Transcriptional Activation Domain Research Articles

Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-Based Transcriptional Activation

Background: CRISPR activation (CRISPRa) uses non-functional Cas9 endonuclease (dCas9) but retains the genome targeting ability through its single guide RNAs (sgRNAs). CRISPRa is widely used as a gene activation system exploiting its ability in recruiting various transcriptional activation domains (TADs) for enhancing expression of the target gene(s). Drought tolerant and resource-efficient crops like millets have the potential for mitigating effects of climate change and for enhancing food security. Objective: This study aimed to use the Setaria italica (foxtail millet) genome sequence in the identification of a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring water logging tolerance that will benefit the future expansion of its cultivation area. Methods and Results: Leveraging existing RNA-seq data and information on functional studies in model plants and from other cereal species, maize and barley, have enabled the identification of candidate ERFVII from the foxtail millet genome sequence in the attempt to engineer water logging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome for the identification and characterization of the ortholog from Setaria italica. Soft berry was used for promoter annotation to obtain the transcription start site (TSS). Subsequently, CRISP-P 2.0 design tools were employed to generate and select a few efficient sgRNAs for CRISPRa that minimize potentially deleterious off-target binding. Conclusion: The study is a useful example of how to advance in genomics research including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species, through the utilization of the available genome sequence.

Reprogrammed lipid metabolism protects inner nuclear membrane against unsaturated fat.

SummaryThe cell nucleus is surrounded by a double membrane. The lipid packing and viscosity of membranes is critical for their function and is tightly controlled by lipid saturation. Circuits regulating the lipid saturation of the outer nuclear membrane (ONM) and contiguous endoplasmic reticulum (ER) are known. However, how lipid saturation is controlled in the inner nuclear membrane (INM) has remained enigmatic. Using INM biosensors and targeted genetic manipulations, we show that increased lipid unsaturation causes a reprogramming of lipid storage metabolism across the nuclear envelope (NE). Cells induce lipid droplet (LD) formation specifically from the distant ONM/ER, whereas LD formation at the INM is suppressed. In doing so, unsaturated fatty acids are shifted away from the INM. We identify the transcription circuits that topologically reprogram LD synthesis and identify seipin and phosphatidic acid as critical effectors. Our study suggests a detoxification mechanism protecting the INM from excess lipid unsaturation.

Activation of gene expression by detergent-like protein domains

SummaryThe mechanisms by which transcriptional activation domains (tADs) initiate eukaryotic gene expression have been an enigma for decades because most tADs lack specificity in sequence, structure, and interactions with targets. Machine learning analysis of data sets of tAD sequences generated in vivo elucidated several functionality rules: the functional tAD sequences should (i) be devoid of or depleted with basic amino acid residues, (ii) be enriched with aromatic and acidic residues, (iii) be with aromatic residues localized mostly near the terminus of the sequence, and acidic residues localized more internally within a span of 20–30 amino acids, (iv) be with both aromatic and acidic residues preferably spread out in the sequence and not clustered, and (v) not be separated by occasional basic residues. These and other more subtle rules are not absolute, reflecting absence of a tAD consensus sequence, enormous variability, and consistent with surfactant-like tAD biochemical properties. The findings are compatible with the paradigm-shifting nucleosome detergent mechanism of gene expression activation, contributing to the development of the liquid-liquid phase separation model and the biochemistry of near-stochastic functional allosteric interactions.

Leukemogenesis via aberrant self-renewal by the MLL/AEP-mediated transcriptional activation system.

