Publications by authors named "Georgi K Marinov"

57 Publications

Transcriptional and chromatin-based partitioning mechanisms uncouple protein scaling from cell size.

Mol Cell 2021 Oct 26. Epub 2021 Oct 26.

Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

Biosynthesis scales with cell size such that protein concentrations generally remain constant as cells grow. As an exception, synthesis of the cell-cycle inhibitor Whi5 "sub-scales" with cell size so that its concentration is lower in larger cells to promote cell-cycle entry. Here, we find that transcriptional control uncouples Whi5 synthesis from cell size, and we identify histones as the major class of sub-scaling transcripts besides WHI5 by screening for similar genes. Histone synthesis is thereby matched to genome content rather than cell size. Such sub-scaling proteins are challenged by asymmetric cell division because proteins are typically partitioned in proportion to newborn cell volume. To avoid this fate, Whi5 uses chromatin-binding to partition similar protein amounts to each newborn cell regardless of cell size. Disrupting both Whi5 synthesis and chromatin-based partitioning weakens G1 size control. Thus, specific transcriptional and partitioning mechanisms determine protein sub-scaling to control cell size.
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http://dx.doi.org/10.1016/j.molcel.2021.10.007DOI Listing
October 2021

An optimized ATAC-seq protocol for genome-wide mapping of active regulatory elements in primary mouse cortical neurons.

STAR Protoc 2021 Dec 30;2(4):100854. Epub 2021 Sep 30.

Department of Genetics, Stanford University, Stanford, CA 94305, USA.

ATAC-seq is a versatile, adaptable, and widely adopted technique for mapping open chromatin regions. However, some biological systems, such as primary neurons, present unique challenges to its application. Conventional ATAC-seq would require the dissociation of the primary neurons after plating but dissociating them leads to rapid cell death and major changes in cell state, affecting ATAC-seq results. We have developed this modified ATAC-seq protocol to address this challenge for primary neurons, providing a high-quality and high-resolution accessible chromatin profile. For complete details on the use and execution of this protocol, please refer to Maor-Nof et al. (2021).
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http://dx.doi.org/10.1016/j.xpro.2021.100854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8496302PMC
December 2021

Identification and characterization of a novel Epstein-Barr Virus-encoded circular RNA from LMP-2 Gene.

Sci Rep 2021 07 13;11(1):14392. Epub 2021 Jul 13.

Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603, Kuala Lumpur, Malaysia.

Epstein-Barr virus (EBV) has been recently found to generate novel circular RNAs (circRNAs) through backsplicing. However, comprehensive catalogs of EBV circRNAs in other cell lines and their functional characterization are still lacking. In this study, we have identified a list of putative EBV circRNAs in GM12878, an EBV-transformed lymphoblastoid cell line, with a significant majority encoded from the EBV latent genes. A novel EBV circRNA derived from the exon 5 of LMP-2 gene which exhibited highest prevalence, was further validated using RNase R assay and Sanger sequencing. This circRNA, which we term circLMP-2_e5, can be universally detected in a panel of EBV-positive cell lines modelling different latency programs. It ranges from lower expression in nasopharyngeal carcinoma (NPC) cells to higher expression in B cells, and is localized to both the cytoplasm and the nucleus. We provide evidence that circLMP-2_e5 is expressed concomitantly with its cognate linear LMP-2 RNA upon EBV lytic reactivation, and may be produced as a result of exon skipping, with its circularization possibly occurring without the involvement of cis elements in the short flanking introns. Furthermore, we show that circLMP-2_e5 is not involved in regulating cell proliferation, host innate immune response, its linear parental transcripts, or EBV lytic reactivation. Taken together, our study expands the current repertoire of putative EBV circRNAs, broadens our understanding of the biology of EBV circRNAs, and lays the foundation for further investigation of their function in the EBV life cycle and disease development.
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http://dx.doi.org/10.1038/s41598-021-93781-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277822PMC
July 2021

Transcription-dependent domain-scale three-dimensional genome organization in the dinoflagellate Breviolum minutum.

