Yeast Transcription Factor Research Articles

Organization of DNA in Mammalian Mitochondria.

As with all organisms that must organize and condense their DNA to fit within the limited volume of a cell or a nucleus, mammalian mitochondrial DNA (mtDNA) is packaged into nucleoprotein structures called nucleoids. In this study, we first introduce the general modes of DNA compaction, especially the role of the nucleoid-associated proteins (NAPs) that structure the bacterial chromosome. We then present the mitochondrial nucleoid and the main factors responsible for packaging of mtDNA: ARS- (autonomously replicating sequence-) binding factor 2 protein (Abf2p) in yeast and mitochondrial transcription factor A (TFAM) in mammals. We summarize the single-molecule manipulation experiments on mtDNA compaction and visualization of mitochondrial nucleoids that have led to our current knowledge on mtDNA compaction. Lastly, we discuss the possible regulatory role of DNA packaging by TFAM in DNA transactions such as mtDNA replication and transcription.

The Role of Transcription Factors and Nuclear Pore Proteins in Controlling the Spatial Organization of the Yeast Genome

Loss of nuclear pore complex (NPC) proteins, transcription factors (TFs), histone modification enzymes, Mediator, and factors involved in mRNA export disrupts the physical interaction of chromosomal sites with NPCs. Conditional inactivation and ectopic tethering experiments support a direct role for the TFs Gcn4 and Nup2 in mediating interaction with the NPC but suggest an indirect role for factors involved in mRNA export or transcription. A conserved "positioning domain" within Gcn4 controls interaction with the NPC and inter-chromosomal clustering and promotes transcription of target genes. Such a function may be quite common; a comprehensive screen reveals that tethering of most yeast TFs is sufficient to promote targeting to the NPC. While some TFs require Nup100, others do not, suggesting two distinct targeting mechanisms. These results highlight an important and underappreciated function of TFs in controlling the spatial organization of the yeast genome through interaction with the NPC.

A conserved role for transcription factor sumoylation in binding-site selection.

Large numbers of eukaryotic transcription factors (TFs) are modified by SUMO post-translational modifications. Whereas the effect of TF sumoylation on the expression of target genes is largely context-dependent, it is not known whether the modification has a common function in regulating TFs throughout eukaryotic species. Here, I highlight four studies that used genome-wide chromatin-immunoprecipitation analysis (ChIP-seq) to examine whether sumoylation affects the selection of sites on the genome that are bound by human and yeast TFs. The studies found that impairing sumoylation led to deregulated binding-site selection for all four of the examined TFs. Predominantly, compared to wild-type forms, the sumoylation-deficient forms of the TFs bound to numerous additional non-specific sites, pointing to a common role for the modification in restricting TF binding to appropriate sites. Evidence from these studies suggests that TF sumoylation influences binding-site selection by modulating protein-protein interactions with other DNA-binding TFs, or by promoting conformational changes in the TFs that alter their DNA-binding specificity or affinity. I propose a model in which, prior to their sumoylation, TFs initially bind to chromatin with reduced specificity, which leads to spurious binding but also ensures that all functional sites become bound. Once the TFs are bound to DNA, sumoylation then acts to increase specificity and promotes release of the TFs from non-specific sites. The similar observations from these four genome-wide studies across divergent species suggest that binding-site selection is a general and conserved function for TF sumoylation.

Predictive models of eukaryotic transcriptional regulation reveals changes in transcription factor roles and promoter usage between metabolic conditions.

Transcription factors (TF) are central to transcriptional regulation, but they are often studied in relative isolation and without close control of the metabolic state of the cell. Here, we describe genome-wide binding (by ChIP-exo) of 15 yeast TFs in four chemostat conditions that cover a range of metabolic states. We integrate this data with transcriptomics and six additional recently mapped TFs to identify predictive models describing how TFs control gene expression in different metabolic conditions. Contributions by TFs to gene regulation are predicted to be mostly activating, additive and well approximated by assuming linear effects from TF binding signal. Notably, using TF binding peaks from peak finding algorithms gave distinctly worse predictions than simply summing the low-noise and high-resolution TF ChIP-exo reads on promoters. Finally, we discover indications of a novel functional role for three TFs; Gcn4, Ert1 and Sut1 during nitrogen limited aerobic fermentation. In only this condition, the three TFs have correlated binding to a large number of genes (enriched for glycolytic and translation processes) and a negative correlation to target gene transcript levels.