Homeostasis of the hematopoietic system is achieved in a hierarchy, with hematopoietic stem cells at the pinnacle. Because only hematopoietic stem cells (HSCs) can self‐renew, the size of the hematopoietic system is strictly controlled. In hematopoietic reconstitution experiments, 1 HSC can reconstitute the entire hematopoietic system, whereas 50 multipotent progenitors cannot. This indicates that only HSCs self‐renew, whereas non‐HSC hematopoietic progenitors are programmed to differentiate or senesce. Oncogenic mutations of the mixed lineage leukemia gene (MLL) overcome this “programmed differentiation” by conferring the self‐renewing ability to non‐HSC hematopoietic progenitors. In leukemia, mutated MLL proteins constitutively activate a broad range of previously transcribed CpG‐rich promoters by an MLL‐mediated transcriptional activation system. This system promotes self‐renewal by replicating an expression profile similar to that of the mother cell in its daughter cells. In this transcriptional activation system, MLL binds to unmethylated CpG‐rich promoters and recruits RNA polymerase II. MLL recruits p300/CBP through its transcriptional activation domain, which acetylates histone H3 at lysines 9, 18, and 27. The AF4 family/ENL family/P‐TEFb complex (AEP) binds to acetylated H3K9/18/27 to activate transcription. Gene rearrangements of MLL with AEP‐ or CBP/p300‐complex components generate constitutively active transcriptional machinery of this transcriptional activation system, which causes aberrant self‐renewal of leukemia stem cells. Inhibitors of the components of this system effectively decrease their leukemogenic potential.

Targeted Therapy for AML Expressing RUNX1 Mutation

RUNX1 is the DNA-binding subunit of the core binding factor (CBF) complex and a transcription factor (TF) involved in normal and malignant hematopoiesis. Transcriptionally active full-length RUNX1b isoform is transcribed from 6 exons, encoding a DNA binding ‘Runt’ homology domain and a more C-terminal transcription activation domain. Within its CTCF-anchored loop or TAD (topologically-associating domain), RUNX1 gene has a highly conserved +24-kb enhancer (eR1 in mouse), which is part of a large super-enhancer (>170 kb) covering the entire intron 1 between proximal P2 and distal P1 promoter of RUNX1 and controlling the transcription of RUNX1. TFs bind to super-enhancers/enhancers that loop over to interact with promoters to transcriptionally regulate oncogenes, such as RUNX1 and its targets, as well as multiple other AML-relevant oncogenes involved in growth and survival of AML blast progenitor cells (BPCs). Heatmap of Hi-C interaction scores within the RUNX1 TAD showed triangle shaped regions of high interaction scores highlighting interactions between +24-kb enhancer and the P2 promoter of RUNX1. To functionally validate these findings, we cloned and transfected into HEK293 cells expressing RUNX1, a luciferase reporter construct containing the DNA of the P2 promoter with or without the +24 kb-enhancer of RUNX1. Our findings confirm that, as compared to the promoter or the enhancer alone, the +24 kb-enhancer-driven P2 promoter exhibited superior luciferase induction representing increased transcriptional activity of RUNX1. Somatic mutations in RUNX1 commonly occur in MDS, as well as in the secondary (s) or de novo AML (~10%). Majority of mutant (mt) RUNX1 are missense, large deletions or truncation-mutations, behaving mostly as loss of function mutations. Presence of mtRUNX1 confers relative therapy-resistance and poorer survival in patients with AML. Lack of specific targeted therapy coupled with resistance to standard chemotherapy may account for the poorer outcome in AML expressing somatic or germline mtRUNX1. Therefore, there is a strong rationale and an unmet need to develop novel targeted therapies for AML expressing somatic or germline mtRUNX1. In the present studies, we demonstrate for the first time that shRNA mediated knockdown of RUNX1 expression inhibits growth and induces differentiation and apoptosis of cultured cell line (OCI-AML5) and patient-derived (PD) AML BPCs expressing mtRUNX1. After engraftment, doxycycline-inducible shRNA-mediated in vivo knockdown of RUNX1 in OCI-AML5 cells, compared to controls, markedly reduced AML burden and improved survival of the immune-depleted (NSG) mice engrafted with luciferase transduced OCI-AML5 cells. Interaction of TFs with DNA and transcriptional co-factors such as the histone acetyltransferase p300 at enhancers and promoters leads to acetylation of histone proteins and TFs. BET (Bromodomain Extra-terminal) protein (BETP) BRD4 binds to acetylated histones and TFs at super-enhancers and recruits transcriptional co-factors to promote RNA pol II (RNAP2)-mediated transcription of oncogenes and their targets. ChIP-Seq analysis with anti-BRD4 and anti-H3K27Ac antibody showed that Runx1 super-enhancer and P2 promoter are occupied by BRD4. shRNA mediated knockdown of BRD4 attenuated expression of RUNX1 and its targets, MYC, PU.1 and myeloperoxidase (MPO), inhibited growth and induced apoptosis of OCI-AML5 cells. We also found that BETP-PROTACs (proteolysis targeted chimera) ARV-825 and its pharmacologically superior version ARV-771 (20 to 250 nM) caused degradation and depletion of BRD4, reduced BRD4 occupancy at the+24-kb-enhancer and P2 promoter of RUNX1, repressing RUNX1 and its targets, and potently induced apoptosis of OCI-AML5, Mono-Mac-1 and PD AML BPCs expressing mtRUNX1. Treatment with ARV-771 (30 mg/kg SQ daily for 3 weeks also inhibited AML growth and improved survival of NSG mice engrafted with luciferase-transduced OCI-AML5 cells. By utilizing the RNA-Seq mRNA signature from RUNX1-depleted AML cells we queried for expression mimickers (EMs) through LINCS1000-CMap (Connectivity Mapping) analyses. This uncovered several novel agents that inhibit RUNX1 expression and induce in vitro lethality and reduce in vivo leukemia burden in AML BPCs expressing mtRUNX1. These findings highlight RUNX1-targeted novel agents to be developed and tested as potential therapy for AML expressing mtRUNX1. DisclosuresDiNardo:AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding. Qian:4Arvinas, LLC. New Haven, CT: Employment. Raina:4Arvinas, LLC. New Haven, CT: Employment. Takahashi:Symbio Pharmaceuticals: Consultancy.

