytochrome and cryptochrome, in response to light cues, are central input pathways for physiological cycles in most photosynthetic organisms. However, among Archaeplastida, red algae are the only taxa that lack phytochromes. Current knowledge about oscillatory rhythms is primarily derived from model species such as Arabidopsis thaliana and Chlamydomonas reinhardtii in the Viridiplantae, whereas little is known about these processes in other clades of the Archaeplastida, such as the red algae (Rhodophyta). We used genome-wide expression profiling of the red seaweed Gracilariopsis chorda and identified 3,098 rhythmic genes. Here, we characterized possible cryptochrome-based regulation and photosynthetic/cytosolic carbon metabolism in this species. We found a large family of cryptochrome genes in G. chorda that display rhythmic expression over the diurnal cycle and may compensate for the lack of phytochromes in this species. The input pathway gates regulatory networks of carbon metabolism which results in a compact and efficient energy metabolism during daylight hours. The system in G. chorda is distinct from energy metabolism in most plants, which activates in the dark. The green lineage, in particular, land plants, balance water loss and CO2 capture in terrestrial environments. In contrast, red seaweeds maintain a reduced set of photoreceptors and a compact cytosolic carbon metabolism to thrive in the harsh abiotic conditions typical of intertidal zones.

The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting.

Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.

STEM-24. SINGLE-CELL ATLAS OF HUMAN FETAL CEREBELLUM DECODING MEDULLOBLASTOMA ORIGIN AND ONCOGENESIS

Abstract Medulloblastomas (MBs) are the most common malignant childhood brain tumors, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatment strategies. Previous cell-type analyses of mouse cerebella or human counterparts from frozen tissue nuclei have not fully defined the compositional heterogeneity of MBs. Here, we undertook an unprecedented single-cell profiling of freshly-isolated human fetal cerebella at different developmental stages to establish a reference map for delineating the hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in human fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group-3 and metastatic tumors. Integrated single-cell multi-omic profiling revealed unique regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in aggressive group-3 MBs. Genomic profiling and Hi-C analyses identified de novo long-range chromatin loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group-3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group-3 MB growth. Together, our integrated single-cell atlases of human fetal cerebella and MBs reveal important cell populations predisposed to transformation and regulatory circuitries underlying tumor cell state evolution and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

Single-cell multiomics identifies clinically relevant mesenchymal stem-like cells and key regulators for MPNST malignancy.

Malignant peripheral nerve sheath tumor (MPNST), a highly aggressive Schwann cell (SC)-derived soft tissue sarcoma, arises from benign neurofibroma (NF); however, the identity, heterogeneity and origins of tumor populations remain elusive. Nestin+ cells have been implicated as tumor stem cells in MPNST; unexpectedly, single-cell profiling of human NF and MPNST and their animal models reveal a broad range of nestin-expressing SC lineage cells and dynamic acquisition of discrete cancer states during malignant transformation. We uncover a nestin-negative mesenchymal neural crest-like subpopulation as a previously unknown malignant stem-like state common to murine and human MPNSTs, which correlates with clinical severity. Integrative multiomics profiling further identifies unique regulatory networks and druggable targets against the malignant subpopulations in MPNST. Targeting key epithelial-mesenchymal transition and stemness regulators including ZEB1 and ALDH1A1 impedes MPNST growth. Together, our studies reveal the underlying principles of tumor cell-state evolution and their regulatory circuitries during NF-to-MPNST transformation, highlighting a hitherto unrecognized mesenchymal stem-like subpopulation in MPNST disease progression.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

How to Use the TDCor Algorithm to Infer Gene Regulatory Networks from Time Series Transcriptomic Data.