Suppression of the yeast elongation factor Spt4 ortholog reduces expanded SCA36 GGCCUG repeat aggregation and cytotoxicity

A hexanucleotide GGCCTG repeat expansion in intron 1 of the nucleolar protein 56 gene causes spinocerebellar ataxia type 36 (SCA36), which is a relatively pure cerebellar ataxia with progressive motor neuron involvement. In this study SCA36 cell models were generated by introducing expanded GGCCTG/CAGGCC repeats into cultured Neuro2A cells. Sense (GGCCUG)exp but not antisense (CAGGCC)exp RNA foci were detected in the cells, consistent with observations in autopsied brains of patients with SCA36. Glycine-proline dipeptide repeat (DPR) formation due to repeat-associated non-ATG translation rarely occurred in cells expressing expanded GGCCTG repeats; in contrast, cells harboring expanded c9orf72 GGGGCC/GGCCCC repeats robustly expressed DPR proteins. There are currently no effective treatments for microsatellite repeat expansion diseases including SCA36. In order to identify potentially useful therapies, we screened five candidate chemical compounds for their ability to diminish the toxicity of expanded SCA36 repeats and evaluated whether small interfering RNA-mediated silencing of Supt4a/Supt5, the murine ortholog of the yeast transcriptional elongation factor Spt4/Spt5, has therapeutic potential based on RNA foci quantification and cytotoxicity assays. Supt4a knockdown and erythromycin treatment suppressed the formation of (GGCCUG)exp RNA foci and DPR protein formation via regulation of (GGCCUG)exp mRNA, thereby ameliorating the cytotoxicity in SCA36 cell models. These data provide a basis for developing effective therapeutic strategies for the treatment of SCA36 and other repeat expansion disorders.

The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris

Rtg1p and Rtg3p are two basic helix-loop-helix, retrograde transcription factors in the budding yeast Saccharomyces cerevisiae Both factors heterodimerize to activate the transcription of nuclear genes in response to mitochondrial dysfunction and glutamate auxotrophy, but are not well characterized in other yeasts. Here, we demonstrate that the Rtg1p/Rtg3p-mediated retrograde signaling pathway is absent in the methylotrophic yeast Pichia pastoris We observed that P. pastoris Rtg1p (PpRtg1p) heterodimerizes with S. cerevisiae Rtg3p and functions as a nuclear, retrograde transcription factor in S. cerevisiae, but not in P. pastoris. We noted that P. pastoris Rtg3p lacks a functional leucine zipper and interacts with neither S. cerevisiae Rtg1p (ScRtg1p) nor PpRtg1p. In the absence of an interaction with Rtg3p, PpRtg1p has apparently acquired a novel function as a cytosolic regulator of multiple P. pastoris metabolic pathways, including biosynthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase required for the utilization of glutamate as the sole carbon source. PpRtg1p also had an essential role in methanol metabolism and regulated alcohol oxidase synthesis and was required for the metabolism of ethanol, acetate, and oleic acid, but not of glucose and glycerol. Although PpRtg1p could functionally complement ScRtg1p, ScRtg1p could not complement PpRtg1p, indicating that ScRtg1p is not a functional PpRtg1p homolog. Thus, PpRtg1p functions as a nuclear, retrograde transcription factor in S. cerevisiae and as a cytosolic, post-transcriptional regulator in P. pastoris We conclude that PpRtg1p is a key component of a signaling pathway that regulates multiple metabolic processes in P. pastoris.

