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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Genome-Wide Transcription Factor DNA Binding Sites and Gene Regulatory Networks in Clostridium thermocellum

Clostridium thermocellum is a thermophilic bacterium recognized for its natural ability to effectively deconstruct cellulosic biomass. While there is a large body of studies on the genetic engineering of this bacterium and its physiology to-date, there is limited knowledge in the transcriptional regulation in this organism and thermophilic bacteria in general. The study herein is the first report of a large-scale application of DNA-affinity purification sequencing (DAP-seq) to transcription factors (TFs) from a bacterium. We applied DAP-seq to > 90 TFs in C. thermocellum and detected genome-wide binding sites for 11 of them. We then compiled and aligned DNA binding sequences from these TFs to deduce the primary DNA-binding sequence motifs for each TF. These binding motifs are further validated with electrophoretic mobility shift assay (EMSA) and are used to identify individual TFs’ regulatory targets in C. thermocellum . Our results led to the discovery of novel, uncharacterized TFs as well as homologues of previously studied TFs including RexA-, LexA-, and LacI-type TFs. We then used these data to reconstruct gene regulatory networks for the 11 TFs individually, which resulted in a global network encompassing the TFs with some interconnections. As gene regulation governs and constrains how bacteria behave, our findings shed light on the roles of TFs delineated by their regulons, and potentially provides a means to enable rational, advanced genetic engineering of C. thermocellum and other organisms alike toward a desired phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Transcriptional Regulation of Drought Response in Arabidopsis and Woody Plants

Within the context of global warming, long-living plants such as perennial woody species endure adverse conditions. Among all of the abiotic stresses, drought stress is one of the most detrimental stresses that inhibit plant growth and productivity. Plants have evolved multiple mechanisms to respond to drought stress, among which transcriptional regulation is one of the key mechanisms. In this review, we summarize recent progress on the regulation of drought response by transcription factor (TF) families, which include abscisic acid (ABA)-dependent ABA-responsive element/ABRE-binding factors (ABRE/ABF), WRKY, and Nuclear Factor Y families, as well as ABA-independent AP2/ERF and NAC families, in the model plant Arabidopsis. We also review what is known in woody species, particularly Populus, due to its importance and relevance in economic and ecological processes. We discuss opportunities for a deeper understanding of drought response in woody plants with the development of high-throughput omics analyses and advanced genome editing techniques.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genome-wide Transcription Factor DNA Binding Sites and Gene Regulatory Networks in Clostridium thermocellum

Clostridium thermocellum is a thermophilic bacterium recognized for its natural ability to effectively deconstruct cellulosic biomass. While there is a large body of studies on the genetic engineering of this bacterium and its physiology to-date, there is limited knowledge in the transcriptional regulation in this organism and thermophilic bacteria in general. The study herein is the first report of a high-throughput application of DNA-affinity purification sequencing (DAP-seq) to transcription factors (TFs) from a thermophile. We applied DAP-seq to >90 TFs in C. thermocellum and detected genome-wide binding sites for 11 of them. We then compiled and aligned DNA binding sequences from these TFs to deduce the primary DNA-binding sequence motifs for each TF. These binding motifs are further validated with electrophoretic mobility shift assay (EMSA) and are used to identify individual TFs’ regulatory targets in C. thermocellum. Our results led to the discovery of novel, uncharacterized TFs as well as homologues of previously studied TFs including RexA-, LexA- and LacI-type TFs. We then used these data to reconstruct gene regulatory networks for the 11 TFs individually, which resulted in a global network encompassing the TFs with some interconnections. As gene regulation governs and constrains how bacteria behave, our findings shed light on the roles of TFs delineated by their regulons, and potentially provides a means to enable rational, advanced genetic engineering of C. thermocellum and other organisms alike towards a desired phenotype.