Nat Genet 2021 05 29;53(5):613-617. Epub 2021 Apr 29.

Department of Genetics, Stanford University, Stanford, CA, USA.

Dinoflagellate chromosomes represent a unique evolutionary experiment, as they exist in a permanently condensed, liquid crystalline state; are not packaged by histones; and contain genes organized into tandem gene arrays, with minimal transcriptional regulation. We analyze the three-dimensional genome of Breviolum minutum, and find large topological domains (dinoflagellate topologically associating domains, which we term 'dinoTADs') without chromatin loops, which are demarcated by convergent gene array boundaries. Transcriptional inhibition disrupts dinoTADs, implicating transcription-induced supercoiling as the primary topological force in dinoflagellates.
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http://dx.doi.org/10.1038/s41588-021-00848-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110477PMC
May 2021

Interrogating the Accessible Chromatin Landscape of Eukaryote Genomes Using ATAC-seq.

Methods Mol Biol 2021 ;2243:183-226

Department of Genetics, Stanford University, Stanford, CA, USA.

The ATAC-seq assay has emerged as the most useful, versatile, and widely adaptable method for profiling accessible chromatin regions and tracking the activity of cis-regulatory elements (cREs) in eukaryotes. Thanks to its great utility, it is now being applied to map active chromatin in the context of a very wide diversity of biological systems and questions. In the course of these studies, considerable experience working with ATAC-seq data has accumulated and a standard set of computational tasks that need to be carried for most ATAC-seq analyses has emerged. Here, we review and provide examples of common such analytical procedures (including data processing, quality control, peak calling, identifying differentially accessible open chromatin regions, and variable transcription factor (TF) motif accessibility) and discuss recommended optimal practices.
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http://dx.doi.org/10.1007/978-1-0716-1103-6_10DOI Listing
April 2021

High-Throughput Discovery and Characterization of Human Transcriptional Effectors.

Cell 2020 12 15;183(7):2020-2035.e16. Epub 2020 Dec 15.

Department of Bioengineering, Stanford University, Stanford, CA 94305, USA. Electronic address:

Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.
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http://dx.doi.org/10.1016/j.cell.2020.11.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178797PMC
December 2020

The changing mouse embryo transcriptome at whole tissue and single-cell resolution.

Nature 2020 07 29;583(7818):760-767. Epub 2020 Jul 29.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

During mammalian embryogenesis, differential gene expression gradually builds the identity and complexity of each tissue and organ system. Here we systematically quantified mouse polyA-RNA from day 10.5 of embryonic development to birth, sampling 17 tissues and organs. The resulting developmental transcriptome is globally structured by dynamic cytodifferentiation, body-axis and cell-proliferation gene sets that were further characterized by the transcription factor motif codes of their promoters. We decomposed the tissue-level transcriptome using single-cell RNA-seq (sequencing of RNA reverse transcribed into cDNA) and found that neurogenesis and haematopoiesis dominate at both the gene and cellular levels, jointly accounting for one-third of differential gene expression and more than 40% of identified cell types. By integrating promoter sequence motifs with companion ENCODE epigenomic profiles, we identified a prominent promoter de-repression mechanism in neuronal expression clusters that was attributable to known and novel repressors. Focusing on the developing limb, single-cell RNA data identified 25 candidate cell types that included progenitor and differentiating states with computationally inferred lineage relationships. We extracted cell-type transcription factor networks and complementary sets of candidate enhancer elements by using single-cell RNA-seq to decompose integrative cis-element (IDEAS) models that were derived from whole-tissue epigenome chromatin data. These ENCODE reference data, computed network components and IDEAS chromatin segmentations are companion resources to the matching epigenomic developmental matrix, and are available for researchers to further mine and integrate.
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http://dx.doi.org/10.1038/s41586-020-2536-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410830PMC
July 2020

Reboot the economy for resilience.

Authors:
Georgi K Marinov

Nature 2020 05;581(7808):262

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http://dx.doi.org/10.1038/d41586-020-01501-7DOI Listing
May 2020

Long-range single-molecule mapping of chromatin accessibility in eukaryotes.

