Transcriptional Feedback Research Articles

CIRCADIAN CLOCK ASSOCIATED 1 and ATAF2 differentially suppress cytochrome P450-mediated brassinosteroid inactivation.

Brassinosteroids (BRs) are a group of steroid hormones regulating plant growth and development. Since BRs do not undergo transport among plant tissues, their metabolism is tightly regulated by transcription factors (TFs) and feedback loops. BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1) are two BR-inactivating cytochrome P450s identified in Arabidopsis thaliana. We previously found that a TF ATAF2 (ANAC081) suppresses BAS1 and SOB7 expression by binding to the Evening Element (EE) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-binding site (CBS) on their promoters. Both the EE and CBS are known binding targets of the circadian regulatory protein CCA1. Here, we confirm that CCA1 binds the EE and CBS motifs on BAS1 and SOB7 promoters, respectively. Elevated accumulations of BAS1 and SOB7 transcripts in the CCA1 null mutant cca1-1 indicate that CCA1 is a repressor of their expression. When compared with either cca1-1 or the ATAF2 null mutant ataf2-2, the cca1-1 ataf2-2 double mutant shows higher SOB7 transcript accumulations and a stronger BR-insensitive phenotype of hypocotyl elongation in white light. CCA1 interacts with ATAF2 at both DNA-protein and protein-protein levels. ATAF2, BAS1, and SOB7 are all circadian regulated with distinct expression patterns. These results demonstrate that CCA1 and ATAF2 differentially suppress BAS1- and SOB7-mediated BR inactivation.

In Vitro Transcriptional Regulation via Nucleic-Acid-Based Transcription Factors.

Cells execute complex transcriptional programs by deploying distinct protein regulatory assemblies that interact with cis-regulatory elements throughout the genome. Using concepts from DNA nanotechnology, we synthetically recapitulated this feature in in vitro gene networks actuated by T7 RNA polymerase (RNAP). Our approach involves engineering nucleic acid hybridization interactions between a T7 RNAP site-specifically functionalized with single-stranded DNA (ssDNA), templates displaying cis-regulatory ssDNA domains, and auxiliary nucleic acid assemblies acting as artificial transcription factors (TFs). By relying on nucleic acid hybridization, de novo regulatory assemblies can be computationally designed to emulate features of protein-based TFs, such as cooperativity and combinatorial binding, while offering unique advantages such as programmability, chemical stability, and scalability. We illustrate the use of nucleic acid TFs to implement transcriptional logic, cascading, feedback, and multiplexing. This framework will enable rapid prototyping of increasingly complex in vitro genetic devices for applications such as portable diagnostics, bioanalysis, and the design of adaptive materials.

An impaired intrinsic microglial clock system induces neuroinflammatory alterations in the early stage of amyloid precursor protein knock-in mouse brain

BackgroundDisturbances in clock genes affect almost all patients with Alzheimer’s disease (AD), as evidenced by their altered sleep/wake cycle, thermoregulation, and exacerbation of cognitive impairment. As microglia-mediated neuroinflammation proved to be a driver of AD rather than a result of the disease, in this study, we evaluated the relationship between clock gene disturbance and neuroinflammation in microglia and their contribution to the onset of AD.MethodsIn this study, the expression of clock genes and inflammatory-related genes was examined in MACS microglia isolated from 2-month-old amyloid precursor protein knock-in (APP-KI) and wild-type (WT) mice using cap analysis gene expression (CAGE) deep sequencing and RT-PCR. The effects of clock gene disturbance on neuroinflammation and relevant memory changes were examined in 2-month-old APP-KI and WT mice after injection with SR9009 (a synthetic agonist for REV-ERB). The microglia morphology was studied by staining, neuroinflammation was examined by Western blotting, and cognitive changes were examined by Y-maze and novel object recognition tests.ResultsCLOCK/BMAL1-driven transcriptional negative feedback loops were impaired in the microglia from 2-month-old APP-KI mice. Pro-inflammatory genes in microglia isolated from APP-KI mice were significantly higher than those isolated from WT mice at Zeitgeber time 14. The expression of pro-inflammatory genes was positively associated with NF-κB activation and negatively associated with the BMAL1 expression. SR9009 induced the activation of microglia, the increased expression of pro-inflammatory genes, and cognitive decline in 2-month-old APP-KI mice.ConclusionClock gene disturbance in microglia is involved in the early onset of AD through the induction of chronic neuroinflammation, which may be a new target for preventing or slowing AD.

