Sequence-specific Binding Protein Research Articles

The Musashi RNA binding proteins direct the translational activation of key pituitary mRNAs

The pituitary functions as a master endocrine gland that secretes hormones critical for regulation of a wide variety of physiological processes including reproduction, growth, metabolism and stress responses. The distinct hormone-producing cell lineages within the pituitary display remarkable levels of cell plasticity that allow remodeling of the relative proportions of each hormone-producing cell population to meet organismal demands. The molecular mechanisms governing pituitary cell plasticity have not been fully elucidated. Our recent studies have implicated a role for the Musashi family of sequence-specific mRNA binding proteins in the control of pituitary hormone production, pituitary responses to hypothalamic stimulation and modulation of pituitary transcription factor expression in response to leptin signaling. To date, these actions of Musashi in the pituitary appear to be mediated through translational repression of the target mRNAs. Here, we report Musashi1 directs the translational activation, rather than repression, of the Prop1, Gata2 and Nr5a1 mRNAs which encode key pituitary lineage specification factors. We observe that Musashi1 further directs the translational activation of the mRNA encoding the glycolipid Neuronatin (Nnat) as determined both in mRNA reporter assays as well as in vivo. Our findings suggest a complex bifunctional role for Musashi1 in the control of pituitary cell function.

RF25 | PMON50 Insight into Integrated Control of Pituitary Function Revealed Through Analysis of Musashi Target mRNAs

Abstract The anterior pituitary controls growth, metabolism, reproduction and stress responses through the synthesis and secretion of specific hormones. Distinct cell lineages within the anterior pituitary generate specific hormones. Interestingly, the anterior pituitary exhibits a high level of adult cell plasticity that allows committed hormone-producing cells to alter their cell fate and produce different hormones to meet altered organismal demands. The molecular mechanisms underlying this adult cell plasticity have not been fully characterized. Our recent work has implicated the stem cell determinant and sequence-specific mRNA binding protein, Musashi, as a regulator of pituitary cell plasticity. The mechanisms by which Musashi controls pituitary cell plasticity are unknown. In this study, we have sought to identify the mRNA regulatory targets of Musashi in the adult mouse pituitary. Through the use of Musashi-RNA-immunoprecipitation we report a cohort of 1192 pituitary mRNAs that specifically interact with Musashi. mRNAs specific to each of the hormone-producing cell lineages were identified, as well as mRNAs specific to immature stem and progenitor cell lineages. The biological pathways encoded by the Musashi-enriched mRNAs include cell adhesion, cellular homeostasis, unfolded protein responses, protein trafficking, endocrine processes and female pregnancy. Functional analysis of validated mRNAs reveals both activating and inhibitory control of mRNA translation by Musashi. Together, our findings indicate a broad and complex role for Musashi in the post-transcriptional regulation of adult pituitary function. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:58 p.m. - 1:03 p.m.

A Genome-Wide Analysis of StTGA Genes Reveals the Critical Role in Enhanced Bacterial Wilt Tolerance in Potato During Ralstonia solanacearum Infection.

TGA is one of the members of TGACG sequence-specific binding protein family, which plays a crucial role in the regulated course of hormone synthesis as a stress-responsive transcription factor (TF). Little is known, however, about its implication in response to bacterial wilt disease in potato (Solanum tuberosum) caused by Ralstonia solanacearum. Here, we performed an in silico identification and analysis of the members of the TGA family based on the whole genome data of potato. In total, 42 StTGAs were predicted to be distributed on four chromosomes in potato genome. Phylogenetic analysis showed that the proteins of StTGAs could be divided into six sub-families. We found that many of these genes have more than one exon according to the conserved motif and gene structure analysis. The heat map inferred that StTGAs are generally expressed in different tissues which are at different stages of development. Genomic collinear analysis showed that there are homologous relationships among potato, tomato, pepper, Arabidopsis, and tobacco TGA genes. Cis-element in silico analysis predicted that there may be many cis-acting elements related to abiotic and biotic stress upstream of StTGA promoter including plant hormone response elements. A representative member StTGA39 was selected to investigate the potential function of the StTGA genes for further analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) assays indicated that the expression of the StTGAs was significantly induced by R. solanacearum infection and upregulated by exogenous salicylic acid (SA), abscisic acid (ABA), gibberellin 3 (GA3), and methyl jasmonate (MeJA). The results of yeast one-hybrid (Y1H) assay showed that StTGA39 regulates S. tuberosum BRI1-associated receptor kinase 1 (StBAK1) expression. Thus, our study provides a theoretical basis for further research of the molecular mechanism of the StTGA gene of potato tolerance to bacterial wilt.