Abstract B34: PD-0332991/palbociclib facilitates the physical interaction between RB1 and BIN1 to increase the vulnerability of pediatric osteosarcoma cells to PARP inhibition

Abstract Osteosarcoma (OS) is a sporadic bone malignancy that is diagnosed mainly in childhood and adolescence. Conventional genotoxic chemotherapy such as cisplatin and doxorubicin has been used as first-line therapy for pediatric OS patients. However, OS cells eventually acquire chemoresistance with poor prognosis. Once OS cells acquire chemoresistance, little or no therapeutic option is available. It is therefore critical to better understand genetic, epigenetic, and signaling alterations in OS cells, by which a novel mechanism-based therapeutic strategy to halt the development of OS may be established. Although there is no single genetic event leading to the development of OS, increased mortality, metastasis, and chemoresistance are frequently observed in pediatric OS patients deficient in the retinoblastoma 1 tumor suppressor protein (RB1), compared to RB1-positive OS cases. These facts suggest that the activation and maintenance of RB1 function may improve the efficacy of OS chemotherapy. In this study, we found that, particularly when RB1 is hypophosphorylated, RB1 physically interacts with the proapoptotic tumor suppressor BIN1 in the human OS cell line U2OS for growth suppression. The adenovirus type 5 E1A oncoprotein disrupted the physical RB1/BIN1 complex, which implies that the protein complex is functionally meaningful as potential anticancer machinery. Acting through the coiled-coil effector domain of BIN1, BIN1 physically interacted with the C pocket domain of RB1 (RB1-C) in vitro. Consistent with this, heterologous expression of BIN1 hindered the effect of serum stimulation on phosphorylation of RB1-C at serine 807/811, which is mediated by cyclin-dependent kinases 4 and 6 (CDK4/6). The transcriptional repression caused by a recombinant BIN1 protein fused with the DNA-binding domain of the yeast transcription factor GAL4 was counterbalanced by suberoylanilide hydroxamic acid (SAHA), a chemical inhibitor of histone deacetylases (HDAC), only in the presence of RB1. This suggests that the BIN1-dependent gene repression greatly requires an HDAC activity employed by RB1. As predicted, in vitro BIN1-induced cancer suppression was compromised by the cotransfection of RB1 short-hairpin RNA. Of note, PD-0332991/palbociclib, a U.S. FDA-approved chemical inhibitor of CDK4/6, which is known to induce hypophosphorylated RB1, stabilized the RB1/BIN1 complex in U2OS cells and markedly increased the susceptibility to olaparib, an FDA-approved inhibitor of poly(ADP-ribose) polymerase (PARP), in a manner dependent on BIN1 and RB1. Because U2OS cells do not carry the molecular features with BRCA1/2-deficient tumors or “BRCAness,” our study suggests a novel BRCAness-independent synthetic lethal possibility activated by the combination of PD-0332991/palbociclib and olaparib in pediatric OS cells. Citation Format: Watson P. Folk, Kelly Homlar, Daitoku Sakamuro. PD-0332991/palbociclib facilitates the physical interaction between RB1 and BIN1 to increase the vulnerability of pediatric osteosarcoma cells to PARP inhibition [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B34.

Chebulinic acid and Boeravinone B act as anti-aging and anti-apoptosis phyto-molecules during oxidative stress