TCF3-Hlf Hijacks ERG to Activate Leukemia-Critical Super-Enhancers in t(17;19) Leukemia

The translocation t(17;19), which results in a fusion of the transcriptional activation domain of the B cell developmental transcription factor TCF3 to the DNA-binding domains of HLF, defines a rare subtype of acute lymphoblastic leukemia (ALL) associated with very poor outcome. The mechanisms by which TCF3-HLF rewires gene expression in leukemia are still unclear.To evaluate the function of TCF3-HLF in leukemia, we deleted the fusion protein by targeting HLF using CRISPR, which resulted in the dropout of targeted ALL cells ex vivo in cell cultures and in vivo using patient derived xenografts (PDX). Serial transcriptome analysis identified c-MYC (MYC) among the top genes that are downregulated early after TCF3-HLF knockout (KO). Several of previously inferred TCF3-HLF target genes including BCL2 and LMO2 were retrieved in this signature. Gene sets that are positively regulated by MYC and reactivation of gene sets that are repressed by MYC were highly enriched by gene set enrichment analysis (GSEA) after TCF3-HLF KO. Importantly, CRISPR knockout of MYC was lethal for TCF3-HLF leukemia. These results reveal MYC as an important target of TCF3-HLF.To gain further insight into the TCF3-HLF regulome, we performed chromatin immunoprecipitation and sequencing (ChIP-seq), using engineered TCF3-HLF ALL cells (HAL01) in which we inserted a FLAG tag at the c-terminal end of the endogenous TCF3-HLF by CRISPR. ChIP-seq identified 484 high confidence binding peaks of TCF3-HLF, with a large fraction occupied introns (44.75%) or distal intergenic regions (44.21%). ChIP-seq of epigenetic marks revealed strong enrichment of active enhancers marks (H3K4me1 and H3K27ac), absence of poised enhancer marks (H3K27me3) and very low detection of promoter marks (H3K4me3) flanking the regions bound by TCF3-HLF. Importantly, 82 of the TCF3-HLF bound regions are associated with high density and long range of active chromatin mark (H3K27ac), which are annotated as super-enhancers. These regions are close to genes that are implicated in leukemia and hematological malignancies (eg. BCL2 and LMO2) and include a distal MYC enhancer that is active in acute myeloid leukemia (AML) and hematopoietic stem cells. Using chromosome conformation capture coupled with qPCR (3C-qPCR), we confirmed that this MYC enhancer is in proximity of MYC transcription start site through an enhancer-promoter loop, which is disrupted when TCF-HLF is knocked out. Furthermore, CRISPR targeting of the TCF3-HLF binding motif in this region abolished MYC expression and strongly reduced ALL cell proliferation. These results identify MYC enhancer as one of the important targets of TCF3-HLF.De novo motif discovery identified high occurrence (399/484) of HLF motif (5'-TTACGTAA-3') enriched in the center of TCF3-HLF binding regions. To evaluate candidate TCF3-HLF binding regions functionally, we cloned 1 KB genomic DNA sequences centered around TCF3-HLF binding sites in an enhancer reporter vector. Mutagenesis of TCF3-HLF binding sites and TCF3-HLF knockout disrupted enhancer function of several candidate enhancers including the MYC enhancer mentioned above. TCF3-HLF KO lead to a marked reduction of H3K27ac abundance at positions flanking TCF3-HLF binding sites, providing functional evidence for a pioneering role of the TCF3-HLF complex in the activation of enhancer regions. Furthermore, we discovered a conserved motif grammar with ETS motifs (5'-GGA(A/T)-3') being closely associated with HLF motifs, in many instances at -1bp upstream of the HLF motif. Deletion of these ETS binding motifs or CRISPR knockout of ERG, but not of other ETS family transcription factors, compromised enhancer activity and leukemia survival. By affinity purification we confirmed that ERG is associated with the TCF3-HLF complex.Collectively, these results brand TCF3-HLF as an oncogenic driver that acts primarily through aberrant activation of enhancer gene regulatory regions. TCF3-HLF likely cooperates with ERG to recruit transcriptional complexes to mediate enhancer activity. Indeed, we retrieved EP300 and BRD4 in the TCF3-HLF complex and show that TCF3-HLF ALL PDX were remarkably sensitive to BRD4 inhibition. Our models will enable further exploration of the TCF3-HLF complex by proteomics and systematic evaluation of candidate therapeutic agents to interfere with the fundamental oncogenic process. DisclosuresSchrappe:Novartis: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Medac: Consultancy, Research Funding; JAZZ Pharma: Consultancy, Research Funding.