09 BIOMASS FUELS↗
DOE OSTI2022

Biomass formation and sugar release efficiency of Populus modified by altered expression of a NAC transcription factor

Woody biomass is an important feedstock for biofuel production. Manipulation of wood properties that enable efficient conversion of biomass to biofuel reduces cost of biofuel production. Wood cell wall composition is regulated at several levels that involve expression of transcription factors such as wood-/secondary cell wall-associated NAC domains (WND or SND). In Arabidopsis thaliana, SND1 regulates cell wall composition through activation of its down-stream targets such as MYBs. The functional aspects of SND1 homologs in the woody Populus have been studied through transgenic manipulation. In this study, we investigated the role of PdWND1B, Populus SND1 sequence ortholog, in wood formation using transgenic manipulation through over-expression or silencing under the control of a vascular-specific 4-coumarate-CoA ligase (4CL) promoter. As compared with control plants, PdWND1B-RNAi plants were shorter in height, with significantly reduced stem diameter and dry biomass, whereas there were no significant differences in growth and productivity of PdWND1B over-expression plants. Conversely, PdWND1B over-expression lines showed a significant reduction in cellulose and increase in lignin content, whereas there was no significant impact on lignin content of downregulated lines. Stem carbohydrate composition analysis revealed a decrease in glucose, mannose, arabinose, and galactose, but an increase in xylose in the over-expression lines. Transcriptome analysis revealed upregulation of several downstream transcription factors and secondary cell wall related structural genes in the PdWND1B over-expression lines, partly explaining the observed phenotypic changes in cell wall chemistry. Relative to the control, glucose release efficiency and ethanol production from stem biomass was significantly reduced in over-expression lines. Our results show that PdWND1B is an important factor determining biomass productivity, cell wall chemistry and its conversion to biofuels in Populus.

09 BIOMASS FUELS↗
DOE OSTI2025

Identification and overexpression of endogenous transcription factors to enhance lipid accumulation in the biotechnologically relevant species Chlamydomonas pacifica

Sustainable low-carbon energy solutions are critical to mitigating global carbon emissions. Algae-based platforms offer potential by converting carbon dioxide into valuable products while aiding carbon sequestration. However, scaling algae cultivation faces challenges like contamination in outdoor systems. Previously, our lab evolved Chlamydomonas pacifica, an extremophile green alga, which tolerates high temperature, pH, salinity, and light, making it ideal for large-scale bioproduct production, including biodiesel. Here, we enhanced lipid accumulation in evolved C. pacifica by identifying and overexpressing key endogenous transcription factors through genome-wide in-silico analysis and in-vivo testing. These factors include Lipid Remodeling Regulator 1 (CpaLRL1), Nitrogen Response Regulator 1 (CpaNRR1), Compromised Hydrolysis of Triacylglycerols 7 (CpaCHT7), and Phosphorus Starvation Response 1 (CpaPSR1). Under nitrogen deprivation, CpaLRL1, CpaNRR1, and CpaCHT7 overexpression enhanced lipid accumulation compared to wild-type. However, CpaPSR1 increased lipid accumulation compared to wild-type in normal media and did not increase further under nitrogen deprivation, highlighting the difference in function based on media conditions. Notably, lipid analysis of CpaPSR1 under normal media conditions revealed a 2.4-fold increase in triglycerides (TAGs) compared to the wild-type, highlighting its potential for biodiesel production. This approach provides a framework for transcription factor-focused metabolic engineering in algae, advancing bioenergy and biomaterial production.

Biofuels↗
DOE OSTI2023

The transcriptional activator ClrB is crucial for the degradation of soybean hulls and guar gum in Aspergillus niger