Over the last few decades, many genes have been functionally characterized and shown to be involved in various metabolic, developmental, and signaling pathways. However it still remains unclear how all these genes and pathways integrate into a unique regulatory network to coordinate the development and the growth, or the response to the environment. This is why unraveling the topology of gene regulatory networks (GRN) has become central to our understanding of all these processes. The recent advancement of high-throughput methods has provided enormous amount of -omics data. These data can now be exploited for rapid network reconstruction with statistical inference methods. We recently published a new GRN inference algorithm called TDCor which reconstructs GRN from time-series transcriptomic data. The algorithm has been released in the form of an R package. Here, I describe into details how to install and use the package.

Abstract LB206: Simultaneous single cell profiling of open chromatin and gene expression in B cell lymphoma highlights tumor-specific regulatory networks

Abstract Profiling multiple analytes from a single cell enables additional dimensions to characterize tumor biology. Simultaneous measurement of epigenetic information and gene expression in single cells provides a clearer readout of the links between regulatory elements and target genes, allowing a more mechanistic interpretation of cell clusters and better cell type distinctions based on a cell's history and its future potential. Here, we describe simultaneous measurement of gene expression and chromatin accessibility from thousands of single nuclei using the Chromium Single Cell Multiome ATAC + Gene Expression. We leverage a microfluidic platform to generate paired barcoded gene expression and ATAC-seq libraries, linking transcriptomic and open chromatin information at the single cell level. Using this method, we jointly profiled gene expression and chromatin accessibility from over 10,000 immune cells from healthy human PBMCs and over 14,000 cells from a human B cell lymphoma sample. In healthy immune cells, we leveraged chromatin information to better parse distinct cell states within a gene expression-defined cell type, showing that the addition of ATAC profiling to gene expression improves resolution of cell type specification. In B cell lymphoma, we used known markers of malignancy to segregate tumor B cells from normal B cells. We constructed regulatory networks in both cell states using the observed transcription factor motif enrichment in open chromatin regions combined with gene expression from the same cells. Comparing regulatory networks in healthy vs. tumor B cells highlighted the presence of unique regulatory networks in tumor B cells. Notably, the PAX5 tumor regulator was observed only in tumor B cells, and its impacts on the regulation of many downstream genes was characterized. Building this regulatory network analysis was only made possible through the joint profiling of open chromatin and gene expression at the single cell level. Our data demonstrate the power of simultaneous profiling of both epigenomic and transcriptomic data in characterizing cells in cancer state and extracting functional information from tumor samples. Citation Format: Shamoni Maheshwari, Li Wang, Vijay Gopalan, Joe Shuga, Sharmila Chatterjee, Kamila Belhocine. Simultaneous single cell profiling of open chromatin and gene expression in B cell lymphoma highlights tumor-specific regulatory networks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB206.

Autophagy-dependent survival is controlled with a unique regulatory network upon various cellular stress events

Although autophagy is a type of programmed cell death, it is also essential for cell survival upon tolerable level of various stress events. For the cell to respond adequately to an external and/or internal stimulus induced by cellular stress, autophagy must be controlled in a highly regulated manner. By using systems biology techniques, here we explore the dynamical features of autophagy induction. We propose that the switch-like characteristic of autophagy induction is achieved by a control network, containing essential feedback loops of four components, so-called autophagy inducer, autophagy controller, mTORC1 and autophagy executor, respectively. We show how an autophagy inducer is capable to turn on autophagy in a cellular stress-specific way. The autophagy controller acts as a molecular switch and not only promotes autophagy but also blocks the permanent hyperactivation of the process via downregulating the autophagy inducer. In this theoretical analysis, we explore in detail the properties of all four proposed controlling elements and their connections. Here we also prove that the kinetic features of this control network can be considered accurate in various stress processes (such as starvation, endoplasmic reticulum stress and oxidative stress), even if the exact components may be different. The robust response of the resulting control network is essential during cellular stress.

Chromatin Structure and Function in Mosquitoes.