Nat Methods 2020 03 10;17(3):319-327. Epub 2020 Feb 10.

Department of Genetics, Stanford University, Stanford, CA, USA.

Mapping open chromatin regions has emerged as a widely used tool for identifying active regulatory elements in eukaryotes. However, existing approaches, limited by reliance on DNA fragmentation and short-read sequencing, cannot provide information about large-scale chromatin states or reveal coordination between the states of distal regulatory elements. We have developed a method for profiling the accessibility of individual chromatin fibers, a single-molecule long-read accessible chromatin mapping sequencing assay (SMAC-seq), enabling the simultaneous, high-resolution, single-molecule assessment of chromatin states at multikilobase length scales. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases with low sequence specificity, in this case EcoGII, an N-methyladenosine (mA) methyltransferase, and the ability of nanopore sequencing to directly read DNA modifications. We demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule nucleosome and transcription factor protection footprints, and quantify the correlation between chromatin states of distal genomic elements.
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http://dx.doi.org/10.1038/s41592-019-0730-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968351PMC
March 2020

Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements.

Nat Commun 2019 09 6;10(1):4063. Epub 2019 Sep 6.

Department of Genetics, Stanford University, Stanford, CA, 94305, USA.

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.
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http://dx.doi.org/10.1038/s41467-019-11955-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731277PMC
September 2019

Deciphering regulatory DNA sequences and noncoding genetic variants using neural network models of massively parallel reporter assays.

PLoS One 2019 17;14(6):e0218073. Epub 2019 Jun 17.

Department of Genetics, Stanford University, Stanford, CA, United States of America.

The relationship between noncoding DNA sequence and gene expression is not well-understood. Massively parallel reporter assays (MPRAs), which quantify the regulatory activity of large libraries of DNA sequences in parallel, are a powerful approach to characterize this relationship. We present MPRA-DragoNN, a convolutional neural network (CNN)-based framework to predict and interpret the regulatory activity of DNA sequences as measured by MPRAs. While our method is generally applicable to a variety of MPRA designs, here we trained our model on the Sharpr-MPRA dataset that measures the activity of ∼500,000 constructs tiling 15,720 regulatory regions in human K562 and HepG2 cell lines. MPRA-DragoNN predictions were moderately correlated (Spearman ρ = 0.28) with measured activity and were within range of replicate concordance of the assay. State-of-the-art model interpretation methods revealed high-resolution predictive regulatory sequence features that overlapped transcription factor (TF) binding motifs. We used the model to investigate the cell type and chromatin state preferences of predictive TF motifs. We explored the ability of our model to predict the allelic effects of regulatory variants in an independent MPRA experiment and fine map putative functional SNPs in loci associated with lipid traits. Our results suggest that interpretable deep learning models trained on MPRA data have the potential to reveal meaningful patterns in regulatory DNA sequences and prioritize regulatory genetic variants, especially as larger, higher-quality datasets are produced.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6576758PMC
February 2020

Population Genetics of Paramecium Mitochondrial Genomes: Recombination, Mutation Spectrum, and Efficacy of Selection.

Genome Biol Evol 2019 05;11(5):1398-1416

Department of Biology, Indiana University, Bloomington.

The evolution of mitochondrial genomes and their population-genetic environment among unicellular eukaryotes are understudied. Ciliate mitochondrial genomes exhibit a unique combination of characteristics, including a linear organization and the presence of multiple genes with no known function or detectable homologs in other eukaryotes. Here we study the variation of ciliate mitochondrial genomes both within and across 13 highly diverged Paramecium species, including multiple species from the P. aurelia species complex, with four outgroup species: P. caudatum, P. multimicronucleatum, and two strains that may represent novel related species. We observe extraordinary conservation of gene order and protein-coding content in Paramecium mitochondria across species. In contrast, significant differences are observed in tRNA content and copy number, which is highly conserved in species belonging to the P. aurelia complex but variable among and even within the other Paramecium species. There is an increase in GC content from ∼20% to ∼40% on the branch leading to the P. aurelia complex. Patterns of polymorphism in population-genomic data and mutation-accumulation experiments suggest that the increase in GC content is primarily due to changes in the mutation spectra in the P. aurelia species. Finally, we find no evidence of recombination in Paramecium mitochondria and find that the mitochondrial genome appears to experience either similar or stronger efficacy of purifying selection than the nucleus.
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http://dx.doi.org/10.1093/gbe/evz081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505448PMC
May 2019

