Cla4A, a Novel Regulator of Gene Expression Networks Required for Asexual and Insect-Pathogenic Lifecycles of Beauveria bassiana.

Cla4, an orthologous p21-activated kinase crucial for non-entomopathogenic fungal lifestyles, has two paralogs (Cla4A/B) functionally unknown in hypocrealean entomopathogens. Here, we report a regulatory role of Cla4A in gene expression networks of Beauveria bassiana required for asexual and entomopathogenic lifecycles while Cla4B is functionally redundant. The deletion of cla4A resulted in severe growth defects, reduced stress tolerance, delayed conidiation, altered conidiation mode, impaired conidial quality, and abolished pathogenicity through cuticular penetration, contrasting with no phenotype affected by cla4B deletion. In ∆cla4A, 5288 dysregulated genes were associated with phenotypic defects, which were restored by targeted gene complementation. Among those, 3699 genes were downregulated, including more than 1300 abolished at the transcriptomic level. Hundreds of those downregulated genes were involved in the regulation of transcription, translation, and post-translational modifications and the organization and function of the nuclear chromosome, chromatin, and protein-DNA complex. DNA-binding elements in promoter regions of 130 dysregulated genes were predicted to be targeted by Cla4A domains. Samples of purified Cla4A extract were proven to bind promoter DNAs of 12 predicted genes involved in multiple stress-responsive pathways. Therefore, Cla4A acts as a novel regulator of genomic expression and stability and mediates gene expression networks required for insect-pathogenic fungal adaptations to the host and environment.

Design, synthesis, and biological evaluation of 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides as a potential EGR-1 inhibitor for targeted therapy of atopic dermatitis.

Atopic dermatitis is a chronic inflammatory skin disease characterized by intense itching and frequent skin barrier dysfunctions. EGR-1 is a transcription factor that aggravates the pathogenesis of atopic dermatitis by promoting the production of various inflammatory cytokines. Three 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides (IT21, IT23, and IT25) were identified as novel inhibitors of EGR-1 DNA-binding activity. In silico docking experiments were performed to elucidate the binding conditions of the EGR-1 zinc-finger (ZnF) DNA-binding domain. Electrophoretic mobility shift assays confirmed the targeted binding effect on the EGR-1 ZnF DNA-binding domain, leading to dose-dependent dissociation of the EGR-1-DNA complex. At the functional cellular level, IT21, IT23, and IT25 effectively reduced mRNA expression of TNFα-induced EGR-1-regulated inflammatory genes, particularly in HaCaT keratinocytes inflamed by TNFα. In the in vivo efficacy study, IT21, IT23, and IT25 demonstrated the potential to alleviate atopic dermatitis-like skin lesions in the ear skin of BALB/c mice. These findings suggest that targeting the EGR-1 ZnF DNA-binding domain with 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide derivatives (IT21, IT23, and IT25) could serve as lead compounds for the development of potential therapeutic agents against inflammatory skin disorders, including atopic dermatitis.

Phytochrome B inhibits the activity of phytochrome-interacting factor 7 involving phase separation.

Shade-intolerant plants sense changes in the light environment and trigger shade-avoidance syndrome in the presence of neighboring vegetation. Phytochrome-interacting factor 7 (PIF7) is an essential regulator that integrates shade signals into plant transcriptional networks. While the regulation of PIF7 under shade conditions has been well studied, the mechanism that represses PIF7 activity under white light remains ambiguous. Here, we report that PIF7 forms nuclear puncta containing phase-separated liquid-like condensates. Phytochrome B (phyB) then binds to dephosphorylated PIF7 and promotes its condensed phase of PIF7 under white light. The phyB-PIF7 condensate subsequently inhibits the DNA-binding activity of PIF7. However, shade inactivation of phyB causes the dissociation of phyB-PIF7 condensates and allows unbound PIF7 to promote the transcription of shade-induced genes. This reversible transcriptional condensation via phase separation provides sessile organisms with the flexibility of gene control to adapt to their surrounding environment.