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence stimulating peptide (CSP) induces bimodal expression of comX, with only a fraction of the population becoming transformable. Curiously, the bimodality of comX is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but CSP elicits no response in defined media lacking small peptides. In addition, growth on certain sugars increases the proportion of the population that activates comX in response to CSP. By investigating the connection between media and comX bimodality, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, where comX bimodality originates. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the XIP/ComS feedback signal. Deletion of pepO eliminates comX bimodality, leading to a unimodal comX response to CSP in both defined and complex media. We also find that CSP stimulates the ComRS feedback system by upregulating comR in a carbohydrate source-dependent fashion. Our data provide mechanistic insight into how S. mutans regulates bimodality and explain the puzzle of growth medium effects on competence induction by CSP.

CRISPRi-Based Downregulation of Transcriptional Feedback Improves Growth and Metabolism of Arginine Overproducing E. coli.

Removing transcriptional feedback regulation of metabolic pathways is a classical approach to enhance overproduction of chemicals in microbes. However, disrupting transcriptional regulation can have broad physiological consequences that decrease cellular growth and productivity. Here, we compared downregulation and deletion of the transcriptional repressor ArgR in arginine overproducing Escherichia coli. Different levels of ArgR downregulation were achieved with CRISPR interference (CRISPRi) and resulted in 2-times higher growth rates compared to deletion of ArgR, while specific arginine production was similar (∼2 mmol gDW-1 h-1). Metabolomics and proteomics data revealed that poor growth of the ArgR deletion strain was caused by a limitation of pyrimidine nucleotide biosynthesis, because a 17-fold overexpression of ornithine carbamoyltransferase (ArgI) perturbed the arginine-pyrimidine branch point. These results demonstrate that overexpression of enzymes in an engineered pathway can impair metabolism of the host, especially in the case of branch-point enzymes. Thus, balancing enzyme levels is important to optimize industrial microbes, and CRISPRi of a transcription factor is a versatile tool for this purpose.

Autokinase Activity of Casein Kinase 1 δ/ε Governs the Period of Mammalian Circadian Rhythms.

Circadian rhythms exist in nearly all organisms. In mammals, transcriptional and translational feedback loops (TTFLs) are believed to underlie the mechanism of the circadian clock. Casein kinase 1δ/ε (CK1δ/ε) are key kinases that phosphorylate clock components such as PER proteins, determining the pace of the clock. Most previous studies of the biochemical properties of the key kinases CK1ε and CK1δ in vitro have focused on the properties of the catalytic domains from which the autoinhibitory C-terminus has been deleted (ΔC); those studies ignored the significance of self-inhibition by autophosphorylation. By comparing the properties of the catalytic domain of CK1δ/ε with the full-length kinase that can undergo autoinhibition, we found that recombinant full-length CK1 showed a sequential autophosphorylation process that induces conformational changes to affect the overall kinase activity. Furthermore, a direct relationship between the period change and the autokinase activity among CK1δ, CK1ε, and CK1ε-R178C was observed. These data implicate the autophosphorylation activity of CK1δ and CK1ε kinases in setting the pace of mammalian circadian rhythms and indicate that the circadian period can be modulated by tuning the autophosphorylation rates of CK1δ/ε.

