Core Cellular Processes Research Articles

Methods for Visualization of RNA and Cytoskeletal Elements in the Early Zebrafish Embryo.

Zebrafish embryos, with their large size (>0.5mm) and accessibility, are valuable tools for investigating core cellular processes. Many of those processes, such as cell division, asymmetric inheritance of cellular components, and structural dynamics involved in cell motility and morphology rely on cytoskeletal rearrangements and associated macromolecules. In addition to the protein-rich cytoskeleton, the early embryo is packed with maternally deposited RNA, which serves essential roles in establishing cell polarity, cell fate, and cell organization. Here, we present methods for visualizing endogenous RNA along with cytoskeletal structures, including microtubules and filamentous actin (F-actin) in the context of an intact vertebrate embryo. Each of the four protocols described herein (embryo fixation, RNA probe design/synthesis, double fluorescent in situ hybridization with tubulin immunofluorescence, and fluorescent in situ hybridization with phalloidin labeling of F-actin) are intended for optimal preservation and visualization of both the cytoskeleton and RNAs of interest. These methods can also be modified and applied to a broad range of other uses.

A Rab escort protein regulates the MAPK pathway that controls filamentous growth in yeast

MAPK pathways regulate different responses yet can share common components. Although core regulators of MAPK pathways are well known, new pathway regulators continue to be identified. Overexpression screens can uncover new roles for genes in biological processes and are well suited to identify essential genes that cannot be evaluated by gene deletion analysis. In this study, a genome-wide screen was performed to identify genes that, when overexpressed, induce a reporter (FUS1-HIS3) that responds to ERK-type pathways (Mating and filamentous growth or fMAPK) but not p38-type pathways (HOG) in yeast. Approximately 4500 plasmids overexpressing individual yeast genes were introduced into strains containing the reporter by high-throughput transformation. Candidate genes were identified by measuring growth as a readout of reporter activity. Fourteen genes were identified and validated by re-testing: two were metabolic controls (HIS3, ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR pathway that plays a role in nutrient signaling. MRS6 overexpression stimulated invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the p38/HOG MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 by two-hybrid analysis. Based on these results, Mrs6 may selectively propagate an ERK-dependent signal. Identifying new regulators of MAPK pathways may provide new insights into signal integration among core cellular processes and the execution of pathway-specific responses.

Sequence Features of Mitochondrial Transporter Protein Families.

Mitochondrial carriers facilitate the transfer of small molecules across the inner mitochondrial membrane (IMM) to support mitochondrial function and core cellular processes. In addition to the classical SLC25 (solute carrier family 25) mitochondrial carriers, the past decade has led to the discovery of additional protein families with numerous members that exhibit IMM localization and transporter-like properties. These include mitochondrial pyruvate carriers, sideroflexins, and mitochondrial cation/H+ exchangers. These transport proteins were linked to vital physiological functions and disease. Their structures and transport mechanisms are, however, still largely unknown and understudied. Protein sequence analysis per se can often pinpoint hotspots that are of functional or structural importance. In this review, we summarize current knowledge about the sequence features of mitochondrial transporters with a special focus on the newly included SLC54, SLC55 and SLC56 families of the SLC solute carrier superfamily. Taking a step further, we combine sequence conservation analysis with transmembrane segment and secondary structure prediction methods to extract residue positions and sequence motifs that likely play a role in substrate binding, binding site gating or structural stability. We hope that our review will help guide future experimental efforts by the scientific community to unravel the transport mechanisms and structures of these novel mitochondrial carriers.

Asymmetric Transcription Factor Partitioning During Yeast Cell Division Requires the FACT Chromatin Remodeler and Cell Cycle Progression.

The polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. The budding yeast Saccharomyces cerevisiae divides asymmetrically, like many other cells, to generate two distinct progeny cells. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We screened a collection of essential genes to analyze the effects of core cellular processes in asymmetric cell division based on Ace2 localization. This screen identified mutations that affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model, we blocked cells from progressing through mitosis and found that prolonged metaphase delay is sufficient to disrupt Ace2 asymmetry after release, and that Ace2 asymmetry is restored after cytokinesis. We also demonstrate that members of the evolutionarily conserved facilitates chromatin transcription (FACT) chromatin-reorganizing complex are required for both asymmetric and cell cycle-regulated localization of Ace2, and for localization of the RAM network components.

Evolutionary Genomics of Niche-Specific Adaptation to the Cystic Fibrosis Lung in Pseudomonas aeruginosa.