IntroductionAquatic pollutant Malachite green (MG) induces oxidative stress by producing intracellular H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals in Saccharomyces cerevisiae. These radicals disturb cellular functions leading to early aging. Exogenous supply of natural antioxidants may play a crucial role as anti-aging by ensuring the cellular survival. MethodsProtective effect of Chebulinic acid (CA) and Boeravinone B (BB) was biochemically evaluated by measuring the expression levels of antioxidant enzymes. Intracellular oxidants generation, nuclear damage, necrosis, apoptosis, reduction in caspase 3/7 activity studied microscopically, spectrofluorometrically and biochemically along with growth dynamics and relative quantitation of Yap1, Sir2 and Bir1 expression using RT-PCR. ResultsMalachite green (MG) showed adverse effect on S. cerevisiae showing 400.83% enhancement in accumulation of intracellular H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals. Independent supplementation of CA (5 μg/ml) and BB (3 μg/ml) significantly reduced the accumulation by 385.78 and 372.68%, respectively. Presence of MG extended the lag phase of growth curve and also reduced colony forming units (CFUs)/ml to 3 × 108 from 15 × 108. Whereas, CA and BB maintained the normal growth curve, CFUs and proved as anti-aging. Elevation in the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) by 241.35, 539.02 and 432.60% was observed after 2 h MG exposure. However, CA and BB significantly reduced the CAT, SOD and GPx activities. Microscopic observation of CA and BB augmented cells revealed protection from H2O2 and associated hydroxyl, hydroxymethyl or hydroperoxide radicals accumulation, nuclear disorganization, morphological distortion, apoptosis and necrosis contrary to MG exposed cells. An enhancement of 112.78% in caspase 3/7 activity was noted in MG exposed cells over control. Both CA and BB supplementation reduced the caspase 3/7 activity by 106.06 and 105.82%, respectively which was almost near normal. MG was found to induce the expression of yeast transcription factor Yap1; while presence of CA and BB restored expression of Yap1. Expression of longevity responsible gene Silent Information Regulator (Sir2) was also found to be reduced during MG exposure. However, CA and BB triggered the expression of Sir2. Similarly, MG lowered the expression of Baculoviral IAP repeat (Bir1) which is the inhibitor of apoptosis while CA and BB aided the over expression of Bir1. ConclusionsCA and BB supplementation could significantly decrease oxidative stress, enhance cell viability and ultimately protected S. cerevisiae cells form aging.

Control of mitotic chromosome condensation by the fission yeast transcription factor Zas1.

Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C2H2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation.

Expanding the role of FDO1 in yeast chromosomal biology

The yeast transcription factor Forkhead 1 (FKH1) has multiple roles in chromosomal biology. Our lab recently discovered a protein‐protein interaction between Fkh1 and Fdo1 that contributes to mating type switching, one of multiple nuclear roles for Fkh1. Little information exists for roles of Fdo1 in yeast, and we were interested in whether Fdo1 has additional roles in chromosomal biology. To this end, we have begun to characterize the yeast cell cycle in a fdo1‐dependent manner. Using flow cytometry, we have uncovered connections between fdo1 and cell cycle progression as well as genome stability.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comprehensive, high-resolution binding energy landscapes reveal context dependencies of transcription factor binding

Transcription factors (TFs) are primary regulators of gene expression in cells, where they bind specific genomic target sites to control transcription. Quantitative measurements of TF-DNA binding energies can improve the accuracy of predictions of TF occupancy and downstream gene expression in vivo and shed light on how transcriptional networks are rewired throughout evolution. Here, we present a sequencing-based TF binding assay and analysis pipeline (BET-seq, for Binding Energy Topography by sequencing) capable of providing quantitative estimates of binding energies for more than one million DNA sequences in parallel at high energetic resolution. Using this platform, we measured the binding energies associated with all possible combinations of 10 nucleotides flanking the known consensus DNA target interacting with two model yeast TFs, Pho4 and Cbf1. A large fraction of these flanking mutations change overall binding energies by an amount equal to or greater than consensus site mutations, suggesting that current definitions of TF binding sites may be too restrictive. By systematically comparing estimates of binding energies output by deep neural networks (NNs) and biophysical models trained on these data, we establish that dinucleotide (DN) specificities are sufficient to explain essentially all variance in observed binding behavior, with Cbf1 binding exhibiting significantly more nonadditivity than Pho4. NN-derived binding energies agree with orthogonal biochemical measurements and reveal that dynamically occupied sites in vivo are both energetically and mutationally distant from the highest affinity sites.

