Components Of Gene Regulatory Networks Research Articles

Living in a noisy world-origins of gene expression noise and its impact on cellular decision-making.

The expression level of a gene can vary between genetically identical cells under the same environmental condition-a phenomenon referred to as gene expression noise. Several studies have now elucidated a central role of transcription factors in the generation of expression noise. Transcription factors, as the key components of gene regulatory networks, drive many important cellular decisions in response to cellular and environmental signals. Therefore, a very relevant question is how expression noise impacts gene regulation and influences cellular decision-making. In this Review, we summarize the current understanding of the molecular origins of expression noise, highlighting the role of transcription factors in this process, and discuss the ways in which noise can influence cellular decision-making. As advances in single-cell technologies open new avenues for studying expression noise as well as gene regulatory circuits, a better understanding of the influence of noise on cellular decisions will have important implications for many biological processes.

TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway.

Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underlies facial shape variation, yet how those networks in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest, even during the late migratory phase, results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both TFAP2 family members dysregulates numerous midface GRN components involved in midface morphogenesis, patterning and differentiation. Notably, Alx1, Alx3 and Alx4 (ALX) transcript levels are reduced, whereas ChIP-seq analyses suggest TFAP2 family members directly and positively regulate ALX gene expression. Tfap2a, Tfap2b and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish implies conservation of this regulatory axis across vertebrates. Consistent with this notion, tfap2a zebrafish mutants present with abnormal alx3 expression patterns, Tfap2a binds ALX loci and tfap2a-alx3 genetic interactions are observed. Together, these data demonstrate TFAP2 paralogs regulate vertebrate midfacial development in part by activating expression of ALX transcription factor genes.

Comparative expression analysis of the Atoh7 gene regulatory network in the mouse and chicken auditory hindbrain

The mammalian and avian auditory brainstem likely arose by independent evolution. To compare the underlying molecular mechanisms, we focused on Atoh7, as its expression pattern in the mammalian hindbrain is restricted to bushy cells in the ventral cochlear nucleus. We thereby took advantage of an Atoh7 centered gene regulatory network (GRN) in the retina including upstream regulators, Hes1 and Pax6, and downstream targets, Ebf3 and Eya2. In situ hybridization demonstrated for the latter four genes broad expression in all three murine cochlear nuclei at postnatal days (P) 4 and P30, contrasting the restricted expression of Atoh7. In chicken, all five transcription factors were expressed in all auditory hindbrain nuclei at embryonic day (E) 13 and P14. Notably, all five genes showed graded expression in the embryonic nucleus magnocellularis (NM). Atoh7 was highly expressed in caudally located neurons, whereas the other four transcription factors were highly expressed in rostrally located neurons. Thus, Atoh7 shows a strikingly different expression between the mammalian and avian auditory hindbrain. This together with the consistent absence of graded expression of GRN components in developing mammalian nuclei provide the first molecular support to the current view of convergent evolution as a major mechanism in the amniote auditory hindbrain. The graded expression of five transcription factors specifically in the developing NM confirms this nucleus as a central organizer of tonotopic features in birds. Finally, the expression of all five retinal GRN components in the auditory system suggests co-options of genes for development of sensory systems of distinct modalities.

Stem cell-based modeling and single-cell multiomics reveal gene-regulatory mechanisms underlying human skeletal development

Although the skeleton is essential for locomotion, endocrine functions, and hematopoiesis, the molecular mechanisms of human skeletal development remain to be elucidated. Here, we introduce an integrative method to model human skeletal development by combining invitro sclerotome induction from human pluripotent stem cells and invivo endochondral bone formation by implanting the sclerotome beneath the renal capsules of immunodeficient mice. Histological and scRNA-seq analyses reveal that the induced bones recapitulate endochondral ossification and are composed of human skeletal cells and mouse circulatory cells. The skeletal cell types and their trajectories are similar to those of human embryos. Single-cell multiome analysis reveals dynamic changes in chromatin accessibility associated with multiple transcription factors constituting cell-type-specific gene-regulatory networks (GRNs). We further identify ZEB2, which may regulate the GRNs in human osteogenesis. Collectively, these results identify components of GRNs in human skeletal development and provide a valuable model for its investigation.