Abstract 1407: Histone marks interplay at LIFR enhancer determines the response to HDAC inhibitors in breast cancer

Abstract The dynamic crosstalk of epigenetic modifications is believed to impose a strong impact on epigenetically-targeted therapies, such as histone deacetylase (HDAC) inhibitors, but current understanding in this regard is extremely limited.Our previous work has revealed that the transcriptional feedback activation of leukemia inhibitory factor receptor (LIFR) limits the response to HDAC inhibitors in breast cancer. However, LIFR transcriptional response appears highly heterogeneous in breast cancer and the reason behind is unclear. This study takes LIFR as a representative to dissect how histone marks interplay determines the transcriptional outcome of epigenetically-targeted therapies. We herein discover that the H3K27 methylation specifically at LIFR enhancer restricts the H3K27 acetylation upon HDAC inhibition and prevents the upregulation of LIFR. The intrinsic level of H3K27 methylation at LIFR enhancer stratifies the responsive subset to HDAC-targeted therapies in a panel of patient-derived breast cancer models. This study provides strong evidence showing how existing histone marks and their specific localization together determine the transcriptional output and the outcome of epigenetically-targeted therapies, highlighting the significance of histone marks crosstalk in epigenetic therapeutics. Note: This abstract was not presented at the meeting. Citation Format: Xiaomin Wang, Jun Xu, Jian Ding, Meiyu Geng, Min Huang. Histone marks interplay at LIFR enhancer determines the response to HDAC inhibitors in breast cancer [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 1407.

Nuclear Envelope Protein MAN1 Regulates the Drosophila Circadian Clock via Period.

Almost all organisms exhibit ~24-h rhythms, or circadian rhythms, in a plentitude of biological processes. These rhythms are driven by endogenous molecular clocks consisting of a series of transcriptional and translational feedback loops. Previously, we have shown that the inner nuclear membrane protein MAN1 regulates this clock and thus the locomotor rhythm in flies, but the mechanism remains unclear. Here, we further confirmed the previous findings and found that knocking down MAN1 in the pacemaker neurons of adult flies is sufficient to lengthen the period of the locomotor rhythm. Molecular analysis revealed that knocking down MAN1 led to reduced mRNA and protein levels of the core clock gene period (per), likely by reducing its transcription. Over-expressing per rescued the long period phenotype caused by MAN1 deficiency whereas per mutation had an epistatic effect on MAN1, indicating that MAN1 sets the pace of the clock by targeting per.

Aging renders desynchronization between clock and immune genes in male Wistar rat kidney: chronobiotic role of curcumin.

Suprachiasmatic nucleus (SCN) contains the central clock that orchestrate circadian rhythms in physiology and behavior in mammals. Tightly interlocked transcriptional and translational feedback loops (TTFLs) comprising of various clock genes such as Clock, Bmal1, Periods, Cryptochromes etc. in the SCN, send the timing signals to peripheral clocks that governs local metabolism with similar TTFLs. Peripheral clocks in kidney regulates several circadian rhythms like blood pressure, immunity etc. However, aging leads to circadian and inflammatory disorders in kidney. Though there are increasing evidences on age associated perturbations, studies elucidating the rhythmic expression of clock and immune genes across aging in kidney are obscure. We therefore studied changes in daily rhythms of clock and immune genes in kidney. In this study we measured mRNA expression of clock genes rBmal1, rPer1, rPer2, rCry1, rCry2, rRev-erbα, rRorα, and inflammatory genes rNfκb1, rTnfα, rIl6, rTlr4 and rTlr9 in 3, 12 and 24months male Wistar rat kidney using qRT-PCR. From our study, we did not observe significant changes in clock genes expression except rRorα, but immune genes showed significant phase alterations as well as increase in mean 24h levels. Pearson correlation analysis of data showed desynchronization between immune and clock genes expression. We further studied the effect of administration of curcumin which has anti-aging, anti-inflammatory, anti-oxidant etc. properties, and evaluated its chronobiotic properties. We here report differential effects of curcumin administration on daily rhythms of clock and immune genes expression.