CYSTEINE-RICH RECEPTOR-LIKE KINASE5 (CRK5) and CRK22 regulate the response to Verticillium dahliae toxins.

Cysteine-rich receptor-like kinases (CRKs) play critical roles in responses to biotic and abiotic stresses. However, the molecular mechanisms of CRKs in plant defense responses remain unknown. Here, we demonstrated that two CRKs, CRK5 and CRK22, are involved in regulating defense responses to Verticillium dahliae toxins (Vd-toxins) in Arabidopsis (Arabidopsis thaliana). Biochemical and genetic analyses showed that CRK5 and CRK22 may act upstream of MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6 to regulate the salicylic acid (SA)-signaling pathway in response to Vd-toxins. In addition, MPK3 and MPK6 interact with the transcription factor WRKY70 to modulate defense responses to Vd-toxins. WRKY70 directly binds the promoter domains of the SA-signaling-related transcription factor genes TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA2) and TGA6 to regulate their expression in response to Vd-toxins. Thus, our study reveals a mechanism by which CRK5 and CRK22 regulate SA signaling through the MPK3/6-WRKY70-TGA2/6 pathway in response to Vd-toxins.

Sequence specificity in DNA binding is mainly determined by association rather than dissociation

Sequence-specific binding of proteins to DNA is essential for gene regulation. We have derived a simple equation for target-site recognition, which sheds light on a previously unrecognized coupling between the macroscopic association and dissociation rates of the searching protein. Importantly, this relationship makes it possible to recover the relevant microscopic rates from experimentally determined macroscopic ones. To test the equation, we have developed a method for doing kinetic measurements on thousands of DNA sequences simultaneously on a protein binding microarray.

Sequence specificity in DNA binding is mainly governed by association.

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.

A Ralstonia solanacearum effector targets TGA transcription factors to subvert salicylic acid signaling.

The bacterial pathogen Ralstonia solanacearum causes wilt disease on Arabidopsis thaliana and tomato (Solanum lycopersicum). This pathogen uses type III effectors to inhibit the plant immune system; however, how individual effectors interfere with plant immune responses, including transcriptional reprograming, remain elusive. Here, we show that the type III effector RipAB targets Arabidopsis TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors, the central regulators of plant immune gene regulation, via physical interaction in the nucleus to dampen immune responses. RipAB was required for R. solanacearum virulence on wild-type tomato and Arabidopsis but not Arabidopsis tga1 tga4 and tga2 tga5 tga6 mutants. Stable expression of RipAB in Arabidopsis suppressed the pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) burst and immune gene induction as well as salicylic acid (SA) regulons including RBOHD and RBOHF, responsible for ROS production, all of which were phenocopied by the tga1 tga4 and tga2 tga5 tga6 mutants. We found that TGAs directly activate RBOHD and RBOHF expression and that RipAB inhibits this through interfering with the recruitment of RNA polymerase II. These results suggest that TGAs are the bona fide and major virulence targets of RipAB, which disrupts SA signaling by inhibiting TGA activity to achieve successful infection.

N-hydroxypipecolic acid-induced transcription requires the salicylic acid signaling pathway at basal SA levels.