The comparative genomics of the transition of the opportunistic pathogen Pseudomonas aeruginosa from a free-living environmental strain to one that causes chronic infection in the airways of cystic fibrosis (CF) patients remain poorly studied. Chronic infections are thought to originate from colonization by a single strain sampled from a diverse, globally distributed population, followed by adaptive evolution to the novel, stressful conditions of the CF lung. However, we do not know whether certain clades are more likely to form chronic infections than others and we lack a comprehensive view of the suite of genes under positive selection in the CF lung. We analyzed whole-genome sequence data from 1,000 P. aeruginosa strains with diverse ecological provenances including the CF lung. CF isolates were distributed across the phylogeny, indicating little genetic predisposition for any one clade to cause chronic infection. Isolates from the CF niche experienced stronger positive selection on core genes than those derived from environmental or acute infection sources, consistent with recent adaptation to the lung environment. Genes with the greatest differential positive selection in the CF niche include those involved in core cellular processes such as metabolism, energy production, and stress response as well as those linked to patho-adaptive processes such as antibiotic resistance, cell wall and membrane modification, quorum sensing, biofilms, mucoidy, motility, and iron homeostasis. Many genes under CF-specific differential positive selection had regulatory functions, consistent with the idea that regulatory mutations play an important role in rapid adaptation to novel environments.

Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study

Thiosemicarbazones (TSC) and their metal complexes display diverse biological activities and are active against multiple pathological conditions ranging from microbial infections to abnormal cell proliferation. Ribonucleotide reductase (RNR) is considered one of the main targets of TSCs, yet, the existence of additional targets, differently responsible for the multifaceted activities of TSCs and their metal complexes has been proposed. To set the basis for a more comprehensive delineation of their mode of action, we chemogenomically profiled the cellular effects of bis(citronellalthiosemicarbazonato)nickel(II) [Ni(S-tcitr)2] using the unicellular eukaryote Saccharomyces cerevisiae as a model organism. Two complementary genomic phenotyping screens led to the identification of 269 sensitive and 56 tolerant deletion mutant strains and of 14 genes that when overexpressed make yeast cells resistant to an otherwise lethal concentration of Ni(S-tcitr)2. Chromatin remodeling, cytoskeleton organization, mitochondrial function and iron metabolism were identified as lead cellular processes responsible for Ni(S-tcitr)2 toxicity. The latter process, and particularly glutaredoxin-mediated iron loading of RNR, was found to be affected by Ni(S-tcitr)2. Given the multiple pathways regulated by glutaredoxins, targeting of these proteins by Ni(S-tcitr)2 can negatively affect various core cellular processes that may critically contribute to Ni(S-tcitr)2 cytotoxicity.

A Physiological Basis for Nonheritable Antibiotic Resistance.

Antibiotics constitute one of the cornerstones of modern medicine. However, individuals may succumb to a bacterial infection if a pathogen survives exposure to antibiotics. The ability of bacteria to survive bactericidal antibiotics results from genetic changes in the preexisting bacterial genome, from the acquisition of genes from other organisms, and from nonheritable phenomena that give rise to antibiotic tolerance. Nonheritable antibiotic tolerance can be exhibited by a large fraction of the bacterial population or by a small subpopulation referred to as persisters. Nonheritable resistance to antibiotics has been ascribed to the activity of toxins that are part of toxin-antitoxin modules, to the universal energy currency ATP, and to the signaling molecule guanosine (penta) tetraphosphate. However, these molecules are dispensable for nonheritable resistance to antibiotics in many organisms. By contrast, nutrient limitation, treatment with bacteriostatic antibiotics, or expression of genes that slow bacterial growth invariably promote nonheritable resistance. We posit that antibiotic persistence results from conditions promoting feedback inhibition among core cellular processes, resulting phenotypically in a slowdown or halt in bacterial growth.

BraInMap Elucidates the Macromolecular Connectivity Landscape of Mammalian Brain

Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.

A Comparative Analysis of Tumors and Plasma Circulating Tumor DNA in 145 Advanced Cancer Patients Annotated by 3 Core Cellular Processes.

