Small Number Of Transcription Factors Research Articles

Dissecting the regulatory logic of specification and differentiation during vertebrate embryogenesis.

The interplay between transcription factors and chromatin accessibility regulates cell type diversification during vertebrate embryogenesis. To systematically decipher the gene regulatory logic guiding this process, we generated a single-cell multi-omics atlas of RNA expression and chromatin accessibility during early zebrafish embryogenesis. We developed a deep learning model to predict chromatin accessibility based on DNA sequence and found that a small number of transcription factors underlie cell-type-specific chromatin landscapes. While Nanog is well-established in promoting pluripotency, we discovered a new function in priming the enhancer accessibility of mesendodermal genes. In addition to the classical stepwise mode of differentiation, we describe instant differentiation, where pluripotent cells skip intermediate fate transitions and terminally differentiate. Reconstruction of gene regulatory interactions reveals that this process is driven by a shallow network in which maternally deposited regulators activate a small set of transcription factors that co-regulate hundreds of differentiation genes. Notably, misexpression of these transcription factors in pluripotent cells is sufficient to ectopically activate their targets. This study provides a rich resource for analyzing embryonic gene regulation and reveals the regulatory logic of instant differentiation.

Identification of mammalian transcription factors that bind to inaccessible chromatin.

Transcription factors (TFs) are proteins that affect gene expression by binding to regulatory regions of DNA in a sequence specific manner. The binding of TFs to DNA is controlled by many factors, including the DNA sequence, concentration of TF, chromatin accessibility and co-factors. Here, we systematically investigated the binding mechanism of hundreds of TFs by analysing ChIP-seq data with our explainable statistical model, ChIPanalyser. This tool uses as inputs the DNA sequence binding motif; the capacity to distinguish between strong and weak binding sites; the concentration of TF; and chromatin accessibility. We found that approximately one third of TFs are predicted to bind the genome in a DNA accessibility independent fashion, which includes TFs that can open the chromatin, their co-factors and TFs with similar motifs. Our model predicted this to be the case when the TF binds to its strongest binding regions in the genome, and only a small number of TFs have the capacity to bind dense chromatin at their weakest binding regions, such as CTCF, USF2 and CEBPB. Our study demonstrated that the binding of hundreds of human and mouse TFs is predicted by ChIPanalyser with high accuracy and showed that many TFs can bind dense chromatin.

Characterization of the TBR1 interactome: variants associated with neurodevelopmental disorders disrupt novel protein interactions.

TBR1 is a neuron-specific transcription factor involved in brain development and implicated in a neurodevelopmental disorder (NDD) combining features of autism spectrum disorder (ASD), intellectual disability (ID) and speech delay. TBR1 has been previously shown to interact with a small number of transcription factors and co-factors also involved in NDDs (including CASK, FOXP1/2/4 and BCL11A), suggesting that the wider TBR1 interactome may have a significant bearing on normal and abnormal brain development. Here, we have identified approximately 250 putative TBR1-interaction partners by affinity purification coupled to mass spectrometry. As well as known TBR1-interactors such as CASK, the identified partners include transcription factors and chromatin modifiers, along with ASD- and ID-related proteins. Five interaction candidates were independently validated using bioluminescence resonance energy transfer assays. We went on to test the interaction of these candidates with TBR1 protein variants implicated in cases of NDD. The assays uncovered disturbed interactions for NDD-associated variants and identified two distinct protein-binding domains of TBR1 that have essential roles in protein-protein interaction.

DNA methylation in human gastric epithelial cells defines regional identity without restricting lineage plasticity

BackgroundEpigenetic modifications in mammalian DNA are commonly manifested by DNA methylation. In the stomach, altered DNA methylation patterns have been observed following chronic Helicobacter pylori infections and in gastric cancer. In the context of epigenetic regulation, the regional nature of the stomach has been rarely considered in detail.ResultsHere, we establish gastric mucosa derived primary cell cultures as a reliable source of native human epithelium. We describe the DNA methylation landscape across the phenotypically different regions of the healthy human stomach, i.e., antrum, corpus, fundus together with the corresponding transcriptomes. We show that stable regional DNA methylation differences translate to a limited extent into regulation of the transcriptomic phenotype, indicating a largely permissive epigenetic regulation. We identify a small number of transcription factors with novel region-specific activity and likely epigenetic impact in the stomach, including GATA4, IRX5, IRX2, PDX1 and CDX2. Detailed analysis of the Wnt pathway reveals differential regulation along the craniocaudal axis, which involves non-canonical Wnt signaling in determining cell fate in the proximal stomach. By extending our analysis to pre-neoplastic lesions and gastric cancers, we conclude that epigenetic dysregulation characterizes intestinal metaplasia as a founding basis for functional changes in gastric cancer. We present insights into the dynamics of DNA methylation across anatomical regions of the healthy stomach and patterns of its change in disease. Finally, our study provides a well-defined resource of regional stomach transcription and epigenetics.