Low-cost plant substrates, such as soybean hulls, are used for various industrial applications. Filamentous fungi are important producers of Carbohydrate Active enZymes (CAZymes) required for the degradation of these plant biomass substrates. CAZyme production is tightly regulated by several transcriptional activators and repressors. One such transcriptional activator is CLR-2/ClrB/ManR, which has been identified as a regulator of cellulase and mannanase production in several fungi. However, the regulatory network governing the expression of cellulase and mannanase encoding genes has been reported to differ between fungal species. Previous studies showed that Aspergillus niger ClrB is involved in the regulation of (hemi-)cellulose degradation, although its regulon has not yet been identified. To reveal its regulon, we cultivated an A. niger ΔclrB mutant and control strain on guar gum (a galactomannan-rich substrate) and soybean hulls (containing galactomannan, xylan, xyloglucan, pectin and cellulose) to identify the genes that are regulated by ClrB. Gene expression data and growth profiling showed that ClrB is indispensable for growth on cellulose and galactomannan and highly contributes to growth on xyloglucan in this fungus. Therefore, we show that A. niger ClrB is crucial for the utilization of guar gum and the agricultural substrate, soybean hulls. Moreover, we show that mannobiose is most likely the physiological inducer of ClrB in A. niger and not cellobiose, which is considered to be the inducer of N. crassa CLR-2 and A. nidulans ClrB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP

7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Further, together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

HNRNPU promotes the progression of hepatocellular carcinoma by enhancing CDK2 transcription

Highlights: • The expression of HNRNPU is upregulated in hepatocellular carcinoma. • HNRNPU knockdown significantly inhibits HCC cell proliferation. • HNRNPU knockdown induces cell cycle arrest. • HNRNPU regulates CDK2 transcription by directly binding to the CDK2 gene locus. The nuclear matrix-associated protein Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), also known as SAF-A, is known to maintain active chromatin structure in mouse hepatocytes. However, the functional roles and molecular mechanisms of HNRNPU in the development of hepatocellular carcinoma (HCC) remain largely unknown. Herein, we found that HNRNPU was upregulated in HCC, and the proliferation of HCC cells was inhibited in vitro and in vivo upon HNRNPU knockdown. Moreover, the upregulation of HNRNPU was correlated with poor prognosis in HCC. Mechanistically, HNRNPU bound to the CDK2 gene locus, a key factor in cell cycle regulation, where it was enriched with H3K27 acetylation (H3K27ac), H3K9 acetylation (H3K9ac), and H3K4 mono-methylation (H3K4me1). Furthermore, HNRNPU knockdown reduced the levels of H3K27ac and H3K9ac at the binding site, where the levels of H3K27 tri-methylation (H3K27me3) were increased, eventually leading to the downregulation of CDK2. Collectively, our results provide a new mechanism whereby HNRNPU promotes HCC development by enhancing the transcription of CDK2.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Transcriptional Regulatory Networks Involved in C3–C4 Alcohol Stress Response and Tolerance in Yeast

Alcohol toxicity significantly impacts the titer and productivity of industrially produced biofuels. To overcome this limitation, we must find and use strategies to improve stress tolerance in production strains. Previously, we developed a multiplex navigation of a global regulatory network (MINR) library that targeted 25 regulatory genes that are predicted to modify global regulation in yeast under different stress conditions. Herein, we expanded this concept to target the active sites of 47 transcriptional regulators using a saturation mutagenesis library. The 47 targeted regulators interact with more than half of all yeast genes. We then screened and selected for C3–C4 alcohol tolerance. We identified specific mutants that have resistance to isopropanol and isobutanol. Notably, the WAR1_K110N variant improved tolerance to both isopropanol and isobutanol. In addition, we investigated the mechanisms for improvement of isopropanol and isobutanol stress tolerance and found that genes related to glycolysis play a role in tolerance to isobutanol, while changes in ATP synthesis and mitochondrial respiration play a role in tolerance to both isobutanol and isopropanol. Overall, this work sheds light on basic mechanisms for isopropanol and isobutanol toxicity and demonstrates a promising strategy to improve tolerance to C3–C4 alcohols by perturbing the transcriptional regulatory network.