A Review of Wrky Transcription Factors in The Regulation of Abiotic and Biotic Stress in Cotton (Gossypium Spp.)

Cotton is an important economic crop that is grown in over 100 countries throughout the world. It provides the textile industry raw materials, cottonseed oil for cooking, and edible and protein-rich oil cake remnants for farm animals. Abiotic and biotic stressors limit its production, which is strongly reliant on climatic circumstances. Cotton plants have several built-in mechanisms for dealing with abiotic and biotic stress. These defense systems are controlled by changes in the expression of stress-responsive genes. Several stress response-related genes have been discovered, including WRKY transcription elements. WRKY proteins bind to the W box (TGACC (A/T)) in the promoter regions of genes to activate or reduce the expression of downstream genes, which regulates the stress response. It also works with other transcription factors to control the defense responses of the plant. The function of WRKY transcription factors in cotton was briefly discussed in this review in terms of drought, salinity, temperature, and oxidative and biotic stress reactions. In conclusion, WRKY genes are thought to be important components of gene regulatory networks involved in plant stress responses and developmental plasticity in response to seasonal variations in environmental conditions and pathogenic infections.

Rapid sequence and functional diversification of a miRNA superfamily targeting calcium signaling components in seed plants.

Summary MicroRNA (miRNA)‐directed posttranscriptional gene silencing (miR‐PTGS) is an integral component of gene regulatory networks governing plant development and responses to the environment. The sequence homology between Sly‐miR4376, a miRNA common to Solanaceae and reported to target autoinhibited Ca2+‐ATPase 10 (ACA10) messenger RNA (mRNA) in tomato, and Arabidopsis miR391 (Ath‐miR391), previously annotated as a nonconserved member of the deeply conserved miR390 family, has prompted us to revisit the function of Ath‐miR391, as well as its regulatory conservation.A combination of genetic, molecular, and bioinformatic analyses revealed a hidden conservation for miR‐PTGS of ACA10 homologs in spermatophytes.We found that the Arabidopsis ACA10 mRNA undergoes miR391‐directed cleavage in vivo. Furthermore, transgenic overexpression of miR391 recapitulated the compact inflorescence (cif) phenotypes characteristic of ACA10 loss‐of‐function mutants, due to miR391‐directed PTGS of ACA10. Significantly, comprehensive data mining revealed robust evidence for widespread PTGS of ACA10 homologs directed by a superfamily of related miRNAs sharing a conserved sequence core. Intriguingly, the ACA‐targeting miRNAs in Poaceae also direct PTGS for calmodulin‐like proteins which are putative Ca2+ sensors.The PTGS of ACA10 homologs is therefore directed by a miRNA superfamily that is of ancient origin and has undergone rapid sequence diversification associated with functional innovation.

The Roles of MADS-Box Genes from Root Growth to Maturity in Arabidopsis and Rice

Rice (Oryza sativa L.) and Arabidopsis thaliana (L.) life cycles involve several major phase changes, throughout which MADS-box genes have a variety of functions. MADS-box genes are well recognized for their functions in floral induction and development, and some have multiple functions in apparently unrelated developmental stages. For example, in Arabidopsis, AGL15 and AGL6 play roles in both vegetative development and floral transition. Similarly, in rice, OsMADS1 is involved in flowering time and seed development, and OsMADS26 is expressed not only in the roots, but also in the leaves, shoots, panicles, and seeds. The roles of other MADS-box genes responsible for the regulation of specific traits in both rice and Arabidopsis are also discussed. Several are key components of gene regulatory networks involved in root development under diverse environmental factors such as drought, heat, and salt stress, and are also involved in the shift from vegetative to flowering growth in response to seasonal changes in environmental conditions. Thus, we argue that MADS-box genes are critical elements of gene regulation that underpin diverse gene expression profiles, each of which is linked to a unique developmental stage that occurs during root development and the shift from vegetative to reproductive growth.