Transcription Factors as Novel Therapeutic Targets and Drivers of Prostate Cancer Progression.

Prostate cancer is the second most diagnosed cancer among men worldwide. Androgen deprivation therapy, the most common targeted therapeutic option, is circumvented as prostate cancer progresses from androgen dependent to castrate-resistant disease. Whilst the nuclear receptor transcription factor, androgen receptor, drives the growth of prostate tumor during initial stage of the disease, androgen resistance is associated with poorly differentiated prostate cancer. In the recent years, increased research has highlighted the aberrant transcriptional activities of a small number of transcription factors. Along with androgen receptors, dysregulation of these transcription factors contributes to both the poorly differentiated phenotypes of prostate cancer cells and the initiation and progression of prostate carcinoma. As master regulators of cell fate decisions, these transcription factors may provide opportunity for the development of novel therapeutic targets for the management of prostate cancer. Whilst some transcriptional regulators have previously been notoriously difficult to directly target, technological advances offer potential for the indirect therapeutic targeting of these transcription factors and the capacity to reprogram cancer cell phenotype. This mini review will discuss how recent advances in our understanding of transcriptional regulators and material science pave the way to utilize these regulatory molecules as therapeutic targets in prostate cancer.

Compensatory growth renders Tcf7l1a dispensable for eye formation despite its requirement in eye field specification.

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.

Abstract 2062: Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry

Abstract Childhood neuroblastomas with MYCN gene amplification form a particularly high-risk subset of this disease and are difficult to treat effectively. This has focused attention on tumor-specific gene dependencies that reflect important pathways in tumorigenesis, and thus could provide valuable targets for the development of novel therapeutics. Using genome-scale CRISPR-Cas9 approaches that allow unbiased detection of genes critically involved in tumor cell growth and survival, we identified 147 candidate genes associated with selective vulnerabilities in nine MYCN-amplified neuroblastoma cell lines, compared to findings in over 300 other human cancer cell lines representing multiple tumor cell types. We then used genome-wide ChIP-seq analysis to test the hypothesis that a small number of transcription factors - MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2, all represented in the selective dependency group - are members of the transcriptional core regulatory circuitry (CRC) that underlies cell state in MYCN-amplified neuroblastoma. We show that these transcription factors bind as dense clusters at defined epicentres within the enhancers of their own genes, as well as those of the other CRC transcription factor genes, creating a positive feed-forward autoregulatory loop that establishes and maintains high levels of gene expression. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which we postulated would act synergistically by targeting both transcriptional initiation and elongation required to synthesize regulatory transcription factors. MYCN-amplified neuroblastoma cells treated with both drugs were killed synergistically, in vitro and in vivo, and accompanied by rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma, a subset of which comprises the oncogenic transcriptional regulatory circuitry that underlies cell state and survival in this tumor. Citation Format: Adam D. Durbin, Mark W. Zimmerman, Neekesh V. Dharia, Brian J. Abraham, Brian J. Abraham, Amanda Balboni-Iniguez, Nina Weichert-Leahey, Shuning He, John M. Krill-Burger, David E. Root, Francisca Vazquez, Aviad Tsherniak, William C. Hahn, Todd R. Golub, Richard A. Young, A. Thomas Look, Kimberly Stegmaier. Selective gene dependencies in MYCN-amplified neuroblastoma include the core transcriptional regulatory circuitry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2062.

Phenotypic heterogeneity: a bacterial virulence strategy

Growing knowledge of the complexity of the host-pathogen interactions during the course of an infection revealed an amazing variability of bacterial pathogens within the same host tissue site. This heterogeneity in bacterial populations is either the result of a different bacterial response to a slightly divergent tissue microenvironment or is caused by a genetic circuit in which small endogenous fluctuations in a small number of transcription factors drive gene expression in combination with a positive feedback loop. As a result host-pathogen encounters can have different outcomes in individual cells, which enables bet-hedging and/or a co-operative behavior that enhance bacterial fitness and virulence, drive different host responses and promote resistance of small subpopulations to antibiotic treatment. This has a strong impact on the progression and control of the infection, which must be considered for the development of successful antimicrobial therapies.