Response to Martin and colleagues: mitochondria do not boost the bioenergetic capacity of eukaryotic cells.

Biol Direct 2018 11 29;13(1):26. Epub 2018 Nov 29.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA.

A recent paper by (Gerlitz et al., Biol Direct 13:21, 2018) questions the validity of the data underlying prior analyses on the bioenergetics capacities of cells, and continues to promote the idea that the mitochondrion endowed eukaryotic cells with energetic superiority over prokaryotes. The former point has been addressed previously, with no resultant changes in the conclusions, and the latter point remains inconsistent with multiple lines of empirical data.
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http://dx.doi.org/10.1186/s13062-018-0228-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822690PMC
November 2018

Author Correction: An NF-κB-microRNA regulatory network tunes macrophage inflammatory responses.

Nat Commun 2018 08 16;9(1):3338. Epub 2018 Aug 16.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA.

Li-Fan Lu and Alexander Y. Rudensky, who supplied miR-146a floxed mice used in this study, were inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article. The generation of the floxed mice has been described in detail by Cho and Lee et al..
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http://dx.doi.org/10.1038/s41467-018-05720-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095848PMC
August 2018

A decade of ChIP-seq.

Authors:
Georgi K Marinov

Brief Funct Genomics 2018 03;17(2):77-79

Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5101.

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http://dx.doi.org/10.1093/bfgp/ely012DOI Listing
March 2018

ChIP-ping the branches of the tree: functional genomics and the evolution of eukaryotic gene regulation.

Brief Funct Genomics 2018 03;17(2):116-137

Advances in the methods for detecting protein-DNA interactions have played a key role in determining the directions of research into the mechanisms of transcriptional regulation. The most recent major technological transformation happened a decade ago, with the move from using tiling arrays [chromatin immunoprecipitation (ChIP)-on-Chip] to high-throughput sequencing (ChIP-seq) as a readout for ChIP assays. In addition to the numerous other ways in which it is superior to arrays, by eliminating the need to design and manufacture them, sequencing also opened the door to carrying out comparative analyses of genome-wide transcription factor occupancy across species and studying chromatin biology in previously less accessible model and nonmodel organisms, thus allowing us to understand the evolution and diversity of regulatory mechanisms in unprecedented detail. Here, we review the biological insights obtained from such studies in recent years and discuss anticipated future developments in the field.
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http://dx.doi.org/10.1093/bfgp/ely004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889016PMC
March 2018

An NF-κB-microRNA regulatory network tunes macrophage inflammatory responses.

Nat Commun 2017 10 11;8(1):851. Epub 2017 Oct 11.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA.

The innate inflammatory response must be tightly regulated to ensure effective immune protection. NF-κB is a key mediator of the inflammatory response, and its dysregulation has been associated with immune-related malignancies. Here, we describe a miRNA-based regulatory network that enables precise NF-κB activity in mouse macrophages. Elevated miR-155 expression potentiates NF-κB activity in miR-146a-deficient mice, leading to both an overactive acute inflammatory response and chronic inflammation. Enforced miR-155 expression overrides miR-146a-mediated repression of NF-κB activation, thus emphasizing the dominant function of miR-155 in promoting inflammation. Moreover, miR-155-deficient macrophages exhibit a suboptimal inflammatory response when exposed to low levels of inflammatory stimuli. Importantly, we demonstrate a temporal asymmetry between miR-155 and miR-146a expression during macrophage activation, which creates a combined positive and negative feedback network controlling NF-κB activity. This miRNA-based regulatory network enables a robust yet time-limited inflammatory response essential for functional immunity.MicroRNAs (miR) are important regulators of gene transcription, with miR-155 and miR-146a both implicated in macrophage activation. Here the authors show that NF-κB signalling, miR-155 and miR-146a form a complex network of cross-regulations to control gene transcription in macrophages for modulating inflammatory responses.
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http://dx.doi.org/10.1038/s41467-017-00972-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636846PMC
October 2017

Transcriptomic analysis of the role of RasGEF1B circular RNA in the TLR4/LPS pathway.