Matched-targeted and immune checkpoint therapies have improved survival in cancer patients, but tumor heterogeneity contributes to drug resistance. Our study categorized gene mutations from next generation sequencing (NGS) into three core processes. This annotation helps decipher complex biologic interactions to guide therapy. We collected NGS data on 145 patients who have failed standard therapy (2016 to 2018). One hundred and forty two patients had data for tissue (Caris MI/X) and plasma cell-free circulating tumor DNA (Guardant360) platforms. The mutated genes were categorized into cell fate (CF), cell survival (CS), and genome maintenance (GM). Comparative analysis was performed for concordance and discordance, unclassified mutations, trends in TP53 alterations, and PD-L1 expression. Two gene mutation maps were generated to compare each NGS platform. Mutated genes predominantly matched to CS with concordance between Guardant360 (64.4%) and Caris (51.5%). TP53 alterations comprised a significant proportion of the mutation pool in Caris and Guardant360, 14.7% and 13.1%, respectively. Twenty-six potentially actionable gene alterations were detected from matching ctDNA to Caris unclassified alterations. The CS core cellular process was the most prevalent in our study population. Clinical trials are warranted to investigate biomarkers for the three core cellular processes in advanced cancer patients to define the next best therapies.

Fabrication of Bioactive Inverted Colloidal Crystal Scaffolds Using Expanded Polystyrene Beads.

Inverted colloidal crystal (ICC) hydrogel scaffolds have emerged as a new class of three-dimensional cell culture matrix that represents a unique opportunity to reproduce lymphoid tissue microenvironments. ICC geometry promotes the formation of stromal cell networks and their interaction with hematopoietic cells, a core cellular process in lymphoid tissues. When subdermally implanted, ICC hydrogel scaffolds direct unique foreign body responses to form a vascularized stromal tissue with prolonged attraction of hematopoietic cells, which together resemble lymphoid tissue microenvironments. While conceptually simple, fabrication of ICC hydrogel scaffold requires multiple steps and laborious handling of delicate materials. Here, we introduce a facile route for ICC hydrogel scaffold fabrication using expanded polystyrene (EPS) beads. EPS beads shrink and fuse in a tunable manner under pressurized thermal conditions, which serves as colloidal crystal templates for ICC scaffold fabrication. Inclusion of collagen in the precursor solution greatly simplified preparation of bioactive hydrogel scaffolds. The resultant EPS-templated bioactive ICC hydrogel scaffolds demonstrate characteristic features required for lymphoid tissue modeling in both in vitro and in vivo settings. We envision that the presented method will facilitate widespread implementation of ICC hydrogel scaffolds for lymphoid tissue engineering and other emerging applications. Impact statement Inverted colloidal crystal (ICC) hydrogel scaffolds have emerged as a new class of three-dimensional cell culture matrix that represents a unique opportunity for lymphoid tissue modeling and other emerging novel bioengineering applications. While conceptually simple, fabrication of the ICC hydrogel scaffold requires multiple steps and laborious handling of delicate materials with highly toxic chemicals. The presented method for ICC hydrogel scaffold fabrication using expanded polystyrene (EPS) beads is simple, cost-effective, and involves less toxic chemicals than conventional methods, while retaining comparable biological significance. We envision that EPS bead-based hydrogel scaffold fabrication will greatly facilitate the widespread implementation of ICC hydrogel scaffolds and their practical applications.

Urbanization without isolation: the absence of genetic structure among cities and forests in the tiny acorn ant Temnothorax nylanderi.

Urban alteration of neutral and adaptive evolutionary processes is still underexplored. Using a genome-wide SNP dataset, we investigated (i) urban-induced modifications of population demography, genetic diversity and population structure and (ii) signature of divergent selection between urban and forest populations in the ant species, Temnothorax nylanderi. Our results did not reveal an impact of urbanization on neutral processes since we observed: (i) analogous genetic diversity among paired urban/forest sites and two control populations; (ii) weak population genetic structure explained neither by habitat (urban versus forest) nor by geography; (iii) a remarkably similar demographic history across populations with an ancestral growth followed by a recent decline, regardless of their current habitat or geographical location. The micro-geographical home range of ants may explain their resilience to urbanization. Finally, we detected 19 candidate loci discriminating urban/forest populations and associated with core cellular components, molecular function or biological process. Two of these loci were associated with a gene ontology term that was previously found to belong to a module of co-expressed genes related to caste phenotype. These results call for transcriptomics analyses to identify genes associated with ant social traits and to infer their potential role in urban adaptation.

Effector-triggered immunity and pathogen sensing in metazoans.