The principles and function of chromatin and nuclear architecture have been extensively studied in model organisms, such as Drosophila melanogaster. However, little is known about the role of these epigenetic processes in transcriptional regulation in other insects including mosquitoes, which are major disease vectors and a worldwide threat for human health. Some of these life-threatening diseases are malaria, which is caused by protozoan parasites of the genus Plasmodium and transmitted by Anopheles mosquitoes; dengue fever, which is caused by an arbovirus mainly transmitted by Aedes aegypti; and West Nile fever, which is caused by an arbovirus transmitted by Culex spp. In this contribution, we review what is known about chromatin-associated mechanisms and the 3D genome structure in various mosquito vectors, including Anopheles, Aedes, and Culex spp. We also discuss the similarities between epigenetic mechanisms in mosquitoes and the model organism Drosophila melanogaster, and advocate that the field could benefit from the cross-application of state-of-the-art functional genomic technologies that are well-developed in the fruit fly. Uncovering the mosquito regulatory genome can lead to the discovery of unique regulatory networks associated with the parasitic life-style of these insects. It is also critical to understand the molecular interactions between the vectors and the pathogens that they transmit, which could hold the key to major breakthroughs on the fight against mosquito-borne diseases. Finally, it is clear that epigenetic mechanisms controlling mosquito environmental plasticity and evolvability are also of utmost importance, particularly in the current context of globalization and climate change.

Abstract 1266: Mutant IDH1 alters epigenetic regulation to promote stemness in low grade glioma: Deciphering signaling pathways altered by mIDH1 inhibition

Abstract Greater than 75% of Low Grade Gliomas (LGG) harbor a mutation in isocitrate dehydrogenase 1 (IDH1) and this molecular subgroup also exhibits mutations in TP53 and ATRX. Mutant IDH1 often recurs and presents with a more aggressive phenotype that is refractory to existing therapies. The production of an oncometabolite, 2-hydroxyglutarate by mutant IDH1 (mIDH1) has been shown to induce histone and DNA hypermethylation. Pharmacological inhibition of mIDH1 (AGI-5198) can restore expression of genes involved in neural differentiation. We hypothesize that mIDH1 remodels chromatin to promote a stem cell-like transcriptional state. Recent single cell RNA-seq data from primary mIDH1 astrocytoma and oligodendroglioma has identified an undifferentiated pool of tumor cells with a neural stem/progenitor transcriptional profile. We aim to identify unique regulatory networks in SF10602, a human primary glioma culture derived from a surgical biopsy, which endogenously expresses mIDH1.We treated SF10602 with AGI-5198 every 2 days for 1 week. We observed a global reduction in histone marks: H3K4me3, H3K36me3, and H3K27me3. This reversibility of histone hypermethylation has previously been described in mIDH1 tumor lines treated with mIDH1 inhibitors that block 2HG production. We confirmed that mIDH1 inhibition reduced 2HG levels by collecting condition media and performing liquid chromatography mass spectrometry. We performed a temporal micrococcal nuclease (Mnase) digestion to assess chromatin accessibility and we observed enhanced chromatin condensation following mIDH1 inhibition. To evaluate if the reversion of chromatin organization resulting from mIDH1 inhibition impacts neural differentiation pathways we performed RNA-seq. We identified 496 differentially up-regulated and 893 down-regulated genes (log2>+/-0.6) comparing SF10602 with/out AGI-5198. Following gene set enrichment analysis, we observed upregulation of pathways involved in epigenetic regulation, DNA conformation and neuronal development. Additionally, we observed downregulation of pathways involved in cell polarity and transcription factor activity. In future work, we will perform ChIP-seq to define enhancers and promoters that regulate stem cell-like characteristics. These findings will reveal novel genomic loci unique to mIDH1 glioma. SC is supported by the T32 Cancer Biology Training Grant, the work is supported by grants from NIH/NINDS to MGC and PRL. Citation Format: Stephen Carney, Brandon Zhu, Padma Kadiyala, Maria Garcia-Fabiani, Preethi Gorla, Felipe Nunez, Pedro R. Lowenstein, Maria G. Castro. Mutant IDH1 alters epigenetic regulation to promote stemness in low grade glioma: Deciphering signaling pathways altered by mIDH1 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1266.