Sci Rep 2017 09 25;7(1):12227. Epub 2017 Sep 25.

Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, 50603, Malaysia.

Circular RNAs (circRNAs) have recently emerged as a large class of novel non-coding RNA species. However, the detailed functional significance of the vast majority of them remains to be elucidated. Most functional characterization studies targeting circRNAs have been limited to resting cells, leaving their role in dynamic cellular responses to stimuli largely unexplored. In this study, we focus on the LPS-induced cytoplasmic circRNA, mcircRasGEF1B, and combine targeted mcircRasGEF1B depletion with high-throughput transcriptomic analysis to gain insight into its function during the cellular response to LPS stimulation. We show that knockdown of mcircRasGEF1B results in altered expression of a wide array of genes. Pathway analysis revealed an overall enrichment of genes involved in cell cycle progression, mitotic division, active metabolism, and of particular interest, NF-κB, LPS signaling pathways, and macrophage activation. These findings expand the set of functionally characterized circRNAs and support the regulatory role of mcircRasGEF1B in immune response during macrophage activation and protection against microbial infections.
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http://dx.doi.org/10.1038/s41598-017-12550-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612941PMC
September 2017

SLC7A11 Overexpression in Glioblastoma Is Associated with Increased Cancer Stem Cell-Like Properties.

Stem Cells Dev 2017 09 27;26(17):1236-1246. Epub 2017 Jul 27.

1 Department of Developmental and Stem Cell Biology, City of Hope National Medical Center and Beckman Research Institute , Duarte, California.

System x is a sodium-independent electroneutral transporter, comprising a catalytic subunit xCT (SLC7A11), which is involved in importing cystine. Certain cancers such as gliomas upregulate the expression of system x, which confers a survival advantage against the detrimental effects of reactive oxygen species (ROS) by increasing generation of the antioxidant glutathione. However, ROS have also been shown to function as targeted, intracellular second messengers in an array of physiological processes such as proliferation. Several studies have implicated ROS in important cancer features such as migration, invasion, and contribution to a cancer stem cell (CSC)-like phenotype. The role of system x in regulating these ROS-sensitive processes in glioblastoma multiforme (GBM), the most aggressive malignant primary brain tumor in adults, remains unknown. Stable SLC7A11 knockdown and overexpressing U251 glioma cells were generated and characterized to understand the role of redox and system x in glioma progression. SLC7A11 knockdown resulted in higher endogenous ROS levels and enhanced invasive properties. On the contrary, overexpression of SLC7A11 resulted in decreased endogenous ROS levels as well as decreased migration and invasion. However, SLC7A11-overexpressing cells displayed actin cytoskeleton changes reminiscent of epithelial-like cells and exhibited an increased CSC-like phenotype. The enhanced CSC-like phenotype may contribute to increased chemoresistance and suggests that overexpression of SLC7A11 in the context of GBM may contribute to tumor progression. These findings have important implications for cancer management where targeting system x in combination with other chemotherapeutics can reduce cancer resistance and recurrence and improve GBM patient survival.
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http://dx.doi.org/10.1089/scd.2017.0123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576215PMC
September 2017

Identification of Candidate Functional Elements in the Genome from ChIP-seq Data.

Authors:
Georgi K Marinov

Methods Mol Biol 2017 ;1543:19-43

Department of Biology, Indiana University, Bloomington, IN, 47405, USA.