DMRT5 Together with DMRT3 Directly Controls Hippocampus Development and Neocortical Area Map Formation.

Mice that are constitutively null for the zinc finger doublesex and mab-3 related (Dmrt) gene, Dmrt5/Dmrta2, show a variety of patterning abnormalities in the cerebral cortex, including the loss of the cortical hem, a powerful cortical signaling center. In conditional Dmrt5 gain of function and loss of function mouse models, we generated bidirectional changes in the neocortical area map without affecting the hem. Analysis indicated that DMRT5, independent of the hem, directs the rostral-to-caudal pattern of the neocortical area map. Thus, DMRT5 joins a small number of transcription factors shown to control directly area size and position in the neocortex. Dmrt5 deletion after hem formation also reduced hippocampal size and shifted the position of the neocortical/paleocortical boundary. Dmrt3, like Dmrt5, is expressed in a gradient across the cortical primordium. Mice lacking Dmrt3 show cortical patterning defects akin to but milder than those in Dmrt5 mutants, perhaps in part because Dmrt5 expression increases in the absence of Dmrt3. DMRT5 upregulates Dmrt3 expression and negatively regulates its own expression, which may stabilize the level of DMRT5. Together, our findings indicate that finely tuned levels of DMRT5, together with DMRT3, regulate patterning of the cerebral cortex.

Prenatal ethanol exposure in mice phenocopies Cdon mutation by impeding Shh function in the etiology of optic nerve hypoplasia.

ABSTRACTSepto-optic dysplasia (SOD) is a congenital disorder characterized by optic nerve, pituitary and midline brain malformations. The clinical presentation of SOD is highly variable with a poorly understood etiology. The majority of SOD cases are sporadic, but in rare instances inherited mutations have been identified in a small number of transcription factors, some of which regulate the expression of Sonic hedgehog (Shh) during mouse forebrain development. SOD is also associated with young maternal age, suggesting that environmental factors, including alcohol consumption at early stages of pregnancy, might increase the risk of developing this condition. Here, we address the hypothesis that SOD is a multifactorial disorder stemming from interactions between mutations in Shh pathway genes and prenatal ethanol exposure. Mouse embryos with mutations in the Shh co-receptor, Cdon, were treated in utero with ethanol or saline at embryonic day 8 (E8.0) and evaluated for optic nerve hypoplasia (ONH), a prominent feature of SOD. We show that both Cdon−/− mutation and prenatal ethanol exposure independently cause ONH through a similar pathogenic mechanism that involves selective inhibition of Shh signaling in retinal progenitor cells, resulting in their premature cell-cycle arrest, precocious differentiation and failure to properly extend axons to the optic nerve. The ONH phenotype was not exacerbated in Cdon−/− embryos treated with ethanol, suggesting that an intact Shh signaling pathway is required for ethanol to exert its teratogenic effects. These results support a model whereby mutations in Cdon and prenatal ethanol exposure increase SOD risk through spatiotemporal perturbations in Shh signaling activity.

HCFC1 is a common component of active human CpG-island promoters and coincides with ZNF143, THAP11, YY1, and GABP transcription factor occupancy

In human transcriptional regulation, DNA-sequence-specific factors can associate with intermediaries that orchestrate interactions with a diverse set of chromatin-modifying enzymes. One such intermediary is HCFC1 (also known as HCF-1). HCFC1, first identified in herpes simplex virus transcription, has a poorly defined role in cellular transcriptional regulation. We show here that, in HeLa cells, HCFC1 is observed bound to 5400 generally active CpG-island promoters. Examination of the DNA sequences underlying the HCFC1-binding sites revealed three sequence motifs associated with the binding of (1) ZNF143 and THAP11 (also known as Ronin), (2) GABP, and (3) YY1 sequence-specific transcription factors. Subsequent analysis revealed colocalization of HCFC1 with these four transcription factors at ∼90% of the 5400 HCFC1-bound promoters. These studies suggest that a relatively small number of transcription factors play a major role in HeLa-cell transcriptional regulation in association with HCFC1.