Microbial pathogens possess an arsenal of strategies to invade their hosts, evade immune defences and promote infection. In particular, bacteria use virulence factors, such as secreted toxins and effector proteins, to manipulate host cellular processes and establish a replicative niche. Survival of eukaryotic organisms in the face of such challenge requires host mechanisms to detect and counteract these pathogen-specific virulence strategies. In this Review, we focus on effector-triggered immunity (ETI) in metazoan organisms as a mechanism for pathogen sensing and distinguishing pathogenic from non-pathogenic microorganisms. For the purposes of this Review, we adopt the concept of ETI formulated originally in the context of plant pathogens and their hosts, wherein specific host proteins 'guard' central cellular processes and trigger inflammatory responses following pathogen-driven disruption of these processes. While molecular mechanisms of ETI are well-described in plants, our understanding of functionally analogous mechanisms in metazoans is still emerging. In this Review, we present an overview of ETI in metazoans and discuss recently described cellular processes that are guarded by the host. Although all pathogens manipulate host pathways, we focus primarily on bacterial pathogens and highlight pathways of effector-triggered immune defence that sense disruption of core cellular processes by pathogens. Finally, we discuss recent developments in our understanding of how pathogens can evade ETI to overcome these host adaptations.

Common Nodes of Virus-Host Interaction Revealed Through an Integrated Network Analysis.

Viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. A collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape.

Landscape of the Plasmodium Interactome Reveals Both Conserved and Species-Specific Functionality

SummaryMalaria represents a major global health issue, and the identification of new intervention targets remains an urgent priority. This search is hampered by more than one-third of the genes of malaria-causing Plasmodium parasites being uncharacterized. We report a large-scale protein interaction network in Plasmodium schizonts, generated by combining blue native-polyacrylamide electrophoresis with quantitative mass spectrometry and machine learning. This integrative approach, spanning 3 species, identifies >20,000 putative protein interactions, organized into 600 protein clusters. We validate selected interactions, assigning functions in chromatin regulation to previously unannotated proteins and suggesting a role for an EELM2 domain-containing protein and a putative microrchidia protein as mechanistic links between AP2-domain transcription factors and epigenetic regulation. Our interactome represents a high-confidence map of the native organization of core cellular processes in Plasmodium parasites. The network reveals putative functions for uncharacterized proteins, provides mechanistic and structural insight, and uncovers potential alternative therapeutic targets.

Abstract 4313: Identifying and characterizing epigenetic «driver» genes («epidrivers») in regulatory pathways involved in tumorigenesis and tumour cell plasticity

Abstract One of the ground-breaking discoveries of the international cancer genome sequencing endeavours is the high frequency of mutational and non-mutational changes in epigenetic regulator genes (ERGs), which constitute a “genetic smoking gun” that epigenetic mechanisms lie in the very heart of cancer biology. However, functional importance of disruption of ERGs in tumorigenesis and cancer phenotype is poorly understood. We hypothesise that these genes are candidates to be drivers («epidrivers») of cancer onset and progression, thus regulating mechanisms underpinning cancer development. Moreover, deregulation of epidrivers may play a role in epithelial-to-mesenchymal transition (EMT) and emergence of cancer resilience. The overarching aim of this study was to identify and functionally characterize “Epidrivers” in tumorigenesis and cancer cell plasticity. To this end, we have set up novel epigenome-wide functional screens in human cultured cells (and organoids) combined with state-of-the-art genome-editing approach (based on CRISPR/Cas9 screen) and multiparametric phenotyping. We designed a custom-made lentiviral CRISPR library that consists of 1,649 gRNAs targeting all known ERGs (426 genes). Lentiviral CRISPR library was used to deliver the ERG gRNAs to target cells expressing the RNA-guided DNA endonuclease Cas9. Independent clones (derived from transduced breast and lung cancer cell lines constitutively expressing Cas9) are screened for acquisition of distinct features of transformed cells. The results of the identification of Epidriver genes based on the analysis of deregulation of core cellular processes, epigenome (ChIP-seq) as well as EMT and multiparametric phenotyping, will be presented. Citation Format: Andrea Halaburkova, Vincent Cahais, Cyrille Cuenin, Rita Khoueiry, Maria Ouzounova, Akram Ghantous, Zdenko Herceg. Identifying and characterizing epigenetic «driver» genes («epidrivers») in regulatory pathways involved in tumorigenesis and tumour cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4313.

Cicada Endosymbionts Have tRNAs That Are Correctly Processed Despite Having Genomes That Do Not Encode All of the tRNA Processing Machinery.