Mutant huntingtin disturbs circadian clock gene expression and sleep patterns in Drosophila

Deficiency of the sleep-wake cycle can accelerate the progression of Huntington’s disease (HD) and exacerbate symptoms making it a target of investigation to better understand the molecular pathology of the disorder. In this study we analyzed sleep defects in a Drosophila model of HD and investigated whether disturbed sleep coincides with alterations in the molecular mechanism controlling circadian rhythm. To analyze sleep defects we recorded the daily activity of flies in 12:12 hours light:dark entrainment and in regard to the underlying molecular mechanism measured circadian “clock” gene expression. In HD flies we observed reduced amount of sleep, sleep fragmentation and prolonged sleep latency. We found changes in gene expression patterns of both transcriptional feedback loops of circadian regulation. We detected prolonged expression of the core feedback loop components period and timeless, whilst the secondary feedback loop member vrille had lower expression rates in general. Our results show that the Drosophila HD model recapitulates most of the sleep related symptoms reported in patients therefore it can be a potential tool to study the molecular background of sleep defects in HD. Altered expression of circadian “clock” genes suggests that disturbed sleep pattern in HD might be the consequence of disturbed circadian regulation.

A period without PER: understanding 24-hour rhythms without classic transcription and translation feedback loops.

Since Ronald Konopka and Seymour Benzer’s discovery of the gene Period in the 1970s, the circadian rhythm field has diligently investigated regulatory mechanisms and intracellular transcriptional and translation feedback loops involving Period, and these investigations culminated in a 2017 Nobel Prize in Physiology or Medicine for Michael W. Young, Michael Rosbash, and Jeffrey C. Hall. Although research on 24-hour behavior rhythms started with Period, a series of discoveries in the past decade have shown us that post-transcriptional regulation and protein modification, such as phosphorylation and oxidation, are alternatives ways to building a ticking clock.

Accuracy of parameter estimation for auto-regulatory transcriptional feedback loops from noisy data.

Bayesian and non-Bayesian moment-based inference methods are commonly used to estimate the parameters defining stochastic models of gene regulatory networks from noisy single cell or population snapshot data. However, a systematic investigation of the accuracy of the predictions of these methods remains missing. Here, we present the results of such a study using synthetic noisy data of a negative auto-regulatory transcriptional feedback loop, one of the most common building blocks of complex gene regulatory networks. We study the error in parameter estimation as a function of (i) number of cells in each sample; (ii) the number of time points; (iii) the highest-order moment of protein fluctuations used for inference; (iv) the moment-closure method used for likelihood approximation. We find that for sample sizes typical of flow cytometry experiments, parameter estimation by maximizing the likelihood is as accurate as using Bayesian methods but with a much reduced computational time. We also show that the choice of moment-closure method is the crucial factor determining the maximum achievable accuracy of moment-based inference methods. Common likelihood approximation methods based on the linear noise approximation or the zero cumulants closure perform poorly for feedback loops with large protein–DNA binding rates or large protein bursts; this is exacerbated for highly heterogeneous cell populations. By contrast, approximating the likelihood using the linear-mapping approximation or conditional derivative matching leads to highly accurate parameter estimates for a wide range of conditions.

SLC1A5 glutamine transporter is a target of MYC and mediates reduced mTORC1 signaling and increased fatty acid oxidation in long-lived Myc hypomorphic mice.

Mice that express reduced levels of the c‐Myc gene (Myc +/− heterozygotes) are long‐lived. Myc hypomorphic mice display reduced rates of protein translation and decreased activity of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Given the prominent effect of mTOR on aging, lower mTORC1 activity could contribute to the exceptional longevity and enhanced healthspan of Myc +/− animals. However, given the downstream position of MYC in these signaling cascades, the mechanism through which mTORC1 activity is downregulated in Myc +/− mice is not understood. We report that the high‐affinity glutamine transporter SLC1A5, which is critical for activation of mTORC1 activity by amino acids, is a transcriptional target of MYC. Myc +/− cells display decreased Slc1a5 gene expression that leads to lower glutamine uptake and consequently reduced mTORC1 activity. Decreased mTORC1 activity in turn mediates an elevation of fatty acid oxidation (FAO) by indirectly upregulating the expression of carnitine palmitoyltransferase 1a (Cpt1a) that mediates the rate‐limiting step of β‐oxidation. Increased FAO has been noted in a number of long‐lived mouse models. Taken together, our results show that transcriptional feedback loops regulated by MYC modulate upstream signaling pathways such as mTOR and impact FAO on an organismal level.