The Development of p53-Targeted Therapies for Human Cancers.

p53 plays a critical role in tumor suppression and is the most frequently mutated gene in human cancers. Most p53 mutants (mutp53) are missense mutations and are thus expressed in human cancers. In human cancers that retain wtp53, the wtp53 activities are downregulated through multiple mechanisms. For example, the overexpression of the negative regulators of p53, MDM2/MDMX, can also efficiently destabilize and inactivate wtp53. Therefore, both wtp53 and mutp53 have become promising and intensively explored therapeutic targets for cancer treatment. Current efforts include the development of small molecule compounds to disrupt the interaction between wtp53 and MDM2/MDMX in human cancers expressing wtp53 and to restore wtp53-like activity to p53 mutants in human cancers expressing mutp53. In addition, a synthetic lethality approach has been applied to identify signaling pathways affected by p53 dysfunction, which, when targeted, can lead to cell death. While an intensive search for p53-targeted cancer therapy has produced potential candidates with encouraging preclinical efficacy data, it remains challenging to develop such drugs with good efficacy and safety profiles. A more in-depth understanding of the mechanisms of action of these p53-targeting drugs will help to overcome these challenges.

Assessing the Activity of Transcription Factor FoxO1.

The transcription factor FoxO1 (forkhead box O1) regulates genes that are involved in development, metabolism, cellular innovation, longevity, and stress responses. Assessment of FoxO1 activity is therefore critical to understand the regulatory network of this transcription factor. FoxO1 transactivation activity relies on its ability to bind to the promoters of target genes, which is controlled by posttranslational modifications (e.g., dephosphorylation or phosphorylation) that may promote nuclear translocation or exclusion of FoxO1. In this chapter we describe the protocols for FoxO1 activity assessment using Western blotting analysis of the posttranslational modification of FoxO1 in whole cell lysates and ELISA of DNA binding activity of FoxO1 in nuclear extracts.

Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins.

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Arabidopsis cryptochrome 1 promotes stomatal development through repression of AGB1 inhibition of SPEECHLESS DNA-binding activity.

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. The heterotrimeric G-protein is known to regulate various physiological processes in plants and mammals. In Arabidopsis, cryptochrome 1 (CRY1) and the G-protein ß subunit AGB1 act antagonistically to regulate stomatal development. The molecular mechanism by which CRY1 and AGB1 regulate this process remains unknown. Here, we show that Arabidopsis CRY1 acts partially through AGB1, and AGB1 acts through SPEECHLESS (SPCH), a master transcription factor that drives stomatal initiation and proliferation, to regulate stomatal development. We demonstrate that AGB1 physically interacts with SPCH to block the bHLH DNA-binding domain of SPCH and inhibit its DNA-binding activity. Moreover, we demonstrate that photoexcited CRY1 represses the interaction of AGB1 with SPCH to release AGB1 inhibition of SPCH DNA-binding activity, leading to the expression of SPCH-target genes promoting stomatal development. Taken together, our results suggest that the mechanism by which CRY1 promotes stomatal development involves positive regulation of the DNA-binding activity of SPCH mediated by CRY1 inhibition of the AGB1-SPCH interaction. We propose that the antagonistic regulation of SPCH DNA-binding activity by CRY1 and AGB1 may allow plants to balance light and G-protein signaling and optimize stomatal density and pattern.

Regulation of Antigenic Variation by Trypanosoma brucei Telomere Proteins Depends on Their Unique DNA Binding Activities.

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, Variant Surface Glycoprotein (VSG), to evade the host immune response. Such antigenic variation is a key pathogenesis mechanism that enables T. brucei to establish long-term infections. VSG is expressed exclusively from subtelomere loci in a strictly monoallelic manner, and DNA recombination is an important VSG switching pathway. The integrity of telomere and subtelomere structure, maintained by multiple telomere proteins, is essential for T. brucei viability and for regulating the monoallelic VSG expression and VSG switching. Here we will focus on T. brucei TRF and RAP1, two telomere proteins with unique nucleic acid binding activities, and summarize their functions in telomere integrity and stability, VSG switching, and monoallelic VSG expression. Targeting the unique features of TbTRF and TbRAP1's nucleic acid binding activities to perturb the integrity of telomere structure and disrupt VSG monoallelic expression may serve as potential therapeutic strategy against T. brucei.

c-Rel Is the Pivotal NF-κB Subunit in Germinal Center Diffuse Large B-Cell Lymphoma: A LYSA Study.