ChIP-seq datasets provide a wealth of information for the identification of candidate regulatory elements in the genome. For this potential to be fully realized, methods for evaluating data quality and for distinguishing reproducible signal from technical and biological noise are necessary. Here, the computational methods for addressing these challenges developed by the ENCODE Consortium are described and the key considerations for analyzing and interpreting ChIP-seq data are discussed.
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http://dx.doi.org/10.1007/978-1-4939-6716-2_2DOI Listing
February 2018

ChIP-seq for the Identification of Functional Elements in the Human Genome.

Authors:
Georgi K Marinov

Methods Mol Biol 2017 ;1543:3-18

Department of Biology, Indiana University, Bloomington, IN, 47405, USA.

Functional elements in the genome express their function through physical association with particular proteins: transcription factors, components of the transcription machinery, specific histone modifications, and others. The genome-wide characterization of the protein-DNA interaction landscape of these proteins is thus a key approach toward the identification of candidate genomic regulatory regions. ChIP-seq (Chromatin Immunoprecipitation coupled with high-throughput sequencing) has emerged as the primary experimental methods for carrying out this task. Here, the ChIP-seq protocol is described together with some of the most important considerations for applying it in practice.
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http://dx.doi.org/10.1007/978-1-4939-6716-2_1DOI Listing
February 2018

On the design and prospects of direct RNA sequencing.

Authors:
Georgi K Marinov

Brief Funct Genomics 2017 Nov;16(6):326-335

Throughout the past nearly a decade, the application of high-throughput sequencing to RNA molecules in the form of RNA sequencing (RNA-seq) and its many variations has revolutionized transcriptomic studies by enabling researchers to take a simultaneously deep and truly global look into the transcriptome. However, there is still considerable scope for improvement on RNA-seq data in its current form, primarily because of the short-read nature of the dominant sequencing technologies, which prevents the completely reliable reconstruction and quantification of full-length transcripts, and the sequencing library building protocols used, which introduce various distortions in the final data sets. The ideal approach toward resolving these remaining issues would involve the direct amplification-free sequencing of full-length RNA molecules. This has recently become practical with the advent of nanopore sequencing, which raises the possibility of yet another revolution in transcriptomics. I discuss the design considerations to be taken into account, the technical challenges that need to be addressed and the biological questions these advances can be expected to resolve.
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http://dx.doi.org/10.1093/bfgp/elw043DOI Listing
November 2017

Membranes, energetics, and evolution across the prokaryote-eukaryote divide.

Elife 2017 03 16;6. Epub 2017 Mar 16.

Department of Biology, Indiana University, Bloomington, United States.

The evolution of the eukaryotic cell marked a profound moment in Earth's history, with most of the visible biota coming to rely on intracellular membrane-bound organelles. It has been suggested that this evolutionary transition was critically dependent on the movement of ATP synthesis from the cell surface to mitochondrial membranes and the resultant boost to the energetic capacity of eukaryotic cells. However, contrary to this hypothesis, numerous lines of evidence suggest that eukaryotes are no more bioenergetically efficient than prokaryotes. Thus, although the origin of the mitochondrion was a key event in evolutionary history, there is no reason to think membrane bioenergetics played a direct, causal role in the transition from prokaryotes to eukaryotes and the subsequent explosive diversification of cellular and organismal complexity.
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http://dx.doi.org/10.7554/eLife.20437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354521PMC
March 2017

Population Genomics of Paramecium Species.

Mol Biol Evol 2017 05;34(5):1194-1216

Department of Biology, Indiana University, Bloomington, IN.