09 BIOMASS FUELS↗
DOE OSTI2022

The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase

Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds–Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Central transcriptional regulator controls photosynthetic growth and carbon storage in response to high light

Carbon capture and biochemical storage are some of the primary drivers of photosynthetic yield and productivity. To elucidate the mechanisms governing carbon allocation, we designed a photosynthetic light response test system for genetic and metabolic carbon assimilation tracking, using microalgae as simplified plant models. The systems biology mapping of high light-responsive photophysiology and carbon utilization dynamics between two variants of the same Picochlorum celeri species, TG1 and TG2 elucidated metabolic bottlenecks and transport rates of intermediates using instationary 13 C-fluxomics. Simultaneous global gene expression dynamics showed 73% of the annotated genes responding within one hour, elucidating a singular, diel-responsive transcription factor, closely related to the CCA1/LHY clock genes in plants, with significantly altered expression in TG2. Transgenic P. celeri TG1 cells expressing the TG2 CCA1/LHY gene, showed 15% increase in growth rates and 25% increase in storage carbohydrate content, supporting a coordinating regulatory function for a single transcription factor.

09 BIOMASS FUELS↗
DOE OSTI2024

Sequence-specific dynamic DNA bending explains mitochondrial TFAM’s dual role in DNA packaging and transcription initiation

Abstract Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent—LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Predicting transcriptional responses to cold stress across plant species

Although genome-sequence assemblies are available for a growing number of plant species, gene-expression responses to stimuli have been cataloged for only a subset of these species. Many genes show altered transcription patterns in response to abiotic stresses. However, orthologous genes in related species often exhibit different responses to a given stress. Accordingly, data on the regulation of gene expression in one species are not reliable predictors of orthologous gene responses in a related species. Here, we trained a supervised classification model to identify genes that transcriptionally respond to cold stress. A model trained with only features calculated directly from genome assemblies exhibited only modest decreases in performance relative to models trained by using genomic, chromatin, and evolution/diversity features. Models trained with data from one species successfully predicted which genes would respond to cold stress in other related species. Cross-species predictions remained accurate when training was performed in cold-sensitive species and predictions were performed in cold-tolerant species and vice versa. Models trained with data on gene expression in multiple species provided at least equivalent performance to models trained and tested in a single species and outperformed single-species models in cross-species prediction. These results suggest that classifiers trained on stress data from well-studied species may suffice for predicting gene-expression patterns in related, less-studied species with sequenced genomes.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

A modERN resource: identification of Drosophila transcription factor candidate target genes using RNAi

Transcription factors (TFs) play a key role in development and in cellular responses to the environment by activating or repressing the transcription of target genes in precise spatial and temporal patterns. In order to develop a catalog of target genes of Drosophila melanogaster TFs, the modERN consortium systematically knocked down the expression of TFs using RNAi in whole embryos followed by RNA-seq. We generated data for 45 TFs which have 18 different DNA-binding domains and are expressed in 15 of the 16 organ systems. The range of inactivation of the targeted TFs by RNAi ranged from log2fold change -3.52 to +0.49. The TFs also showed remarkable heterogeneity in the numbers of candidate target genes identified, with some generating thousands of candidates and others only tens. We present detailed analysis from five experiments, including those for three TFs that have been the focus of previous functional studies (ERR, sens, and zfh2) and two previously uncharacterized TFs (sens-2 and CG32006), as well as short vignettes for selected additional experiments to illustrate the utility of this resource. The RNA-seq datasets are available through the ENCODE DCC (http://encodeproject.org) and the Sequence Read Archive (SRA). TF and target gene expression patterns can be found here: https://insitu.fruitfly.org. These studies provide data that facilitate scientific inquiries into the functions of individual TFs in key developmental, metabolic, defensive, and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks during embryogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Hepatitis B e Antigen-Negative Single Hepatocyte Analysis Shows Transcriptional Silencing and Slow Decay of Infected Cells With Treatment