Derivation of notochordal cells from human embryonic stem cells reveals unique regulatory networks by single cell-transcriptomics.

Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.

Mutational Landscape of the BAP1 Locus Reveals an Intrinsic Control to Regulate the miRNA Network and the Binding of Protein Complexes in Uveal Melanoma.

The BAP1 (BRCA1-associated protein 1) gene is associated with a variety of human cancers. With its gene product being a nuclear ubiquitin carboxy-terminal hydrolase with deubiquitinase activity, BAP1 acts as a tumor suppressor gene with potential pleiotropic effects in multiple tumor types. Herein, we focused specifically on uveal melanoma (UM) in which BAP1 mutations are associated with a metastasizing phenotype and decreased survival rates. We identified the ubiquitin carboxyl hydrolase (UCH) domain as a major hotspot region for the pathogenic mutations with a high evolutionary action (EA) score. This also includes the mutations at conserved catalytic sites and the ones overlapping with the phosphorylation residues. Computational protein interaction studies revealed that distant BAP1-associated protein complexes (FOXK2, ASXL1, BARD1, BRCA1) could be directly impacted by this mutation paradigm. We also described the conformational transition related to BAP1-BRCA-BARD1 complex, which may pose critical implications for mutations, especially at the docking interfaces of these three proteins. The mutations affect - independent of being somatic or germline - the binding affinity of miRNAs embedded within the BAP1 locus, thereby altering the unique regulatory network. Apart from UM, BAP1 gene expression and survival associations were found to be predictive for the prognosis in several (n = 29) other cancer types. Herein, we suggest that although BAP1 is conceptually a driver gene in UM, it might contribute through its interaction partners and its regulatory miRNA network to various aspects of cancer. Taken together, these findings will pave the way to evaluate BAP1 in a variety of other human cancers with a shared mutational spectrum.

Chemotherapy-Induced Distal Enhancers Drive Transcriptional Programs to Maintain the Chemoresistant State in Ovarian Cancer.

Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small-molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers. SIGNIFICANCE: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.

Virulent duck enteritis virus infected DEF cells generate a unique pattern of viral microRNAs and a novel set of host microRNAs

BackgroundDuck enteritis virus (DEV) belongs to the family Herpesviridae and is an important epornitic agent that causes economic losses in the waterfowl industry. The Chinese virulent (CHv) and attenuate vaccines (VAC) are two different pathogenic DEV strains. MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene expression in viral infection. Nonetheless, there is little information on virulent duck enteritis virus (DEV)-encoded miRNAs.ResultsUsing high-throughput sequencing, we identified 39 mature viral miRNAs from CHv-infected duck embryo fibroblasts cells. Compared with the reported 33 VAC-encoded miRNAs, only 13 miRNA sequences and 22 “seed sequences” of miRNA were identical, and 8 novel viral miRNAs were detected and confirmed by stem-loop RT-qPCR in this study. Using RNAhybrid and PITA software, 38 CHv-encoded miRNAs were predicted to target 41 viral genes and formed a complex regulatory network. Dual luciferase reporter assay (DLRA) confirmed that viral dev-miR-D8-3p can directly target the 3’-UTR of CHv US1 gene (p < 0.05). Gene Ontology analysis on host target genes of viral miRNAs were mainly involved in biological regulation, cellular and metabolic processes. In addition, 598 novel duck-encoded miRNAs were detected in this study. Thirty-eight host miRNAs showed significant differential expression after CHv infection: 13 miRNAs were up-regulated, and 25 miRNAs were down-regulated, which may affect viral replication in the host cell.ConclusionsThese data suggested that CHv encoded a different set of microRNAs and formed a unique regulatory network compared with VAC. This is the first report of DEF miRNAs expression profile and an analysis of these miRNAs regulatory mechanisms during DEV infection. These data provide a basis for further exploring miRNA regulatory roles in the pathogenesis of DEV infection and contribute to the understanding of the CHv-host interaction at the miRNA level.