Nuclear ADP-ribosylation drives IFN\u03b3-dependent STAT1\u03b1 enhancer formation in macrophages

STAT1α is a key transcription factor driving pro-inflammatory responses in macrophages. We found that the interferon gamma (IFNγ)-regulated transcriptional program in macrophages is controlled by ADP-ribosylation (ADPRylation) of STAT1α, a post-translational modification resulting in the site-specific covalent attachment of ADP-ribose moieties. PARP-1, the major nuclear poly(ADP-ribose) polymerase (PARP), supports IFNγ-stimulated enhancer formation by regulating the genome-wide binding and IFNγ-dependent transcriptional activation of STAT1α. It does so by ADPRylating STAT1α on specific residues in its DNA-binding domain (DBD) and transcription activation (TA) domain. ADPRylation of the DBD controls STAT1α binding to its cognate DNA elements, whereas ADPRylation of the TA domain regulates enhancer activation by modulating STAT1α phosphorylation and p300 acetyltransferase activity. Loss of ADPRylation at either site leads to diminished IFNγ-dependent transcription and downstream pro-inflammatory responses. We conclude that PARP-1-mediated ADPRylation of STAT1α drives distinct enhancer activation mechanisms and is a critical regulator of inflammatory responses in macrophages.

Inhibition of the Oxygen-Sensing Asparaginyl Hydroxylase Factor Inhibiting Hypoxia-Inducible Factor: A Potential Hypoxia Response Modulating Strategy

Factor inhibiting hypoxia-inducible factor (FIH) is a JmjC domain 2-oxogluarate and Fe(II)-dependent oxygenase that catalyzes hydroxylation of specific asparagines in the C-terminal transcriptional activation domain of hypoxia-inducible factor alpha (HIF-α) isoforms. This modification suppresses the transcriptional activity of HIF by reducing its interaction with the transcriptional coactivators p300/CBP. By contrast with inhibition of the HIF prolyl hydroxylases (PHDs), inhibitors of FIH, which accepts multiple non-HIF substrates, are less studied; they are of interest due to their potential ability to alter metabolism (either in a HIF-dependent and/or -independent manner) and, provided HIF is upregulated, to modulate the course of the HIF-mediated hypoxic response. Here we review studies on the mechanism and inhibition of FIH. We discuss proposed biological roles of FIH including its regulation of HIF activity and potential roles of FIH-catalyzed oxidation of non-HIF substrates. We highlight potential therapeutic applications of FIH inhibitors.