Systemic acquired resistance (SAR) is a plant immune response established in uninfected leaves after colonization of local leaves with biotrophic or hemibiotrophic pathogens. The amino acid-derived metabolite N-hydroxypipecolic acid (NHP) travels from infected to systemic leaves, where it activates salicylic acid (SA) biosynthesis through the isochorismate pathway. The resulting increased SA levels are essential for induction of a large set of SAR marker genes and full SAR establishment. In this study, we show that pharmacological treatment of Arabidopsis thaliana with NHP induces a subset of SAR-related genes even in the SA induction-deficient2 (sid2/isochorismate synthase1) mutant, which is devoid of NHP-induced SA. NHP-mediated induction is abolished in sid2-1 NahG plants, in which basal SA levels are degraded. The SA receptor NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and its interacting TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors are required for the NHP-mediated induction of SAR genes at resting SA levels. Isothermal titration analysis determined a KD of 7.9 ± 0.5 µM for the SA/NPR1 complex, suggesting that basal levels of SA would not bind to NPR1 unless yet unknown potentially NHP-induced processes increase the affinity. Moreover, the nucleocytoplasmic protein PHYTOALEXIN DEFICIENT4 is required for a slight NHP-mediated increase in NPR1 protein levels and NHP-induced expression of SAR-related genes. Our experiments have unraveled that NHP requires basal SA and components of the SA signaling pathway to induce SAR genes. Still, the mechanism of NHP perception remains enigmatic.

Understanding the expression of signalling pathway marker genes associated with bacterial wilt in susceptible and resistant ginger sps

Ralstonia solanacearum causes bacterial wilt in ginger. Here the role of signalling pathways such as salicylic acid (SA), ethylene (ET) and jasmonic acid (JA) was studied by comparing the differential expression of the marker genes for these pathways during bacterial wilt in both susceptible (ginger, Zingiber officinale) and resistant (mango ginger, Curcuma amada) ginger species. When expression of marker genes was compared among susceptible and resistant ginger sps., SA pathway marker genes, non-expressor of PR1 genes (NPR1) and TGACG sequence-specific binding protein (TGA6) and the ET pathway marker genes, AP2 domain class transcription factor (AP2) and 4-Coumarate: CoA ligase (4CL1), were expressed with a statistically different (p < 0.05) fold change among susceptible and resistant ginger sps in all time intervals. Expression of marker genes in resistant ginger showed higher expression in the first-hour post inoculation itself; however, marker genes of susceptible ginger had a slow pace in expression. Even though the marker genes of JA such as allene oxide cyclase (AOC) and lipoxygenase (LOX2) were activated in the initial hour post-inoculation in both ginger sps, their expression of these marker genes were significantly at par (p < 0.05) among ginger species in almost all time intervals. These results indicate that even though JA is activated upon infection, SA and ET signalling pathways play an essential role in giving resistance to resistant ginger. Moreover, the time and magnitude of expression play a higher impact on host-pathogen resistance. Understanding the role of signalling pathways involved in bacterial wilt resistance in resistant ginger can provide insights into host-pathogen resistance in ginger, which can help develop wilt-resistant ginger.

Origin, Diversity, and Evolution of Telomere Sequences in Plants.

Telomeres are basic structures of eukaryote genomes. They distinguish natural chromosome ends from double-stranded breaks in DNA and protect chromosome ends from degradation or end-to-end fusion with other chromosomes. Telomere sequences are usually tandemly arranged minisatellites, typically following the formula (TxAyGz)n. Although they are well conserved across large groups of organisms, recent findings in plants imply that their diversity has been underestimated. Changes in telomeres are of enormous evolutionary importance as they can affect whole-genome stability. Even a small change in the telomere motif of each repeat unit represents an important interference in the system of sequence-specific telomere binding proteins. Here, we provide an overview of telomere sequences, considering the latest phylogenomic evolutionary framework of plants in the broad sense (Archaeplastida), in which new telomeric sequences have recently been found in diverse and economically important families such as Solanaceae and Amaryllidaceae. In the family Lentibulariaceae and in many groups of green algae, deviations from the typical plant telomeric sequence have also been detected recently. Ancestry and possible homoplasy in telomeric motifs, as well as extant gaps in knowledge are discussed. With the increasing availability of genomic approaches, it is likely that more telomeric diversity will be uncovered in the future. We also discuss basic methods used for telomere identification and we explain the implications of the recent discovery of plant telomerase RNA on further research about the role of telomerase in eukaryogenesis or on the molecular causes and consequences of telomere variability.

I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

Diverse DNA Sequence Motifs Activate Meiotic Recombination Hotspots Through a Common Chromatin Remodeling Pathway.