The downregulation of putative anticancer target BORIS/CTCFL in an addicted myeloid cancer cell line modulates the expression of multiple protein coding and ncRNA genes

The BORIS/CTCFL gene, is a testis-specific CTCF paralog frequently erroneously activated in cancer, although its exact role in cancer remains unclear. BORIS is both a transcription factor and an architectural chromatin protein. BORIS’ normal role is to establish a germline-like gene expression and remodel the epigenetic landscape in testis; it similarly remodels chromatin when activated in human cancer. Critically, at least one cancer cell line, K562, is dependent on BORIS for its self-renewal and survival.Here, we downregulate BORIS expression in the K562 cancer cell line to investigate downstream pathways regulated by BORIS. RNA-seq analyses of both mRNA and small ncRNAs, including miRNA and piRNA, in the knock-down cells revealed a set of differentially expressed genes and pathways, including both testis-specific and general proliferation factors, as well as proteins involved in transcription regulation and cell physiology. The differentially expressed genes included important transcriptional regulators such as SOX6 and LIN28A. Data indicate that both direct binding of BORIS to promoter regions and locus-control activity via long-distance chromatin domain regulation are involved. The sum of findings suggests that BORIS activation in leukemia does not just recapitulate the germline, but creates a unique regulatory network.

FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae.

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated.

PHA-4/FOXA-regulated microRNA feed forward loops during Caenorhabditis elegans dietary restriction.

Dietary restriction (DR) increases life span and delays the onset of age-related diseases across species. However, the molecular mechanisms have remained relatively unexplored in terms of gene regulation. InC. elegans, a popular model for aging studies, the FOXA transcription factor PHA-4 is a robust genetic regulator of DR, although little is known about how it regulates gene expression. We profiled the transcriptome and miRNAome of an eat-2 mutant, a genetic surrogate of DR, by Next Generation sequencing and find that most of the miRNAs are upregulated in the young-adult worms, none significantly downregulated. Interestingly, PHA-4 can potentially regulate the expression of most of these miRNA genes. Remarkably, many of the PHA-4-regulated genes that are induced during DR are also targets of the PHA-4-upregulated miRNAs, forming a large feed-forward gene regulatory network. The genes targeted by the feed-forward loops (FFLs) are enriched for functions related to ubiquitin-mediated decay, lysosomal autophagy, cellular signalling, protein folding etc., processes that play critical roles in DR and longevity. Together our data provides a framework for understanding the complex and unique regulatory network employed during DR, suggesting that PHA-4 employs such FFLs to fine-tune gene expression and instil robustness in the system during energy crisis.

Tissue specificity and differential expression of transcription factors in tomato provide hints of unique regulatory networks during fruit ripening

Tissue specificity or dramatically different expression levels of transcription factors in different tissue types allows differential regulation of tissue development as well as alternate modes of metabolic regulation. Recently we reported (Rohrmann et al., 2011) the development of a quantitative real-time PCR platform (qRT-PCR) that allows accurate quantification of the expression level of approximately 1000 tomato transcription factors. Application of this platform to samples collected during a ripening time course of wild type tomato and the high pigment mutant hp1 allowed us to identify transcription factors of importance both to ripening per se and to the metabolic shifts that occur during this critical biological process. Here we extend the quantitative real-time PCR analyses to include samples from flower, leaf, stem and root of wild type tomato. Co-expression network analysis to identify both conserved and unique regulatory networks both between individual tissues of tomato and also in cross-species comparisons of specific tissues, suggested some key TF genes which are involved in photosynthesis and fruit development.