Gene loss and genome reduction are defining characteristics of endosymbiotic bacteria. The most highly reduced endosymbiont genomes have lost numerous essential genes related to core cellular processes such as replication, transcription, and translation. Computational gene predictions performed for the genomes of the two bacterial symbionts of the cicada Diceroprocta semicincta, "Candidatus Hodgkinia cicadicola" (Alphaproteobacteria) and "Ca Sulcia muelleri" (Bacteroidetes), have found only 26 and 16 tRNA genes and 15 and 10 aminoacyl tRNA synthetase genes, respectively. Furthermore, the original "Ca Hodgkinia cicadicola" genome annotation was missing several essential genes involved in tRNA processing, such as those encoding RNase P and CCA tRNA nucleotidyltransferase as well as several RNA editing enzymes required for tRNA maturation. How these cicada endosymbionts perform basic translation-related processes remains unknown. Here, by sequencing eukaryotic mRNAs and total small RNAs, we show that the limited tRNA set predicted by computational annotation of "Ca Sulcia muelleri" and "Ca Hodgkinia cicadicola" is likely correct. Furthermore, we show that despite the absence of genes encoding tRNA processing activities in the symbiont genomes, symbiont tRNAs have correctly processed 5' and 3' ends and seem to undergo nucleotide modification. Surprisingly, we found that most "Ca Hodgkinia cicadicola" and "Ca Sulcia muelleri" tRNAs exist as tRNA halves. We hypothesize that "Ca Sulcia muelleri" and "Ca Hodgkinia cicadicola" tRNAs function in bacterial translation but require host-encoded enzymes to do so.IMPORTANCE The smallest bacterial genomes, in the range of about 0.1 to 0.5 million base pairs, are commonly found in the nutritional endosymbionts of insects. These tiny genomes are missing genes that encode proteins and RNAs required for the translation of mRNAs, one of the most highly conserved and important cellular processes. In this study, we found that the bacterial endosymbionts of cicadas have genomes which encode incomplete tRNA sets and lack genes required for tRNA processing. Nevertheless, we found that endosymbiont tRNAs are correctly processed at their 5' and 3' ends and, surprisingly, that mostly exist as tRNA halves. We hypothesize that the cicada host must supply its symbionts with these missing tRNA processing activities.

Transcription Is Just the Beginning of Gene Expression Regulation: The Functional Significance of RNA-Binding Proteins to Post-transcriptional Processes in Plants.

Plants have developed sophisticated mechanisms to compensate and respond to ever-changing environmental conditions. Research focus in this area has recently shifted towards understanding the post-transcriptional mechanisms that contribute to RNA transcript maturation, abundance and function as key regulatory steps in allowing plants to properly react and adapt to these never-ending shifts in their environments. At the center of these regulatory mechanisms are RNA-binding proteins (RBPs), the functional mediators of all post-transcriptional processes. In plants, RBPs are becoming increasingly appreciated as the critical modulators of core cellular processes during development and in response to environmental stimuli. With the majority of research on RBPs and their functions historically in prokaryotic and mammalian systems, it has more recently been unveiled that plants have expanded families of conserved and novel RBPs compared with their eukaryotic counterparts. To better understand the scope of RBPs in plants, we present past and current literature detailing specific roles of RBPs during stress response, development and other fundamental transition periods. In this review, we highlight examples of complex regulation coordinated by RBPs with a focus on the diverse mechanisms of plant RBPs and the unique processes they regulate. Additionally, we discuss the importance for additional research into understanding global interactions of RBPs on a systems and network-scale, with genome mining and annotation providing valuable insight for potential uses in improving crop plants in order to maintain high-level production in this era of global climate change.

Meta-analysis of drought-tolerant genotypes in Oryza sativa: A network-based approach.