Inflammatory regulatory network mediated by the joint action of NF-kB, STAT3, and AP-1 factors is involved in many human cancers

Using an inducible, inflammatory model of breast cellular transformation, we describe the transcriptional regulatory network mediated by STAT3, NF-κB, and AP-1 factors on a genomic scale. These proinflammatory regulators form transcriptional complexes that directly regulate the expression of hundreds of genes in oncogenic pathways via a positive feedback loop. This transcriptional feedback loop and associated network functions to various extents in many types of cancer cells and patient tumors, and it is the basis for a cancer inflammation index that defines cancer types by functional criteria. We identify a network of noninflammatory genes whose expression is well correlated with the cancer inflammatory index. Conversely, the cancer inflammation index is negatively correlated with the expression of genes involved in DNA metabolism, and transformation is associated with genome instability. We identify drugs whose efficacy in cell lines is correlated with the cancer inflammation index, suggesting the possibility of using this index for personalized cancer therapy. Inflammatory tumors are preferentially associated with infiltrating immune cells that might be recruited to the site of the tumor via inflammatory molecules produced by the cancer cells.

The Plant Circadian Oscillator.

It has been nearly 300 years since the first scientific demonstration of a self-sustaining circadian clock in plants. It has become clear that plants are richly rhythmic, and many aspects of plant biology, including photosynthetic light harvesting and carbon assimilation, resistance to abiotic stresses, pathogens, and pests, photoperiodic flower induction, petal movement, and floral fragrance emission, exhibit circadian rhythmicity in one or more plant species. Much experimental effort, primarily, but not exclusively in Arabidopsis thaliana, has been expended to characterize and understand the plant circadian oscillator, which has been revealed to be a highly complex network of interlocked transcriptional feedback loops. In addition, the plant circadian oscillator has employed a panoply of post-transcriptional regulatory mechanisms, including alternative splicing, adjustable rates of translation, and regulated protein activity and stability. This review focuses on our present understanding of the regulatory network that comprises the plant circadian oscillator. The complexity of this oscillatory network facilitates the maintenance of robust rhythmicity in response to environmental extremes and permits nuanced control of multiple clock outputs. Consistent with this view, the clock is emerging as a target of domestication and presents multiple targets for targeted breeding to improve crop performance.

Myeloid Folliculin balances mTOR activation to maintain innate immunity homeostasis.

The mTOR pathway is central to most cells. How mTOR is activated in macrophages and modulates macrophage physiology remain poorly understood. The tumor suppressor Folliculin (FLCN) is a GAP for RagC/D, a regulator of mTOR. We show here that LPS potently suppresses FLCN in macrophages, allowing nuclear translocation of the transcription factor TFE3, leading to lysosome biogenesis, cytokine production, and hypersensitivity to inflammatory signals. Nuclear TFE3 additionally activates a transcriptional RagD positive feedback loop that stimulates FLCN-independent canonical mTOR signaling to S6K and increases cellular proliferation. LPS thus simultaneously suppresses the TFE3 arm and activates the S6K arm of mTOR. In vivo, mice lacking myeloid FLCN reveal chronic macrophage activation, leading to profound histiocytic infiltration and tissue disruption, with hallmarks of human histiocytic syndromes like Erdheim-Chester Disease. Our data thus identify a critical FLCN-mTOR-TFE3 axis in myeloid cells, modulated by LPS, that balances mTOR activation and curbs innate immune responses.