The exoribonuclease Xrn1 is a post-transcriptional negative regulator of autophagy

ABSTRACTMacroautophagy/autophagy is a conserved catabolic process that promotes survival during stress. Autophagic dysfunction is associated with pathologies such as cancer and neurodegenerative diseases. Thus, autophagy must be strictly modulated at multiple levels (transcriptional, post-transcriptional, translational and post-translational) to prevent deregulation. Relatively little is known about the post-transcriptional control of autophagy. Here we report that the exoribonuclease Xrn1/XRN1 functions as a negative autophagy factor in the yeast Saccharomyces cerevisiae and in mammalian cells. In yeast, chromosomal deletion of XRN1 enhances autophagy and the frequency of autophagosome formation. Loss of Xrn1 results in the upregulation of autophagy-related (ATG) transcripts under nutrient-replete conditions, and this effect is dependent on the ribonuclease activity of Xrn1. Xrn1 expression is regulated by the yeast transcription factor Ash1 in rich conditions. In mammalian cells, siRNA depletion of XRN1 enhances autophagy and the replication of 2 picornaviruses. This work provides insight into the role of the RNA decay factor Xrn1/XRN1 as a post-transcriptional regulator of autophagy.

Super Resolution Imaging of Start Transcription Factors in Yeast

The balance between growth and division in budding yeast (Saccharomyces cerevisiae) is critical for proliferation. Commitment to division occurs at Start and is controlled by the transcription factor complexes SBF and MBF. In poor nutrient (glycerol) environments, cells divide at a smaller size, and have a slower growth rate compared to rich nutrients (glucose). We have recently found that the SBF and MBF copy numbers are small and increase in G1 phase, prior to Start, which might result in the titration of their target sites in the G1/S regulon to trigger the transition. In this scenario, upregulation of SBF and MBF in poor carbon nutrient sources would explain why cells pass Start at a smaller size. However, with the small SBF/MBF copy numbers when compared to their target DNA sequences, it is not known what role the 3-dimensional structural organization of these transcription clusters facilitate in the upregulation-titration mechanism. In the present study, single-molecule localization microscopy was used to resolve sub-diffraction limited, 50-100 nm sized, nucleation site structures of the SBF/MBF complexes within the nucleus of budding yeast in rich and poor nutrient environments. Interestingly, poor carbon nutrients increased the nucleation site density compared to rich nutrient sources in fixed cells. In live cells, single-particle tracking revealed discrete molecular diffusion dynamics of the SBF and MBF components, one fast, and one slowly diffusing component, which we interpret as non-specifically and specifically bound proteins. No nutrient dependence of the dynamics was observed. These observations support our previous work, indicating increasing copy number of SBF and MBF as a key factor controlling Start and its modulation by nutrients.

Coupled feedback loops control the stimulus-dependent dynamics of the yeast transcription factor Msn2

Information about environmental stimuli often can be encoded by the dynamics of signaling molecules or transcription factors. In the yeast Saccharomyces cerevisiae, different types of stresses induce distinct nuclear translocation dynamics of the general stress-responsive transcription factor Msn2, but the underlying mechanisms remain unclear. Using deterministic and stochastic modeling, we reproduced in silico the different dynamic responses of Msn2 to glucose limitation and osmotic stress observed in vivo and found that a positive feedback loop on protein kinase A mediated by the AMP-activated protein kinase Snf1 is coupled with a negative feedback loop to generate the characteristic pulsatile dynamics of Msn2. The model predicted that the stimulus-specific positive feedback loop could be responsible for the difference between Msn2 dynamics induced by glucose limitation and osmotic stress. This prediction was further verified experimentally by time-lapse microscopic examinations of the snf1Δ strain. In this mutant lacking the Snf1-mediated positive feedback loop, Msn2 responds similarly to glucose limitation and osmotic stress, and its pulsatile translocation is largely abrogated. Our combined computational and experimental analysis reveals a regulatory mechanism by which cells can encode information about environmental cues into distinct signaling dynamics through stimulus-specific network architectures.

Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains weres-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4).The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity ofs-tag-BMP-2 in vivo was shown in various experimental animal models.

Facilitated dissociation of transcription factors from single DNA binding sites

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate [Formula: see text], establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.

Mitochondrial AtTrxo1 is transcriptionally regulated by AtbZIP9 and AtAZF2 and affects seed germination under saline conditions.