Relationships between c-Rel and GCB-DLBCLs remain unclear. We found that strong c-Rel DNA-binding activity was mostly found in GCBs on two independent series of 48 DLBCLs and 66 DLBCLs, the latter issued from the GHEDI series. c-Rel DNA-binding activity was associated with increased REL mRNA expression. Extending the study to the whole GHEDI and Lenz DLBCL published series of 202 and 233 cases, it was found that the c-Rel gene expression profile (GEP) overlapped partially (12%) but only with the GCB GEP and not with the GEP of ABC-DLBCLs. Cases with both overexpression of REL mRNA and c-Rel GEP were defined as those having a c-Rel signature. These cases were GCBs in 88 and 83% of the GHEDI or Lenz's DLBCL series respectively. The c-Rel signature was also associated with various recurrent GCB-DLBCL genetic events, including REL gains, BCL2 translocation, MEF2B, EZH2, CREBBP, and TNFRSF14 mutations and with the EZB GCB genetic subtype. By CGH array, the c-Rel signature was specifically correlated with 2p15-16.1 amplification that includes XPO1, BCL11A, and USP34 and with the 22q11.22 deletion that covers IGLL5 and PRAME. The total number of gene copy number aberrations, so-called genomic imbalance complexity, was decreased in cases with the c-Rel signature. These cases exhibited a better overall survival. Functionally, overexpression of c-Rel induced its constitutive nuclear localization and protected cells against apoptosis while its repression tended to increase cell death. These results show that, clinically and biologically, c-Rel is the pivotal NF-κB subunit in the GCB-DLBCL subgroup. Functionally, c-Rel overexpression could directly promote DLBCL tumorigenesis without need for further activation signals.

Changes in DNA-binding activity of transcription factors in the kidney of mice exposed to cadmium.

Cadmium (Cd) is a toxic heavy metal, long-term exposure to which causes renal damage associated with disruption in gene expression. Transcription factors whose activities were altered in the kidneys of mice exposed to Cd for 3 months were assessed using protein/DNA-binding assays. Female C57BL/6J mice were exposed to 300 ppm Cd in the diet for 3 months. Nuclear extracts of kidney were used for protein/DNA-binding assays. The concentration of Cd was approximately 100 ppm in mouse kidney, a level that did not induce renal toxicity. Among the 345 transcription factors evaluated, five transcription factors showed over a two-fold increase in their activities and 14 transcription factors showed a half-fold change in their activities after Cd exposure. These findings may provide new information about the causative transcription factors associated with Cd renal toxicity.

Identification and functional characterization of the transcription factor coding Dp1 gene in large yellow croaker Pseudosciaena crocea.

The transcription factor Dp1, as a binding partner, often forms a dimerization complex with typical E2F to play a central role in regulating gene expression during G1/S cell cycle progression. In this study, a full-length dp1 cDNA (Pcdp1) was successfully cloned and characterized from the large yellow croaker Pseudosciaena crocea. The nucleotidic sequence of Pcdp1 is 1,427 bp long with an open reading frame (ORF) of 1,239 bp encoding a putative protein of 412 amino acids, a 5'-untranslated region of 116 bp and a 3'-untranslated region of 70 bp. Prediction of protein domains showed that PcDp1 contains a DNA-binding domain (DBD) with a DEF box, a dimerization domain and an acidic region at C terminus with transcription activity. Homology comparisons indicated that PcDp1 shared the highest sequence identity of 98.55% with Oreochromis niloticus dp1, followed by 88.72% identity with Danio rerio dp1 and a relatively low identity of 78.91-80.55% with its mammalian and amphibian counterparts. The mRNA of Pcdp1 showed ubiquitously expression in all analyzed tissues, with the highest level of expression in the body kidney. Moderate expression levels of Pcdp1 was found in several immune-related tissues including the gills, head kidney and liver, indicating that PcDp1 might play an important role in osmotic pressure regulation and immune response of the large yellow croaker. The subcellular localization of PcDp1 revealed that it is mainly distributed in the cytoplasm both in COS-7 and parenchymal cells of the spleen, head kidney and kidney tissues. Furthermore, the recombinant PcDp1 exhibited DNA-binding activity to E2F site in vitro. In conclusion, these results indicated that PcDp1 may participate in immune regulation and provide a foundation for further study of the regulatory mechanism of Dp1 in teleosts.