SHORT-ROOT Controls Cell Elongation in the Etiolated Arabidopsis Hypocotyl.

Transcriptional regulation, a core component of gene regulatory networks, plays a key role in controlling individual organism’s growth and development. To understand how plants modulate cellular processes for growth and development, the identification and characterization of gene regulatory networks are of importance. The SHORT-ROOT (SHR) transcription factor is known for its role in cell divisions in Arabidopsis (Arabidopsis thaliana). However, whether SHR is involved in hypocotyl cell elongation remains unknown. Here, we reveal that SHR controls hypocotyl cell elongation via the transcriptional regulation of XTH18, XTH22, and XTH24, which encode cell wall remodeling enzymes called xyloglucan endotransglucosylase/hydrolases (XTHs). Interestingly, SHR activates transcription of the XTH genes, independently of its partner SCARECROW (SCR), which is different from the known mode of action. In addition, overexpression of the XTH genes can promote cell elongation in the etiolated hypocotyl. Moreover, confinement of SHR protein in the stele still induces cell elongation, despite the aberrant organization in the hypocotyl ground tissue. Therefore, it is likely that SHR-mediated growth is uncoupled from SHR-mediated radial patterning in the etiolated hypocotyl. Our findings also suggest that intertissue communication between stele and endodermis plays a role in coordinating hypocotyl cell elongation of the Arabidopsis seedling. Taken together, our study identifies SHR as a new crucial regulator that is necessary for cell elongation in the etiolated hypocotyl.

Candidate positive targets of LHX6 and LHX8 transcription factors in the developing upper jaw

Craniofacial development is controlled by a large number of genes, which interact with one another to form a complex gene regulatory network (GRN). Key components of GRN are signaling molecules and transcription factors. Therefore, identifying targets of core transcription factors is an important part of the overall efforts toward building a comprehensive and accurate model of GRN. LHX6 and LHX8 are transcription factors expressed in the oral mesenchyme of the first pharyngeal arch (PA1), and they are crucial regulators of palate and tooth development. Previously, we performed genome-wide transcriptional profiling and chromatin immunoprecipitation to identify target genes of LHX6 and LHX8 in PA1, and described a set of genes repressed by LHX. However, there has not been any discussion of the genes positively regulated by LHX6 and LHX8. In this paper, we revisited the above datasets to identify candidate positive targets of LHX in PA1. Focusing on those with known connections to craniofacial development, we performed RNA in situ hybridization to confirm the changes in expression in Lhx6;Lhx8 mutant. We also confirmed the binding of LHX6 to several putative enhancers near the candidate target genes. Together, we have uncovered novel connections between Lhx and other important regulators of craniofacial development, including Eya1, Barx1, Rspo2, Rspo3, and Wnt11.

Transcription Factor-Based Genetic Engineering in Microalgae.

Sequence-specific DNA-binding transcription factors (TFs) are key components of gene regulatory networks. Advances in high-throughput sequencing have facilitated the rapid acquisition of whole genome assembly and TF repertoires in microalgal species. In this review, we summarize recent advances in gene discovery and functional analyses, especially for transcription factors in microalgal species. Specifically, we provide examples of the genome-scale identification of transcription factors in genome-sequenced microalgal species and showcase their application in the discovery of regulators involved in various cellular functions. Herein, we highlight TF-based genetic engineering as a promising framework for designing microalgal strains for microalgal-based bioproduction.

Temporal dynamics of a CSF1R signaling gene regulatory network involved in epilepsy.