Characterization of a transcription factor SlNAC7 gene from Suaeda liaotungensis and its role in stress tolerance.

NAC (NAM, ATAF1/2, CUC2) transcription factors play important roles in plant growth, development, and responses to abiotic stress. In this study, we cloned an NAC2 subfamily transcription factor gene (SlNAC7) from the halophyte Suaeda liaotungensis K., and conducted a series of studies to determine the characteristics and functions of this gene. The SlNAC7 coding region contains 1719 base pairs that encode a 573 amino acid long protein. SlNAC7 is expressed in the roots, stems, and leaves of S. liaotungensis, with the highest expression in the leaves. We found that SlNAC7 expression can be induced by drought, salt, cold, and abscisic acid. Transient expression in onion epidermal cells revealed that SlNAC7 is located in both the nucleus and cytoplasm. A transcriptional activation experiment in yeast showed that the transcriptional activation domain of SlNAC7 is located at the C terminus. When SlNAC7 was transformed into Arabidopsis under the control of a CaMV 35S promoter its overexpression was found to enhance the ability of transgenic plants to resist drought, salt, and cold stress. Moreover, these plants showed multiple changes in growth characteristics and physiological and biochemical indices in response to different stresses, as well as the upregulation of numerous stress-related genes. We have thus characterized a new halophyte-derived NAC transcription factor, SlNAC7, which can regulate plant growth and physiological and biochemical changes under adverse conditions by regulating the expression of stress-related genes, thereby enhancing plant stress resistance. SlNAC7 is a promising candidate for breeding new varieties of stress-tolerant crops.

Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (“fuzzy” binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism of viral spike protein-mediated fusion or screening for viral inhibitors under biosafety level 1 conditions.

Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator.

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Rational engineering of a modular bacterial CRISPR-Cas activation platform with expanded target range.

CRISPR–Cas activator (CRISPRa) systems that selectively turn on transcription of a target gene are a potentially transformative technology for programming cellular function. While in eukaryotes versatile CRISPRa systems exist, in bacteria these systems suffer from a limited ability to activate different genes due to strict distance-dependent requirements of functional target binding sites, and require greater customization to optimize performance in different genetic and cellular contexts. To address this, we apply a rational protein engineering approach to create a new CRISPRa platform that is highly modular to allow for easy customization and has increased targeting flexibility through harnessing engineered Cas proteins. We first demonstrate that transcription activation domains can be recruited by CRISPR–Cas through noncovalent protein-protein interactions, which allows each component to be encoded on separate and easily interchangeable plasmid elements. We then exploit this modularity to rapidly screen a library of different activation domains, creating new systems with distinct regulatory properties. Furthermore, we demonstrate that by harnessing a library of circularly permuted Cas proteins, we can create CRISPRa systems that have different target binding site requirements, which together, allow for expanded target range.

Genome Editing in iPSC-Based Neural Systems: From Disease Models to Future Therapeutic Strategies