Stemness-related Factor Sall4 Interacts with Transcription Factors Oct-3/4 and Sox2 and Occupies Oct-Sox Elements in Mouse Embryonic Stem Cells

A small number of transcription factors, including Oct-3/4 and Sox2, constitute the transcriptional network that maintains pluripotency in embryonic stem (ES) cells. Previous reports suggested that some of these factors form a complex that binds the Oct-Sox element, a composite sequence consisting of closely juxtaposed Oct-3/4 binding and Sox2 binding sites. However, little is known regarding the components of the complex. In this study we show that Sall4, a member of the Spalt-like family of proteins, directly interacts with Sox2 and Oct-3/4. Sall4 in combination with Sox2 or Oct-3/4 simultaneously occupies the Oct-Sox elements in mouse ES cells. Overexpression of Sall4 in ES cells increased reporter activities in a luciferase assay when the Pou5f1- or Nanog-derived Oct-Sox element was included in the reporter. Microarray analyses revealed that Sall4 and Sox2 bound to the same genes in ES cells significantly more frequently than expected from random coincidence. These factors appeared to bind the promoter regions of a subset of the Sall4 and Sox2 double-positive genes in precisely similar distribution patterns along the promoter regions, suggesting that Sall4 and Sox2 associate with such Sall4/Sox2-overlapping genes as a complex. Importantly, gene ontology analyses indicated that the Sall4/Sox2-overlapping gene set is enriched for genes involved in maintaining pluripotency. Sall4/Sox2/Oct-3/4 triple-positive genes identified by referring to a previous study identifying Oct-3/4-bound genes in ES cells were further enriched for pluripotency genes than Sall4/Sox2 double-positive genes. These results demonstrate that Sall4 contributes to the transcriptional network operating in pluripotent cells together with Oct-3/4 and Sox2.

Spatial expression of transcription factors in Drosophilaembryonic organ development

BackgroundSite-specific transcription factors (TFs) bind DNA regulatory elements to control expression of target genes, forming the core of gene regulatory networks. Despite decades of research, most studies focus on only a small number of TFs and the roles of many remain unknown.ResultsWe present a systematic characterization of spatiotemporal gene expression patterns for all known or predicted Drosophila TFs throughout embryogenesis, the first such comprehensive study for any metazoan animal. We generated RNA expression patterns for all 708 TFs by in situ hybridization, annotated the patterns using an anatomical controlled vocabulary, and analyzed TF expression in the context of organ system development. Nearly all TFs are expressed during embryogenesis and more than half are specifically expressed in the central nervous system. Compared to other genes, TFs are enriched early in the development of most organ systems, and throughout the development of the nervous system. Of the 535 TFs with spatially restricted expression, 79% are dynamically expressed in multiple organ systems while 21% show single-organ specificity. Of those expressed in multiple organ systems, 77 TFs are restricted to a single organ system either early or late in development. Expression patterns for 354 TFs are characterized for the first time in this study.ConclusionsWe produced a reference TF dataset for the investigation of gene regulatory networks in embryogenesis, and gained insight into the expression dynamics of the full complement of TFs controlling the development of each organ system.

Stem cells in the context of evolution and development

Stem cells in the context of evolution and development

Both PAX6 and MITF are required for pigment epithelium development in vivo

Both PAX6 and MITF are required for pigment epithelium development <i>in vivo</i>

Stochastic simulation for the inference of transcriptional control network of yeast cyclins genes

Cell cycle is controlled by the activity of protein family of cyclins and cyclin-dependent kinases that are periodically expressed during cell cycle and that are conserved among different species. Genome-wide location analysis found that cyclins are controlled by a small number of transcription factors that form closed network of genes controlling each other. To investigate gene expression dynamics of this network, we developed a general procedure for stochastic simulation of gene expression process. Using the binding data, we simulated gene expression of all genes of the network for all possible combinations of regulatory interactions and by statistical comparison with experimentally measured time series excluded those interactions that formed gene expression temporal profiles significantly different from the measured ones. These experiments led to a new definition of the cyclins regulatory network coherent with the binding experiments which are kinetically plausible. Level of influence of individual regulators in control of the regulated genes is defined. Simulation results indicate particular mechanism of regulatory activity of protein complexes involved in the control of cyclins.