AtMPK6-induced phosphorylation of AtERF72 enhances its DNA binding activity and interaction with TGA4/OBF4 in Arabidopsis.

The ethylene-responsive element binding factor (ERF) family is a large family of transcription factors involved in plant development and environmental stress responses. We previously reported the identification of 29 putative substrates of Mitogen-activated Protein Kinase3 (AtMPK3), AtMPK4 and AtMPK6, based on a solid-phase phosphorylation screening using a lambda phage expression library in Arabidopsis thaliana. In this study, a putative MPK substrate, AtERF72 (At3g16770), was strongly phosphorylated by AtMPK6 on the serine residue at position 151 (Ser151). AtERF72 binds to the GCC box (AGCCGCC) in the promoters of several pathogenesis-related (PR) genes and activates their transcription. We also show that the DNA-binding activity of AtERF72 is enhanced upon phosphorylation by AtMPK6 in vitro. In addition, transient co-expression experiments in Arabidopsis protoplasts revealed that effector constructs expressing a mutant variant of AtERF72, AtERF72S151D (carrying a Ser to aspartic acid [Asp] substitution at amino acid position 151) showed higher expression of the ß-glucuronidase (GUS) reporter gene driven by the GCC box element than effector constructs expressing the wild-type AtERF72. Furthermore, yeast two-hybrid assays revealed that the interaction between AtERF72S151D and TGA4/OBF4 was stronger than that between wild-type AtERF72 and TGA4/OBF4. Since AtERF72S151D is equivalent to AtERF72 phosphorylated by AtMPK6 at Ser151, these results suggest that the phosphorylation of AtERF72 by AtMPK6 triggers an event of transcriptional regulation from defence signalling in Arabidopsis.

Novel functional characterization of the insecticidal protein Vip3Aa on DNA binding activity.

The toxicity of Vip3Aa protein on insect pests is known, however, it remains unclear underlying the structure-dependent molecular function of the Vip3Aa protein. To investigate the novel function of the Vip3Aa protein, we isolated recombinant Vip3Aa protein. The recombinant Vip3Aa protein was mostly present as oligomeric form depending on the hydrophobic amino acid residue. We found that the oligomeric Vip3Aa protein specifically binds to nucleic acids, including single-stranded (ssDNA) and double-stranded DNA (dsDNA). The conformational and functional domains of the Vip3Aa protein were confirmed by separating the Vip3Aa full and Vip3Aa active (actVip3Aa) forms using size exclusion chromatography and nucleic acid binding activity. Interestingly, actVip3Aa protein had a conformational change and decreased DNA binding activity compared to that of the Vip3Aa full, suggesting that N-terminal part of the Vip3Aa play an important role in maintaining the conformation and nucleic acid binding activity. These studies highlight novel functional characterization of the insecticidal protein Vip3Aa on DNA binding activity and may be attributed to the protection of DNA from the damage caused by oxidative stress.

OsMFT2 is involved in the regulation of ABA signaling-mediated seed germination through interacting with OsbZIP23/66/72 in rice.