Colony Stimulating Factor 1 Receptor (CSF1R) is a potential target for anti-epileptic drugs. However, inhibition of CSF1R is not well tolerated by patients, thereby prompting the need for alternative targets. To develop a framework for identification of such alternatives, we here develop a mathematical model of a pro-inflammatory gene regulatory network (GRN) involved in epilepsy and centered around CSF1R. This GRN comprises validated transcriptional and post-transcriptional regulations involving STAT1, STAT3, NFκB, IL6R, CSF3R, IRF8, PU1, C/EBPα, TNFR1, CSF1 and CSF1R. The model was calibrated on mRNA levels of all GRN components in lipopolysaccharide (LPS)-treated mouse microglial BV-2 cells, and allowed to predict that STAT1 and STAT3 have the strongest impact on the expression of the other GRN components. Microglial BV-2 cells were selected because, the modules from which the GRN was deduced are enriched for microglial marker genes. The function of STAT1 and STAT3 in the GRN was experimentally validated in BV-2 cells. Further, in silico analysis of the GRN dynamics predicted that a pro-inflammatory stimulus can induce irreversible bistability whereby the expression level of GRN components occurs as two distinct states. The irreversibility of the switch may enforce the need for chronic inhibition of the CSF1R GRN in order to achieve therapeutic benefit. The cell-to-cell heterogeneity driven by the bistability may cause variable therapeutic response. In conclusion, our modeling approach uncovered a GRN controlling CSF1R that is predominantly regulated by STAT1 and STAT3. Irreversible inflammation-induced bistability and cell-to-cell heterogeneity of the GRN provide a theoretical foundation to the need for chronic GRN control and the limited potential for disease modification via inhibition of CSF1R.

Sea Urchin as a Universal Model for Studies of Gene Networks.

The purple sea urchin Strongylocentrotus purpuratus has been used for over 150 years as a model organism in developmental biology. Using this model species, scientists have been able to describe, in detail, the mechanisms of cell cycle control and cell adhesion, fertilization, calcium signaling, cell differentiation, and death. Massive parallel sequencing of the sea urchin genome enabled the deciphering of the main components of gene regulatory networks during the activation of embryonic signaling pathways. This knowledge helped to extrapolate aberrations in somatic cells that may lead to diseases, including cancer in humans. Furthermore, since many, if not all, developmental signaling pathways were shown to be controlled by non-coding RNAs (ncRNAs), the sea urchin organism represents an attractive experimental model. In this review, we discuss the main discoveries in the genetics, genomics, and transcriptomics of sea urchins during embryogenesis with the main focus on the role of ncRNAs. This information may be useful for comparative studies between different organisms, and may help identify new regulatory networks controlled by ncRNAs.

Controlling spatiotemporal pattern formation in a concentration gradient with a synthetic toggle switch.

The formation of spatiotemporal patterns of gene expression is frequently guided by gradients of diffusible signaling molecules. The toggle switch subnetwork, composed of two cross‐repressing transcription factors, is a common component of gene regulatory networks in charge of patterning, converting the continuous information provided by the gradient into discrete abutting stripes of gene expression. We present a synthetic biology framework to understand and characterize the spatiotemporal patterning properties of the toggle switch. To this end, we built a synthetic toggle switch controllable by diffusible molecules in Escherichia coli. We analyzed the patterning capabilities of the circuit by combining quantitative measurements with a mathematical reconstruction of the underlying dynamical system. The toggle switch can produce robust patterns with sharp boundaries, governed by bistability and hysteresis. We further demonstrate how the hysteresis, position, timing, and precision of the boundary can be controlled, highlighting the dynamical flexibility of the circuit.

MiR-6086 inhibits ovarian cancer angiogenesis by downregulating the OC2/VEGFA/EGFL6 axis

miRNAs have emerged as a pivotal component of gene regulatory networks, mediating cytokines secretion, cell cycle, and differentiation regulation. However, how miRNAs collaborate with transcription factors and downstream effector proteins that determine the fate of ovarian cancer cells remains to be understood, especially regarding to mechanism of tumor angiogenesis regulation. Based on the qRT-PCR and IHC analysis, we found that miR-6086 was maintained a very low level both in ovarian cancer cell lines and tissues. Further, we identified OC2 and EGFL6 as the direct targets of miR-6086 by luciferase assay and we observed an inverse relationship between the expression of miR-6086 and the OC2/VEGFA/EGFL6 axis. The Western blotting analysis suggested that OC2 could directly upregulate VEGFA and indirectly up-regulate EGFL6 through VEGFA. Moreover, miR-6086 could indirectly downregulate VEGFA through OC2. In addition, miR-6086, siOC2 and siEGFL6 could negatively regulate the tumor growth and angiogenesis of ovarian cancer (Skov3) in the animal studies, with the inhibition rates of 77.07%, 69.89%, and 73.62%, respectively (**p < 0.01). Moreover, the tumor cell proliferation, migration, and invasion of ovarian cancer cell lines (Caov3 and Skov3) and vascular formation (HUVECs) were significantly suppressed in vitro, by decreasing the AKT/MAPK pathways (*p < 0.05). Taken together, our results reveal that miR-6086 can suppress the angiogenesis networks in ovarian cancer by down-regulating the OC2/VEGFA/EGFL6 axis, directly or indirectly, which may provide potential targets for tumor therapeutics.