In meiosis, multiple different DNA sequence motifs help to position homologous recombination at hotspots in the genome. How do the seemingly disparate cis-acting regulatory modules each promote locally the activity of the basal recombination machinery? We defined molecular mechanisms of action for five different hotspot-activating DNA motifs (M26, CCAAT, Oligo-C, 4095, 4156) located independently at the same site within the ade6 locus of the fission yeast Schizosaccharomyces pombe Each motif promoted meiotic recombination (i.e., is active) within this context, and this activity required the respective binding proteins (transcription factors Atf1, Pcr1, Php2, Php3, Php5, Rst2). High-resolution analyses of chromatin structure by nucleosome scanning assays revealed that each motif triggers the displacement of nucleosomes surrounding the hotspot motif in meiosis. This chromatin remodeling required the respective sequence-specific binding proteins, was constitutive for two motifs, and was enhanced meiotically for three others. Hotspot activity of each motif strongly required the ATP-dependent chromatin remodeling enzyme Snf22 (Snf2/Swi2), with lesser dependence on Gcn5, Mst2, and Hrp3. These findings support a model in which most meiotic recombination hotspots are positioned by the binding of transcription factors to their respective DNA sites. The functional redundancy of multiple, sequence-specific protein-DNA complexes converges upon shared chromatin remodeling pathways that help provide the basal recombination machinery (Spo11/Rec12 complex) access to its DNA substrates within chromatin.

Human Artificial Chromosomes that Bypass Centromeric DNA

SummaryRecent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere—the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B. We report the development of a type of HAC that functions independently of these constraints. Formed by an initial CENP-A nucleosome seeding strategy, a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts.

Investigating the effect of charged amino acids on DNA conformation in EcoRI–DNA complex: a molecular dynamics simulation study

Sequence-specific binding of proteins to DNA is essential for almost all the cellular processes like transcription, translation, replication, etc. One among the various mechanisms that has been identified so far that contributes to the specificity in protein–DNA interaction is the DNA conformational change. Electrostatic neutralization of the phosphate groups by the positively charged amino acid residues in proteins is thought to bring about such conformational changes in DNA. Here, we employ molecular dynamics simulations to examine the effect of charge on amino acids Lys113, Arg145, and Asp91 which are attached to the scissile phosphate on the conformation of DNA in EcoRI–DNA complex. The results indicate that the charge of these amino acids is essential for maintaining the local conformation of DNA in the EcoRI-bound form. Interestingly, we observe that the positively charged amino acids Lys113 and Arg145 have a long-range influence on the DNA conformation, whereas the negatively charged amino acid Asp91 has only a localized effect on the DNA conformation. The charge on the amino acids also alters the collective dynamics of EcoRI. Collectively, the results shed light on the diversity of the effect of charges on DNA conformation as well as on protein dynamics.

RNA Binding: Getting Specific about Specificity

Sequence-specific nucleic acid binding proteins do not recognize one sequence out of all possibilities; rather, they bind to many sequences with a range of affinities. In this issue of Cell Chemical Biology, Lin etal. (2016), describe the entire landscape of affinities between different RNA molecules and an RNA-binding protein, thus providing a comprehensive description of the factors affecting specificity.

Preparation of Chromatin Templates to Study RNA Polymerase I Transcription In Vitro.

Cellular DNA is packaged into chromatin, which is the substrate of all DNA-dependent processes in eukaryotes. The regulation of chromatin requires specialized enzyme activities to allow the access of sequence-specific binding proteins and RNA polymerases. In order to dissect chromatin-dependent features of transcription regulation in detail, in vitro systems to generate defined chromatin templates for transcription are required. I present a protocol that allows the assembly of nucleosomes on ribosomal RNA (rRNA) minigenes by salt gradient dialysis and subsequent sucrose gradient centrifugation. This procedure yields high nucleosome occupancy and high dynamic response in subsequent transcriptional analysis. It provides an invaluable tool to study rRNA gene transcription, as transcription on free DNA is clearly different from the more in vivo-like transcription on reconstituted chromatin templates.