Therapeutic advances for neurological disorders are challenging due to limited accessibility of the human central nervous system and incomplete understanding of disease mechanisms. Many neurological diseases lack precision treatments, leading to significant disease burden and poor outcome for affected patients. Induced pluripotent stem cell (iPSC) technology provides human neuronal cells that facilitate disease modeling and development of therapies. The use of genome editing, in particular CRISPR-Cas9 technology, has extended the potential of iPSCs, generating new models for a number of disorders, including Alzheimers and Parkinson Disease. Editing of iPSCs, in particular with CRISPR-Cas9, allows generation of isogenic pairs, which differ only in the disease-causing mutation and share the same genetic background, for assessment of phenotypic differences and downstream effects. Moreover, genome-wide CRISPR screens allow high-throughput interrogation for genetic modifiers in neuronal phenotypes, leading to discovery of novel pathways, and identification of new therapeutic targets. CRISPR-Cas9 has now evolved beyond altering gene expression. Indeed, fusion of a defective Cas9 (dCas9) nuclease with transcriptional repressors or activation domains allows down-regulation or activation of gene expression (CRISPR interference, CRISPRi; CRISPR activation, CRISPRa). These new tools will improve disease modeling and facilitate CRISPR and cell-based therapies, as seen for epilepsy and Duchenne muscular dystrophy. Genome engineering holds huge promise for the future understanding and treatment of neurological disorders, but there are numerous barriers to overcome. The synergy of iPSC-based model systems and gene editing will play a vital role in the route to precision medicine and the clinical translation of genome editing-based therapies.

FIH-1-modulated HIF-1α C-TAD promotes acute kidney injury to chronic kidney disease progression via regulating KLF5 signaling.

Incomplete recovery from episodes of acute kidney injury (AKI) can predispose patients to develop chronic kidney disease (CKD). Although hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the response to hypoxia/ischemia, the role of HIF-1α in CKD progression following incomplete recovery from AKI is poorly understood. Here, we investigated this issue using moderate and severe ischemia/reperfusion injury (I/RI) mouse models. We found that the outcomes of AKI were highly associated with the time course of tubular HIF-1α expression. Sustained activation of HIF-1α, accompanied by the development of renal fibrotic lesions, was found in kidneys with severe AKI. The AKI to CKD progression was markedly ameliorated when PX-478 (a specific HIF-1α inhibitor, 5 mg· kg-1·d-1, i.p.) was administered starting onday5 after severe I/RI for 10 consecutive days. Furthermore, we demonstrated that HIF-1α C-terminal transcriptional activation domain (C-TAD) transcriptionally stimulated KLF5, which promoted progression of CKD following severe AKI. The effect of HIF-1α C-TAD activation on promoting AKI to CKD progression was also confirmed in in vivo and in vitro studies. Moreover, we revealed that activation of HIF-1α C-TAD resulted in the loss of FIH-1, which was the key factor governing HIF-1α-driven AKI to CKD progression. Overexpression of FIH-1 inhibited HIF-1α C-TAD and prevented AKI to CKD progression. Thus, FIH-1-modulated HIF-1α C-TAD activation was the key mechanism of AKI to CKD progression by transcriptionally regulating KLF5 pathway. Our results provide new insights into the role of HIF-1α in AKI to CKD progression and also the potential therapeutic strategy for the prevention of renal diseases progression.

The Correlation Between Tuberous Sclerosis Complex Genotype and Renal Angiomyolipoma Phenotype.

Tuberous sclerosis complex (TSC) is a rare multisystem autosomal dominant genetic disease that occurs between 1 in 6,000 and 1 in 10,000 live births. Additionally, renal angiomyolipoma is the most common form of renal disease in patients affected by TSC. Although a genetic mutation analysis of TSC is not rare, the correlation between the TSC gene mutation and renal angiomyolipoma phenotype is poorly understood. This study aims to analyze the mutation sites in 261 types of selected TSC patients. The results reveal that: (1) female patients develop more renal angiomyolipoma than male patients [p = 0.008, OR = 2.474, 95%CI (1.258–4.864)]; (2). The missense mutation of TSC1 led to a higher risk of renal angiomyolipoma [p < 0.01, OR = 15, 95%CI (2.859–78.691)], and in contrast, showed a reduced risk in patients with frameshift mutation [p = 0.03, OR = 0.252, 95%CI (0.07–0.912)]; (3). Patients with TSC2 mutations in the transcription activation domain 1 coding genes, had increased renal angiomyolipoma [p = 0.019, OR = 3.519, 95%CI (1.226–10.101)]. Therefore, our genotype-phenotype correlation study might shed light on the early monitoring and evaluation of renal angiomyolipoma in TSC patients.