OTX2 represses sister cell fate choices in the developing retina to promote photoreceptor specification.

During vertebrate retinal development, subsets of progenitor cells generate progeny in a non-stochastic manner, suggesting that these decisions are tightly regulated. However, the gene-regulatory network components that are functionally important in these progenitor cells are largely unknown. Here we identify a functional role for the OTX2 transcription factor in this process. CRISPR/Cas9 gene editing was used to produce somatic mutations of OTX2 in the chick retina and identified similar phenotypes to those observed in human patients. Single cell RNA sequencing was used to determine the functional consequences OTX2 gene editing on the population of cells derived from OTX2-expressing retinal progenitor cells. This confirmed that OTX2 is required for the generation of photoreceptors, but also for repression of specific retinal fates and alternative gene regulatory networks. These include specific subtypes of retinal ganglion and horizontal cells, suggesting that in this context, OTX2 functions to repress sister cell fate choices.

Gene Regulatory Networks in Peripheral Mononuclear Cells Reveals Critical Regulatory Modules and Regulators of Multiple Sclerosis

Multiple sclerosis (MS) is a complex, demyelinating disease with the involvement of autoimmunity and neurodegeneration. Increasing efforts have been made towards identifying the diagnostic markers to differentiate the classes of MS from other similar neurological conditions. Using a systems biology approach, we constructed four types of gene regulatory networks (GRNs) involved in peripheral blood mononuclear cells (PBMCs). The regulatory strength of each GRN across primary progressive MS (PPMS), relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), and control were evaluated by an integrity algorithm. Among the constructed GRNs (referred as TF_gene_miRNA), POU3F2_CDK6_hsa-miR-590-3p, MEIS1_CASC3_hsa-miR-1261, STAT3_OGG1_hsa-miR-298, and TCF4_FMR1_hsa-miR-301b were top-ranked and differentially regulated in all classes of MS compared to control. These GRNs showed potential involvement in regulating various molecular pathways such as interleukin, integrin, glypican, sphingosine phosphate, androgen, and Wnt signaling pathways. For validation, the qPCR analysis of the GRN components (TFs, gene, and miRNAs) in PBMCs of healthy controls (n = 30), RRMS (n = 14), PPMS (n = 13) and SPMS (n = 12) were carried out. Real-time expression analysis of GRNs showed a similar regulatory pattern as derived from our systems biology approach. Also, our study provided several novel GRNs that regulate unique and common molecular mechanisms between MS conditions. Hence, these regulatory components of GRNs will help to understand the disease mechanism across MS classes and further insight may though light towards diagnosis.

Evolutionary genetics in insect phenotypic radiations: the value of a comparative genomic approach

Expanding genome sequencing and transgenic technologies are enabling the discovery of genes driving phenotypic diversity across insect taxa. Limitations in downstream functional genetic approaches, however, have been an obstacle for developing non-model systems for evolutionary genetics. Phenotypically diverse radiations, such as those exhibiting convergence and divergence as a result of mimicry, are ideal for evolutionary genetics as they can lead to insights using comparative genomic approaches alone. The varied and repeated instances of phenotypes in highly polymorphic systems allow assessment of whether similar loci are repeatedly targeted by selection and can inform how alleles sort across lineages. Comparative genomics of these taxa can be used to decipher components of gene regulatory networks, dissect regulatory regions, and validate genes.