Identification of MEF2B , EBF1 , and IL6R as Direct Gene Targets of Epstein-Barr Virus (EBV) Nuclear Antigen 1 Critical for EBV-Infected B-Lymphocyte Survival

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the EBV-encoded nuclear antigen and sequence-specific DNA binding protein required for viral origin binding and episome maintenance during latency. EBNA1 can also bind to numerous sites in the cellular genome and can provide a host cell survival function, but it is not yet known how EBNA1 sequence-specific binding is responsible for host cell survival. Here, we integrate EBNA1 chromatin immunoprecipitation sequencing (ChIP-Seq) with transcriptome sequencing (RNA-Seq) after EBNA1 depletion to identify cellular genes directly regulated by EBNA1 that are also essential for B-cell survival. We first compared EBNA1 ChIP-Seq patterns in four different EBV-positive cell types, including Burkitt lymphoma (BL) cells, nasopharyngeal carcinoma (NPC) cells, and lymphoblastoid cell lines (LCLs). EBNA1 binds to ~1,000 sites that are mostly invariant among cell types and share a consensus recognition motif. We found that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R, and EBF1. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1 depletion by decreasing the cell growth and viability of cells latently infected with EBV. These findings suggest that EBNA1 binds to a large cohort of cellular genes important for cell viability and implicates EBNA1 as a critical regulator of transcription of host cell genes important for enhanced survival of latently infected cells. Epstein-Barr virus (EBV) latent infection is responsible for a variety of lymphoid and epithelial cell malignancies. EBNA1 is the EBV-encoded nuclear antigen that is consistently expressed in all EBV-associated cancers. EBNA1 is known to provide a host cell survival function, but the mechanism is not known. EBNA1 is a sequence-specific binding protein important for viral genome maintenance during latency. Here, by integrating ChIP-Seq and RNA-Seq, we demonstrate that EBNA1 binds directly to the promoter regulatory regions and upregulates the transcription of host genes that are important for the survival of EBV-infected cells. Identification of EBNA1 target genes provides potential new targets for therapeutic intervention in EBV-associated disease.

DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity of Human Centromere Function

Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere position and function through a two-step mechanism relying on self-templating centromeric chromatin assembled with the histone H3 variant CENP-A, followed by CENP-A-dependent nucleation of kinetochore assembly. Nevertheless, natural human centromeres are positioned within specific megabase chromosomal regions containing α-satellite DNA repeats, which contain binding sites for the DNA sequence-specific binding protein CENP-B. We now demonstrate that CENP-B directly binds both CENP-A's amino-terminal tail and CENP-C, a key nucleator of kinetochore assembly. DNA sequence-dependent binding of CENP-B within α-satellite repeats is required to stabilize optimal centromeric levels of CENP-C. Chromosomes bearing centromeres without bound CENP-B, including the human Y chromosome, are shown to mis-segregate in cells at rates several-fold higher than chromosomes withCENP-B-containing centromeres. These data demonstrate a DNA sequence-specific enhancement by CENP-B of the fidelity of epigenetically defined human centromere function.

Specific minor groove solvation is a crucial determinant of DNA binding site recognition.

The DNA sequence preferences of nearly all sequence specific DNA binding proteins are influenced by the identities of bases that are not directly contacted by protein. Discrimination between non-contacted base sequences is commonly based on the differential abilities of DNA sequences to allow narrowing of the DNA minor groove. However, the factors that govern the propensity of minor groove narrowing are not completely understood. Here we show that the differential abilities of various DNA sequences to support formation of a highly ordered and stable minor groove solvation network are a key determinant of non-contacted base recognition by a sequence-specific binding protein. In addition, disrupting the solvent network in the non-contacted region of the binding site alters the protein's ability to recognize contacted base sequences at positions 5–6 bases away. This observation suggests that DNA solvent interactions link contacted and non-contacted base recognition by the protein.

Cytoplasmic polyadenylation element binding proteins in development, health, and disease.

The cytoplasmic polyadenylation element binding (CPEB) proteins are sequence-specific mRNA binding proteins that control translation in development, health, and disease. CPEB1, the founding member of this family, has become an important model for illustrating general principles of translational control by cytoplasmic polyadenylation in gametogenesis, cancer etiology, synaptic plasticity, learning, and memory. Although the biological functions of the other members of this protein family in vertebrates are just beginning to emerge, it is already evident that they, too, mediate important processes, such as cancer etiology and higher cognitive function. In Drosophila, the CPEB proteins Orb and Orb2 play key roles in oogenesis and in neuronal function, as do related proteins in Caenorhabditis elegans and Aplysia. We review the biochemical features of the CPEB proteins, discuss their activities in several biological systems, and illustrate how understanding CPEB activity in model organisms has an important impact on neurological disease.