BackgroundDrought is a severe environmental stress. It is estimated that about 50% of the world rice production is affected mainly by drought. Apart from conventional breeding strategies to develop drought-tolerant crops, innovative computational approaches may provide insights into the underlying molecular mechanisms of stress response and identify drought-responsive markers. Here we propose a network-based computational approach involving a meta-analytic study of seven drought-tolerant rice genotypes under drought stress.ResultsCo-expression networks enable large-scale analysis of gene-pair associations and tightly coupled clusters that may represent coordinated biological processes. Considering differentially expressed genes in the co-expressed modules and supplementing external information such as resistance/tolerance QTLs, transcription factors, network-based topological measures, we identify and prioritize drought-adaptive co-expressed gene modules and potential candidate genes. Using the candidate genes that are well-represented across the datasets as ‘seed’ genes, two drought-specific protein-protein interaction networks (PPINs) are constructed with up- and down-regulated genes. Cluster analysis of the up-regulated PPIN revealed ABA signalling pathway as a central process in drought response with a probable crosstalk with energy metabolic processes. Tightly coupled gene clusters representing up-regulation of core cellular respiratory processes and enhanced degradation of branched chain amino acids and cell wall metabolism are identified. Cluster analysis of down-regulated PPIN provides a snapshot of major processes associated with photosynthesis, growth, development and protein synthesis, most of which are shut down during drought. Differential regulation of phytohormones, e.g., jasmonic acid, cell wall metabolism, signalling and posttranslational modifications associated with biotic stress are elucidated. Functional characterization of topologically important, drought-responsive uncharacterized genes that may play a role in important processes such as ABA signalling, calcium signalling, photosynthesis and cell wall metabolism is discussed. Further transgenic studies on these genes may help in elucidating their biological role under stress conditions.ConclusionCurrently, a large number of resources for rice functional genomics exist which are mostly underutilized by the scientific community. In this study, a computational approach integrating information from various resources such as gene co-expression networks, protein-protein interactions and pathway-level information is proposed to provide a systems-level view of complex drought-responsive processes across the drought-tolerant genotypes.

Inferring the Evolutionary History of Your Favorite Protein: A Guide for Molecular Biologists.

Comparative genomics has proven a fruitful approach to acquire many functional and evolutionary insights into core cellular processes. Here it is argued that in order to perform accurate and interesting comparative genomics, one first and foremost has to be able to recognize, postulate, and revise different evolutionary scenarios. After all, these studies lack a simple protocol, due to different proteins having different evolutionary dynamics and demanding different approaches. The authors here discuss this challenge from a practical (what are the observations?) and conceptual (how do these indicate a specific evolutionary scenario?) viewpoint, with the aim to guide investigators who want to analyze the evolution of their protein(s) of interest. By sharing how the authors draft, test, and update such a scenario and how it directs their investigations, the authors hope to illuminate how to execute molecular evolution studies and how to interpret them. Also see the video abstract here https://youtu.be/VCt3l2pbdbQ.

Investigating the Mechanisms by which the Methyl‐CpG Binding Protein ZBTB38 Identifies Interacting Partners and Mediates Transcription

Cytosine methylation in CpG dinucleotides is an essential epigenetic modification utilized for regulating transcriptional processes within the eukaryotic genome. Consequently, misappropriation of DNA methylation patterns is associated with many diseases, including cancer. At many gene promoters, DNA methylation mediates expression through recruitment of methyl‐CpG binding proteins (MBPs). These specialized transcription factors read the methylated DNA signal and subsequently recruit chromatin remodeling complexes; leading to alterations in chromatin architecture and transcriptional responses. Thus, MBPs represent important functional intermediaries between these two key epigenetic events. ZBTB38 is the largest member of the ZBTB family of MBPs and contains two sets of five Cys2His2 zinc finger (ZF) domains distributed into N‐ and C‐terminal clusters, as well as an N‐terminal BTB/POZ protein interaction domain. There is increasing evidence that ZBTB38 regulates transcription for several core cellular processes in both normal cells as well as disease states. However, full mechanistic insight for how ZBTB38 identifies binding partners to elicit cellular function remains to be determined. Using combined structural and biochemical approaches, specific DNA binding partners have been identified and characterized for both the N‐ and C‐terminal ZF domains of ZBTB38. Importantly, we recently discovered that in addition to the N‐terminal ZFs, the C‐terminal ZFs also exhibit methyl‐selective DNA binding through the use of an alternative mode for methylated DNA recognition. Cell based studies have been utilized to begin delineating how these two ZF domains may function together or independently to modulate transcription. In addition to DNA interactions, we have begun to evaluate protein interacting partners for ZBTB38 using a combination of biophysical and cell based methodologies. Together, these findings expand our understanding for ZF recognition of methylated DNA, as well as illuminate the molecular basis by which ZBTB38 preferentially recognizes its biomolecular binding partners to translate transcriptional outcomes into specific cellular responses.Support or Funding InformationThis work was supported in part by MCB‐1715370 from the National Science Foundation and by RSG‐14‐185‐01‐DMC from the American Cancer Society.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.