PERIOD-controlled deadenylation of the timeless transcript in the Drosophila circadian clock

The Drosophila circadian oscillator relies on a negative transcriptional feedback loop, in which the PERIOD (PER) and TIMELESS (TIM) proteins repress the expression of their own gene by inhibiting the activity of the CLOCK (CLK) and CYCLE (CYC) transcription factors. A series of posttranslational modifications contribute to the oscillations of the PER and TIM proteins but few posttranscriptional mechanisms have been described that affect mRNA stability. Here we report that down-regulation of the POP2 deadenylase, a key component of the CCR4-NOT deadenylation complex, alters behavioral rhythms. Down-regulating POP2 specifically increases TIM protein and tim mRNA but not tim pre-mRNA, supporting a posttranscriptional role. Indeed, reduced POP2 levels induce a lengthening of tim mRNA poly(A) tail. Surprisingly, such effects are lost in per0 mutants, supporting a PER-dependent inhibition of tim mRNA deadenylation by POP2. We report a deadenylation mechanism that controls the oscillations of a core clock gene transcript.

Principles of the animal molecular clock learned from Neurospora.

Study of Neurospora, a model system evolutionarily related to animals and sharing a circadian system having nearly identical regulatory architecture to that of animals, has advanced our understanding of all circadian rhythms. Work on the molecular bases of the Oscillator began in Neurospora before any clock genes were cloned and provided the second example of a clock gene, frq, as well as the first direct experimental proof that the core of the Oscillator was built around a transcriptional translational negative feedback loop (TTFL). Proof that FRQ was a clock component provided the basis for understanding how light resets the clock, and this in turn provided the generally accepted understanding for how light resets all animal and fungal clocks. Experiments probing the mechanism of light resetting led to the first identification of a heterodimeric transcriptional activator as the positive element in a circadian feedback loop, and to the general description of the fungal/animal clock as a single step TTFL. The common means through which DNA damage impacts the Oscillator in fungi and animals was first described in Neurospora. Lastly, the systematic study of Output was pioneered in Neurospora, providing the vocabulary and conceptual framework for understanding how Output works in all cells. This model system has contributed to the current appreciation of the role of Intrinsic Disorder in clock proteins and to the documentation of the essential roles of protein post-translational modification, as distinct from turnover, in building a circadian clock.

Sirt6 deacetylase activity regulates circadian rhythms via Per2

Circadian clock relies on a transcription and translation feedback loop (TTFL). Two transcription factors, i.e. Bmal1 and Clock, activate the transcription of Period (Per) and Cryptochrome (Cry), which inhibit their own transcription when accumulated to a critical concentration. NAD+-dependent deacylase Sirt1 deacetylates Bmal1 and Per2 to regulate circadian rhythms. Sirt6 interacts with Bmal1 to regulate clock-controlled gene (CCG) expression by local chromatin remodeling. Whether Sirt6 directly modify clock components is elusive. Here, we found that loss of Sirt6 jeopardizes circadian phase. At molecular level, Sirt6 interacts with and deacetylates Per2, thus preventing its proteasomal degradation. These data highlight an important function of Sirt6 in the direct regulation of TTFL and circadian rhythms.

Metabolic engineering for the production of l-phenylalanine in Escherichia coli.

As one of the three proteinogenic aromatic amino acids, l-phenylalanine is widely applied in the food, chemical and pharmaceutical industries, especially in production of the low-calorie sweetener aspartame. Microbial production of l-phenylalanine has become attractive as it possesses the advantages of environmental friendliness, low cost, and feedstock renewability. With the progress of metabolic engineering, systems biology and synthetic biology, production of l-phenylalanine from glucose in Escherichia coli with relatively high titer has been achieved by improving the intracellular levels of precursors, alleviating transcriptional repression and feedback inhibition of key enzymes, increasing the export of l-phenylalanine, engineering of global regulators, and overexpression of rate-limiting enzymes. In this review, successful metabolic engineering strategies for increasing l-phenylalanine accumulation from glucose in E. coli are described. In addition, perspectives for further improvement of production of l-phenylalanine are discussed.