Seed germination is a complex process involving various physical and biochemical cues, determined by exogenous and endogenous factors. Here, we identified a gene, OsMFT2, that negatively regulates seed germination in rice. OsMFT2 knock-out lines exhibited pre-harvest sprouting, whereas OsMFT2 overexpression lines showed delayed germination. RNA expression profiling showed that OsMFT2 was specifically expressed in seeds. Subcellular localization indicated that OsMFT2 was a nuclear protein. Exogenous abscisic acid (ABA) treatment of imbibed seeds and seedlings indicated that OsMFT2 altered ABA sensitivity during seed germination and post-germination growth. In vivo and in vitro assays showed that three bZIP transcription factors, OsbZIP23, OsbZIP66 and OsbZIP72, interacted with OsMFT2. OsbZIP23/66/72 bound to the promoter of Rab16A, a typical gene containing the ABA-responsive element, and OsMFT2 enhanced the binding to the Rab16A promoter. Moreover, several ABA-responsive genes were differentially expressed in the imbibed seeds of OsMFT2 transgenic lines and the wild type. The performance of the transgenic plants demonstrated that overexpressing OsbZIP23 rescued the pre-harvest sprouting phenotype and the decrease in ABA-signaling genes expression caused by OsMFT2 knock-out. All of these results demonstrate that OsMFT2 positively regulates ABA-responsive genes through interacting with OsbZIP23/66/72 and functions in seed germination.

The Helicobacter pylori HspR-Modulator CbpA Is a Multifunctional Heat-Shock Protein.

The medically important human pathogen Helicobacter pylori relies on a collection of highly conserved heat-shock and chaperone proteins to preserve the integrity of cellular polypeptides and to control their homeostasis in response to external stress and changing environmental conditions. Among this set of chaperones, the CbpA protein has been shown to play a regulatory role in heat-shock gene regulation by directly interacting with the master stress-responsive repressor HspR. Apart from this regulatory role, little is known so far about CbpA functional activities. Using biochemistry and molecular biology approaches, we have started the in vitro functional characterization of H. pylori CbpA. Specifically, we show that CbpA is a multifunctional protein, being able to bind DNA and to stimulate the ATPase activity of the major chaperone DnaK. In addition, we report a preliminary observation suggesting that CbpA DNA-binding activity can be affected by the direct interaction with the heat-shock master repressor HspR, supporting the hypothesis of a reciprocal crosstalk between these two proteins. Thus, our work defines novel functions for H. pylori CbpA and stimulates further studies aimed at the comprehension of the complex regulatory interplay among chaperones and heat-shock transcriptional regulators.

Consensus protein engineering on the thermostable histone-like bacterial protein HUs significantly improves stability and DNA binding affinity.

Consensus-based protein engineering strategy has been applied to various proteins and it can lead to the design of proteins with enhanced biological performance. Histone-like HUs comprise a protein family with sequence variety within a highly conserved 3D-fold. HU function includes compacting and regulating bacterial DNA in a wide range of biological conditions in bacteria. To explore the possible impact of consensus-based design in the thermodynamic stability of HU proteins, the approach was applied using a dataset of sequences derived from a group of 40 mesostable, thermostable, and hyperthermostable HUs. The consensus-derived HU protein was named HUBest, since it is expected to perform best. The synthetic HU gene was overexpressed in E. coli and the recombinant protein was purified. Subsequently, HUBest was characterized concerning its correct folding and thermodynamic stability, as well as its ability to interact with plasmid DNA. A substantial increase in HUBest stability at high temperatures is observed. HUBest has significantly improved biological performance at ambience temperature, presenting very low Kd values for binding plasmid DNA as indicated from the Gibbs energy profile of HUBest. This Kd may be associated to conformational changes leading to decreased thermodynamic stability and, therefore, higher flexibility at ambient temperature.