Population-genomic analyses are essential to understanding factors shaping genomic variation and lineage-specific sequence constraints. The dearth of such analyses for unicellular eukaryotes prompted us to assess genomic variation in Paramecium, one of the most well-studied ciliate genera. The Paramecium aurelia complex consists of ∼15 morphologically indistinguishable species that diverged subsequent to two rounds of whole-genome duplications (WGDs, as long as 320 MYA) and possess extremely streamlined genomes. We examine patterns of both nuclear and mitochondrial polymorphism, by sequencing whole genomes of 10-13 worldwide isolates of each of three species belonging to the P. aurelia complex: P. tetraurelia, P. biaurelia, P. sexaurelia, as well as two outgroup species that do not share the WGDs: P. caudatum and P. multimicronucleatum. An apparent absence of global geographic population structure suggests continuous or recent dispersal of Paramecium over long distances. Intergenic regions are highly constrained relative to coding sequences, especially in P. caudatum and P. multimicronucleatum that have shorter intergenic distances. Sequence diversity and divergence are reduced up to ∼100-150 bp both upstream and downstream of genes, suggesting strong constraints imposed by the presence of densely packed regulatory modules. In addition, comparison of sequence variation at non-synonymous and synonymous sites suggests similar recent selective pressures on paralogs within and orthologs across the deeply diverging species. This study presents the first genome-wide population-genomic analysis in ciliates and provides a valuable resource for future studies in evolutionary and functional genetics in Paramecium.
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http://dx.doi.org/10.1093/molbev/msx074DOI Listing
May 2017

Increased Expression of System xc- in Glioblastoma Confers an Altered Metabolic State and Temozolomide Resistance.

Mol Cancer Res 2016 12 22;14(12):1229-1242. Epub 2016 Sep 22.

Department of Neurosciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California.

Glioblastoma multiforme is the most aggressive malignant primary brain tumor in adults. Several studies have shown that glioma cells upregulate the expression of xCT (SLC7A11), the catalytic subunit of system x, a transporter involved in cystine import, that modulates glutathione production and glioma growth. However, the role of system x in regulating the sensitivity of glioma cells to chemotherapy is currently debated. Inhibiting system x with sulfasalazine decreased glioma growth and survival via redox modulation, and use of the chemotherapeutic agent temozolomide together with sulfasalazine had a synergistic effect on cell killing. To better understand the functional consequences of system x in glioma, stable SLC7A11-knockdown and -overexpressing U251 glioma cells were generated. Modulation of SLC7A11 did not alter cellar proliferation but overexpression did increase anchorage-independent cell growth. Knockdown of SLC7A11 increased basal reactive oxygen species (ROS) and decreased glutathione generation resulting in increased cell death under oxidative and genotoxic stress. Overexpression of SLC7A11 resulted in increased resistance to oxidative stress and decreased chemosensitivity to temozolomide. In addition, SLC7A11 overexpression was associated with altered cellular metabolism including increased mitochondrial biogenesis, oxidative phosphorylation, and ATP generation. These results suggest that expression of SLC7A11 in the context of glioma contributes to tumorigenesis, tumor progression, and resistance to standard chemotherapy.

Implications: SLC7A11, in addition to redox modulation, appears to be associated with increased cellular metabolism and is a mediator of temozolomide resistance in human glioma, thus making system x a potential therapeutic target in glioblastoma multiforme. Mol Cancer Res; 14(12); 1229-42. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237285PMC
December 2016

Inducible RasGEF1B circular RNA is a positive regulator of ICAM-1 in the TLR4/LPS pathway.

RNA Biol 2016 09 30;13(9):861-71. Epub 2016 Jun 30.

a Institute of Biological Sciences, Faculty of Science, University of Malaya , Kuala Lumpur , Malaysia.

Circular RNAs (circRNAs) constitute a large class of RNA species formed by the back-splicing of co-linear exons, often within protein-coding transcripts. Despite much progress in the field, it remains elusive whether the majority of circRNAs are merely aberrant splicing by-products with unknown functions, or their production is spatially and temporally regulated to carry out specific biological functions. To date, the majority of circRNAs have been cataloged in resting cells. Here, we identify an LPS-inducible circRNA: mcircRasGEF1B, which is predominantly localized in cytoplasm, shows cell-type specific expression, and has a human homolog with similar properties, hcircRasGEF1B. We show that knockdown of the expression of mcircRasGEF1B reduces LPS-induced ICAM-1 expression. Additionally, we demonstrate that mcircRasGEF1B regulates the stability of mature ICAM-1 mRNAs. These findings expand the inventory of functionally characterized circRNAs with a novel RNA species that may play a critical role in fine-tuning immune responses and protecting cells against microbial infection.
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http://dx.doi.org/10.1080/15476286.2016.1207036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014010PMC
September 2016

Conservation and divergence of the histone code in nucleomorphs.