Functional interaction between ELL transcription elongation factor and Epe1 reveals the role of Epe1 in the regulation of transcription outside heterochromatin.

Eleven-nineteen lysine-rich leukemia (ELL) is a eukaryotic RNA polymerase II transcription elongation factor. In Schizosaccharomyces pombe, it is important for survival under genotoxic stress conditions. However, the molecular basis underlying this function of ELL in S. pombe is yet to be deciphered. Here, we carried out a genetic screen to identify multicopy suppressor(s) that could restore normal growth of ell1 deletion mutant in the presence of DNA damaging agent. Sequence analysis of the identified suppressors revealed the anti-silencing protein, Epe1, as one of the suppressors of ell1 deletion associated genotoxic stress sensitivity. Our results further demonstrate that the overexpression of Epe1 could suppress all other phenotypes associated with the absence of Ell1. Moreover, transcriptional defect of ell1Δ strain could also be alleviated by the overexpression of Epe1. Epe1 also showed a physical interaction with Ell1. Interestingly, we also observed that the region of Epe1 encompassing 403-948 amino acids was indispensable for all the above functions. Furthermore, our results show that the overexpression of Epe1 causes increased H3K9 acetylation and RNA polymerase II recruitment. Taken together, our results show a functional interaction between Epe1 and Ell1, and this function is independent of the well-known JmjC and N-terminal transcriptional activation domains of Epe1 in S. pombe.

Multiplex and optimization of dCas9-TV-mediated gene activation in plants.

Synthetic gene activators consisting of nuclease-dead Cas9 (dCas9) for single-guide RNA (sgRNA)-directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9-TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9-TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA-sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000-fold in rice. Moreover, the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9-TV-refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants.

YAP promotes ocular neovascularization by modifying PFKFB3-driven endothelial glycolysis.

Ocular neovascularization is the leading cause of vision impairment in a variety of ocular diseases, such as age-related macular degeneration and retinopathy of prematurity. Emerging studies have suggested that the yes-associated protein (YAP), a downstream effector of the Hippo pathway, is involved in the pathological angiogenesis, but the mechanism are largely unknown. Here, we demonstrated that hypoxic treatment triggered YAP expression and nuclear translocation in human umbilical vein endothelial cells (HUVECs). YAP acted as a transcriptional co-activator working together with transcriptional enhancer activator domain 1 (TEAD1) to binds the promoter of the key glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3), and thereby increases PFKFB3 expression. Moreover, silencing of YAP inhibited glycolysis as well as proliferation, migration, sprouting and tube formation of HUVECs under hypoxia, all of which could be reversed by enforced expression of PFKFB3. Finally, our animal study also showed that intravitreal injection of small interfering RNA of YAP or PFKFB3 dramatically suppressed the neovascular growth in mouse models of choroidal neovascularization and oxygen-induced retinopathy. These findings provide new insights into a previously unrecognized effect of YAP on endothelial glycolysis and highlight the potential of targeting YAP/PFKFB3 axis in the treatment of ocular neovascularization.

MeMYB26, a drought-responsive transcription factor in cassava (Manihot esculenta Crantz)

Abstract Cassava (Manihot esculenta Crantz) is worldwide the sixth-largest food crop and highly drought resistant. The expression of the myeloblastosis (MYB) family protein MeMYB26 in cassava was previously reported to be significantly up-regulated under water deficit. To study the basic characterization, natural variations and potential functions of MeMYB26 for drought resistance in cassava, the protein was subjected to bioinformatics analysis, multiplex-PCR with next-generation sequencing and candidate association studies. The results indicated that MeMYB26 is a typical transcription factor, with two MYB DNA-binding and transcriptional activation domains. One stop-gained and five nonsynonymous variations in the genomic region of MeMYB26 were significantly associated with drought resistance traits. The results of the scale-free coexpression network showed that the MeMYB26 gene plays a critical role in plant stress resistance, growth and biomass development. It was concluded that MeMYB26 is a reliable candidate gene associated with drought tolerance and biomass storage in cassava.