Combinatorial Activation and Repression by Seven Transcription Factors Specify Drosophila Odorant Receptor Expression

Author SummaryOur nervous system has a daunting diversity; it contains 100 billion neurons that all have defined functions and connections. To address how neuronal diversity is produced, we have turned to a complex but defined set of neurons, the Drosophila olfactory system located in the antenna. This system contains 34 neuron classes with different functions and connections, each defined by the unique odorant receptor they express. We set out to identify the transcription factors (regulatory genes) that are required for each class to express the correct odorant receptor. We find that seven transcription factors are continuously required in different combinations for the expression of all tested 32 odorant receptors. We also show that these transcription factors can both turn on and turn off odorant receptor genes, making the expression regulation more economical. We conclude that dual use of a small set of factors, which are always on in the neuron, can define its functional class and thereby produce diversity in the nervous system.

Identification and Functional Analysis of CT069 as a Novel Transcriptional Regulator in Chlamydia

Only a small number of transcription factors have been predicted in Chlamydia spp., which are obligate intracellular bacteria that include a number of important human pathogens. We used a bioinformatics strategy to identify novel transcriptional regulators from the Chlamydia trachomatis genome by predicting proteins with the general structure and characteristic functional domains of a bacterial transcription factor. With this approach, we identified CT069 as a candidate transcription factor with sequence similarity at its C terminus to Treponema pallidum TroR. Like TroR, the gene for CT069 belongs to an operon that encodes components of a putative ABC transporter for importing divalent metal cations. However, CT069 has been annotated as YtgC because of sequence similarity at its N terminus to TroC, a transmembrane component of this metal ion transporter. Instead, CT069 appears to be a fusion protein composed of YtgC and a TroR ortholog that we have called YtgR. Although it has not been previously reported, a similar YtgC-YtgR fusion protein is predicted to be encoded by other Chlamydia spp. and several other bacteria, including Bacillus subtilis. We show that recombinant YtgR polypeptide bound specifically to an operator sequence upstream of the ytg operon and that binding was enhanced by Zn(2+). We also demonstrate that YtgR repressed transcription from the ytg promoter in a heterologous in vivo reporter assay. These results provide evidence that CT069 is a negative regulator of the ytg operon, which encodes a putative metal ion transporter in C. trachomatis.

The Arabidopsis RWP-RK Protein RKD4 Triggers Gene Expression and Pattern Formation in Early Embryogenesis

Morphogenesis of seed plants commences with highly stereotypical cell division sequences in early embryogenesis [1, 2]. Although a small number of transcription factors and a mitogen-activated protein (MAP) kinase cascade have been implicated in this process [3-8], pattern formation in early embryogenesis remains poorly understood. We show here that the Arabidopsis RKD4, a member of the RWP-RK motif-containing putative transcription factors [9], is required for this process. Loss-of-function rkd4 mutants were defective in zygotic cell elongation, as well as subsequent cell division patterns. As expected from this mutant phenotype, RKD4 was transcribed preferentially in early embryos. RKD4 possessed functional characteristics of transcription factors and was able to ectopically induce early embryo-specific genes when overexpressed in seedlings. Strikingly, induced overexpression of RKD4 primed somatic cells for embryogenesis independently of external growth regulators. These results reveal that RKD4 is a novel key regulator of the earliest stage of plant development.

Dissecting the expression patterns of transcription factors across conditions using an integrated network-based approach

In prokaryotes, regulation of gene expression is predominantly controlled at the level of transcription. Transcription in turn is mediated by a set of DNA-binding factors called transcription factors (TFs). In this study, we map the complete repertoire of ∼300 TFs of the bacterial model, Escherichia coli, onto gene expression data for a number of nonredundant experimental conditions and show that TFs are generally expressed at a lower level than other gene classes. We also demonstrate that different conditions harbor varying number of active TFs, with an average of about 15% of the total repertoire, with certain stress and drug-induced conditions exhibiting as high as one-third of the collection of TFs. Our results also show that activators are more frequently expressed than repressors, indicating that activation of promoters might be a more common phenomenon than repression in bacteria. Finally, to understand the association of TFs with different conditions and to elucidate their dynamic interplay with other TFs, we develop a network-based framework to identify TFs which act as markers, defined as those which are responsible for condition-specific transcriptional rewiring. This approach allowed us to pinpoint several marker TFs as being central in various specialized conditions such as drug induction or growth condition variations, which we discuss in light of previously reported experimental findings. Further analysis showed that a majority of identified markers effectively control the expression of their regulons and, in general, transcriptional programs of most conditions can be effectively rewired by a very small number of TFs. It was also found that closeness is a key centrality measure which can aid in the successful identification of marker TFs in regulatory networks. Our results suggest the utility of the network-based approaches developed in this study to be applicable for understanding other interactomic data sets.