Coordinated Conformational Processing of the Tumor Suppressor Protein p53 by the Hsp70 and Hsp90 Chaperone Machineries.

p53, the guardian of the genome, requires chaperoning by Hsp70 and Hsp90. However, how the two chaperone machineries affect p53 conformation and regulate its function remains elusive. We found that Hsp70, together with Hsp40, unfolds p53 in an ATP-dependent reaction. This unfolded state of p53 is susceptible to aggregation after release induced by the nucleotide exchange factor Bag-1. However, when Hsp90 and the adaptor protein Hop are present, p53 is transferred from Hsp70 to Hsp90, allowing restoration of the native state upon ATP hydrolysis. Our results suggest that the p53 conformation is constantly remodeled by the two major chaperone machineries. This connects p53 activity to stress, and the levels of free molecular chaperones are important factors regulating p53 activity. Together, our findings reveal an intricate interplay and cooperation of Hsp70 and Hsp90 in regulating the conformation of a client.

DNA-binding activity of STAT3 increased in hypothalamus of DIO mice; the reduction of STAT3 phosphorylation may facilitate leptin signaling.

Leptin-mediated DNA-binding activity of STAT3 in hypothalamus plays crucial roles in the maintenance of energy homeostasis in lean mice; however its effects still remains unclear in case of leptin resistance in mice with diet induced obesity (DIO). In this study significant elevation of both basal and exogenously leptin-treated DNA-binding activity of STAT3 was detected using EMSA in the hypothalamus of male C57BL/6J mice fed high-fat diet for 10 wks, in concomitant with hyperleptinemia, high body weight, high fat mass, and hyperphagia as well as decreased POMC expression. The studies in vitro showed that both DNA binding activity and the proximal SBE of POMC promoter was essential to leptin-mediated POMC expression. However, the diminution of STAT3 phosphorylation, achieved by S3I-201 or a FoxO1 mutant, facilitated leptin-mediated POMC expression. The findings here demonstrated excess STAT3 activity negatively regulated POMC expression in hypothalamus of DIO mice, and suggested the limitation of STAT3 activity may promote leptin signaling.

Determining the Roles of a Conserved α-Helix in a Global Virulence Regulator from Staphylococcus aureus.

SarA, a pleiotropic transcription regulator, is encoded by Staphylococcus aureus, a pathogenic bacterium. The expression of many virulence and non-virulence genes in S. aureus is modulated by this regulator. Structural studies have shown it to be a winged-helix DNA-binding protein carrying two monomers. Each SarA monomer is composed of five α-helices (α1-α5), three ß-strands (ß1-ß3) and multiple loops. The putative DNA binding region of SarA is constituted with α3, α4, ß2, and ß3, whereas, its dimerization seems to occur using α1, α2, and α5. Interestingly, many SarA-like proteins are dimeric and use three or more helices for their dimerization. To clearly understand the roles of helix α1 in the dimerization, we have constructed and purified a SarA mutant (Δα1) that lacks helix α1. Our in-depth studies with Δα1 indicate that the helix α1 is critical for preserving the structure, DNA binding activity and thermodynamic stability of SarA. However, the helix has little affected its dimerization ability. Possible reasons for such anomaly have been discussed at length.

Identification and characterization of a CI binding operator at a distant location in the temperate staphylococcal phage ф11.

Bacteriophage ф11 encodes repressors CI and Cro for executing its growth in Staphylococcus aureus, a human pathogen. There are three homologous operators O1, O2 and O3 between the repressor-expressing genes. While CI binds to O1 and O2, Cro interacts only with O3. To locate additional CI binding operators in ф11, we searched its genome using the O1/O2 sequence as a probe. The results show the presence of a putative CI binding operator (O4) at the 3΄ end of the cro. O4 differs from O2 and O1 by one base and five bases, respectively. A specific interaction was noticed between O4 and rCI, a recombinant CI. However, O4 shows no interaction with rCro, a chimeric Cro. Additionally, six guanine bases, situated in and around O4, have interacted with rCI. Interestingly, the rCI binding affinity of O4 or O1 is about 15 times higher than that of O2. A comparative study indicates that some bases and structural alteration, unique to O1 and O4, may contribute to their enhanced rCI binding affinity. Collectively, the study has not only broadened the distinct gene regulatory circuit of ф11 but also suggested that it possibly employs a complex mechanism for its development in S. aureus.