MiR-22 Regulates Invasion, Gene Expression and Predicts Overall Survival in Patients with Clear Cell Renal Cell Carcinoma.

Background:Clear cell renal cell carcinoma (ccRCC) is molecularly diverse and distinct molecular subtypes show different clinical outcomes. MicroRNAs (miRNAs) are essential components of gene regulatory networks and play a crucial role in progression of many cancer types including ccRCC.Objective:Identify prognostic miRNAs and determine the role of miR-22 in ccRCC.Methods:Hierarchical clustering was done in R using gene expression profiles of over 450 ccRCC cases in The Cancer Genome Atlas (TCGA). Kaplan-Meier analysis was performed to identify prognostic miRNAs in the TCGA dataset. RNA-Seq was performed to identify miR-22 target genes in primary ccRCC cells and Matrigel invasion assay was performed to assess the effects of miR-22 overexpression on cell invasion.Results:Hierarchical clustering analysis using 2,621 prognostic genes previously identified by our group demonstrated that ccRCC patients with longer overall survival expressed lower levels of genes promoting proliferation or immune responses, while better maintaining gene expression associated with cortical differentiation and cell adhesion. Targets of 26 miRNAs were significantly enriched in the 2,621 prognostic genes and these miRNAs were prognostic by themselves. MiR-22 was associated with poor overall survival in the TCGA dataset. Overexpression of miR-22 promoted invasion of primary ccRCC cells in vitro and modulated transcriptional programs implicated in cancer progression including DNA repair, cell proliferation and invasion.Conclusions:Our results suggest that ccRCCs with differential clinical outcomes have distinct transcriptomes for which miRNAs could serve as master regulators. MiR-22, as a master regulator, promotes ccRCC progression at least in part by enhancing cell invasion.

MADS-Box Genes Are Key Components of Genetic Regulatory Networks Involved in Abiotic Stress and Plastic Developmental Responses in Plants.

Plants, as sessile organisms, adapt to different stressful conditions, such as drought, salinity, extreme temperatures, and nutrient deficiency, via plastic developmental and growth responses. Depending on the intensity and the developmental phase in which it is imposed, a stress condition may lead to a broad range of responses at the morphological, physiological, biochemical, and molecular levels. Transcription factors are key components of regulatory networks that integrate environmental cues and concert responses at the cellular level, including those that imply a stressful condition. Despite the fact that several studies have started to identify various members of the MADS-box gene family as important molecular components involved in different types of stress responses, we still lack an integrated view of their role in these processes. In this review, we analyze the function and regulation of MADS-box gene family members in response to drought, salt, cold, heat, and oxidative stress conditions in different developmental processes of several plants. In addition, we suggest that MADS-box genes are key components of gene regulatory networks involved in plant responses to stress and plant developmental plasticity in response to seasonal changes in environmental conditions.

The IncRNA BORG: A novel inducer of TNBC metastasis, chemoresistance, and disease recurrence.

Although greater than 90% of breast cancer-related mortality can be attributed to metastases, the molecular mechanisms underpinning the dissemination of primary breast tumor cells and their ability to establish malignant lesions in distant tissues remain incompletely understood. Genomic and transcriptomic analyses identified a class of transcripts called long noncoding RNA (lncRNA), which interact both directly and indirectly with key components of gene regulatory networks to alter cell proliferation, invasion, and metastasis. We identified a pro-metastatic lncRNA BORG whose aberrant expression promotes metastatic relapse by reactivating proliferative programs in dormant disseminated tumor cells (DTCs). BORG expression is broadly and strongly induced by environmental and chemotherapeutic stresses, a transcriptional response that facilitates the survival of DTCs. Transcriptomic reprogramming in response to BORG resulted in robust signaling via survival and viability pathways, as well as decreased activation of cell death pathways. As such, BORG expression acts as a (i) marker capable of predicting which breast cancer patients are predisposed to develop secondary metastatic lesions, and (ii) unique therapeutic target to maximize chemosensitivity of DTCs. Here we review the molecular and cellular factors that contribute to the pathophysiological activities of BORG during its regulation of breast cancer metastasis, chemoresistance, and disease recurrence.