Background Nucleos(t)ide analogues (NUCs) rarely cure chronic hepatitis B (CHB) because they do not eliminate covalently closed circular deoxyribonucleic acid, the stable replication template. In hepatitis B e antigen (HBeAg)-positive CHB during NUCs, HBV-infected cells decline slowly and are transcriptionally silenced. Whether these occur in HBeAg-negative CHB is unknown. Methods Using paired liver biopsies separated by 2.7–3.7 years in 4 males with HIV and HBeAg-negative CHB at both biopsies and 1 male with HIV who underwent HBeAg seroconversion between biopsies, we quantified amounts of viral nucleic acids in hundreds of individual hepatocytes. Results In the 4 persistently HBeAg-negative participants, HBV-infected hepatocytes ranged from 6.2% to 17.7% (biopsy 1) and significantly declined in 3 of 4 by biopsy 2. In the HBeAg seroconverter, the proportion was 97.4% (biopsy 1) and declined to 81.9% at biopsy 2 (P < .05). Here, we extrapolated that HBV eradication with NUCs would take >100 years. At biopsy 1 in the persistently HBeAg-negative participants, 23%–56.8% of infected hepatocytes were transcriptionally inactive—higher than we observed in HBeAg-positive CHB—and significantly declined in 1 of 4 at biopsy 2. Conclusions In HBeAg-negative CHB on NUCs, the negligible decline in infected hepatocytes is similar to HBeAg-positive CHB, supporting the need for more potent therapeutics to achieve functional cure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

ssDNA is an allosteric regulator of the C. crescentus SOS-independent DNA damage response transcription activator, DriD

DNA damage repair systems are critical for genomic integrity. However, they must be coordinated with DNA replication and cell division to ensure accurate genomic transmission. In most bacteria, this coordination is mediated by the SOS response through LexA, which triggers a halt in cell division until repair is completed. Recently, an SOS-independent damage response system was revealed in Caulobacter crescentus. This pathway is controlled by the transcription activator, DriD, but how DriD senses and signals DNA damage is unknown. To address this question, we performed biochemical, cellular, and structural studies. We show that DriD binds a specific promoter DNA site via its N-terminal HTH domain to activate transcription of genes, including the cell division inhibitor didA. A structure of the C-terminal portion of DriD revealed a WYL motif domain linked to a WCX dimerization domain. Strikingly, we found that DriD binds ssDNA between the WYL and WCX domains. Comparison of apo and ssDNA-bound DriD structures reveals that ssDNA binding orders and orients the DriD domains, indicating a mechanism for ssDNA-mediated operator DNA binding activation. Biochemical and in vivo studies support the structural model. Our data thus reveal the molecular mechanism underpinning an SOS-independent DNA damage repair pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Novosphingobium aromaticivorans LigR coordinates transcription of genes involved in metabolism of multiple types of aromatics

Aromatic compounds are a ubiquitous and diverse family of chemicals with functions as biomolecules, natural products, industrial chemicals, and pollutants. Novosphingobium aromaticivorans DSM 12444 uses multiple inducible pathways to catabolize H-, G-, and S-type aromatics that contain zero, one, or two methoxy groups, respectively. Here, we obtain a systems-level view of the transcriptional control of its aromatic metabolic pathways. Several in vitro analyses found that a N. aromaticivorans homolog of the Sphingobium lignivorans SYK-6 transcription factor LigR bound genomic DNA upstream of genes involved in metabolism of multiple aromatic types. We found that a ΔLigR mutant had growth defects on all three types of aromatics as sole carbon sources. Transcriptomic analysis revealed that LigR was required to increase expression of gene products that function in metabolism of all three aromatic types. We also found that, in media containing both glucose and an aromatic carbon source, the ΔLigR mutant directed intermediates through alternative aromatic metabolic pathways. Protein-DNA binding assays showed that N. aromaticivorans LigR binds immediately upstream of promoters of genes involved in aromatic metabolism. We found that N. aromaticivorans LigR coordinates the expression of enzymes that function in the catabolism of H-, G-, and S-type aromatics, and that there are differences in the role of LigR in N. aromaticivorans and S. lignivorans. A comparative genomic analysis predicted that LigR homologs and the aromatic-metabolizing genes that it directly regulates are often co-localized in the genomes of Sphingomonadales, but often not found in this arrangement in many other known aromatic metabolizing bacteria.

Aromatic Compound Degradation↗