Biol Direct 2016 Apr 5;11(1):18. Epub 2016 Apr 5.

Department of Biology, Indiana University, Bloomington, 47405, IN, United States.

Background: Nucleomorphs, the remnant nuclei of photosynthetic algae that have become endosymbionts to other eukaryotes, represent a unique example of convergent reductive genome evolution in eukaryotes, having evolved independently on two separate occasions in chlorarachniophytes and cryptophytes. The nucleomorphs of the two groups have evolved in a remarkably convergent manner, with numerous very similar features. Chief among them is the extreme reduction and compaction of nucleomorph genomes, with very small chromosomes and extremely short or even completely absent intergenic spaces. These characteristics pose a number of intriguing questions regarding the mechanisms of transcription and gene regulation in such a crowded genomic context, in particular in terms of the functioning of the histone code, which is common to almost all eukaryotes and plays a central role in chromatin biology.

Results: This study examines the sequences of nucleomorph histone proteins in order to address these issues. Remarkably, all classical transcription- and repression-related components of the histone code seem to be missing from chlorarachniophyte nucleomorphs. Cryptophyte nucleomorph histones are generally more similar to the conventional eukaryotic state; however, they also display significant deviations from the typical histone code. Based on the analysis of specific components of the code, we discuss the state of chromatin and the transcriptional machinery in these nuclei.

Conclusions: The results presented here shed new light on the mechanisms of nucleomorph transcription and gene regulation and provide a foundation for future studies of nucleomorph chromatin and transcriptional biology.
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http://dx.doi.org/10.1186/s13062-016-0119-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822330PMC
April 2016

Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila.

Genes Dev 2016 Apr;30(7):840-55

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA;

The conserved THO/TREX (transcription/export) complex is critical for pre-mRNA processing and mRNA nuclear export. In metazoa, TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent fashion; however, how TREX functions is poorly understood. Here we show that Thoc5 and other TREX components are essential for the biogenesis of piRNA, a distinct class of small noncoding RNAs that control expression of transposable elements (TEs) in the Drosophila germline. Mutations in TREX lead to defects in piRNA biogenesis, resulting in derepression of multiple TE families, gametogenesis defects, and sterility. TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and colocalize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depend on Cutoff, a protein associated with chromatin of piRNA clusters. Finally, we show that TREX is required for accumulation of nascent piRNA precursors. Our study reveals a novel splicing-independent mechanism for TREX loading on nascent RNA and its importance in piRNA biogenesis.
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http://dx.doi.org/10.1101/gad.276030.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826399PMC
April 2016

Draft Whole-Genome Sequence of Haemophilus ducreyi Strain AUSPNG1, Isolated from a Cutaneous Ulcer of a Child from Papua New Guinea.

Genome Announc 2016 Feb 4;4(1). Epub 2016 Feb 4.

Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA Center for Immunobiology, Indiana University School of Medicine, Indianapolis, Indiana, USA

Haemophilus ducreyi has recently emerged as a leading cause of cutaneous ulcers in the yaws-endemic areas of Papua New Guinea and other South Pacific islands. Here, we report the draft genome sequence of the H. ducreyi strain AUSPNG1, isolated from a cutaneous ulcer of a child from Papua New Guinea.
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http://dx.doi.org/10.1128/genomeA.01661-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742684PMC
February 2016

Reply to Lane and Martin: Mitochondria do not boost the bioenergetic capacity of eukaryotic cells.

Proc Natl Acad Sci U S A 2016 Feb 25;113(6):E667-8. Epub 2016 Jan 25.

Department of Biology, Indiana University, Bloomington, IN 47401.

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http://dx.doi.org/10.1073/pnas.1523394113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760791PMC
February 2016
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