Mitochondrial thioredoxin-o (AtTrxo1) was characterized and its expression examined in different organs of Arabidopsis thaliana. AtTrxo1 transcript levels were particularly high in dry seeds and cotyledons where they reached a maximum 36 h after imbibition with water, coinciding with 50% germination. Expression was lower in seeds germinating in 100 mM NaCl. To gain insight into the transcriptional regulation of the AtTrxo1 gene, a phylogenomic analysis was coupled with the screening of an arrayed library of Arabidopsis transcription factors in yeast. The basic leucine zipper AtbZIP9 and the zinc finger protein AZF2 were identified as putative transcriptional regulators. Transcript regulation of AtbZIP9 and AtAFZ2 during germination was compatible with the proposed role in transcriptional regulation of AtTrxo1. Transient over-expression of AtbZIP9 and AtAZF2 in Nicotiana benthamiana leaves demonstrated an activation effect of AtbZIP9 and a repressor effect of AtAZF2 on AtTrxo1 promoter-driven reporter expression. Although moderate concentrations of salt delayed germination in Arabidopsis wild-type seeds, those of two different AtTrxo1 knock-out mutants germinated faster and accumulated higher H2O2 levels than the wild-type. All these data indicate that AtTrxo1 has a role in redox homeostasis during seed germination under salt conditions.

The OsHAPL1-DTH8-Hd1 complex functions as the transcription regulator to repress heading date in rice.

Heading date is an important agronomic trait related to crop yield. Many genes related to heading date have already been identified in rice (Oryza sativa), and a complicated, preliminary regulatory genetic network has also already been established, but the protein regulatory network is poorly understood. We have identified a novel heading date regulator, Heme Activator Protein like 1 (OsHAPL1), which inhibits flowering under long-day conditions. OsHAPL1 is a nuclear-localized protein that is highly expressed in leaves in a rhythmic manner. OsHAPL1 can physically interact with Days To Heading on chromosome 8 (DTH8), which physically interacts with Heading date 1 (Hd1) both in vitro and in vivo. OsHAPL1 forms a complex with DTH8 and Hd1 in Escherichia coli. OsHAPL1, DTH8, and Hd1 physically interact with the HAP complex, and also with general transcription factors in yeast (Saccharomyces cerevisiae). Further studies showed that OsHAPL1 represses the expression of the florigen genes and FLOWERING LOCUS T 1 (RFT1) and Hd3a through Early heading date 1 (Ehd1). We propose that OsHAPL1 functions as a transcriptional regulator and, together with DTH8, Hd1, the HAP complex, and general transcription factors, regulates the expression of target genes and then affects heading date by influencing the expression of Hd3a and RFT1 through Ehd1.

The Information Content of Glutamine-Rich Sequences Define Protein Functional Characteristics.

The presence of abnormally expanded glutamine (Q) repeats within specific proteins (e.g., huntingtin) are the well-established cause of several neurogenerative diseases, including Huntington disease and spinocerebellar ataxias. However, the impact of "expanded Q" stretches on the protein function is not well-understood, mostly due to lack of knowledge about the physiological role of Q repeats and the mechanism by which these repeats achieve functional-specificity. Indeed, is intriguing that regions with such low complexity (low information content) can display exquisite functional specificity. Prompting the question: where is this information stored? Applying biochemical/structural constraints and statistical analysis of protein composition we identified Q-rich (QR) regions present in coiled coils of yeast transcription factors and endocytic proteins. Our analysis indicated the existence of non-Q amino-acids differentially enriched or excluded from QR regions in one protein group versus the other. Importantly, when the non-Q amino-acids from an endocytic protein were exchanged by the ones enriched in QR from transcription factors, the resulting protein was unable to localize to the plasma membrane and was instead found in the nucleus. These results indicate that while QR repeats can efficiently engage in binding, the non-Q amino-acids provide essential specificity information. We speculate that coupling low complexity regions with information-intensive determinants might be a strategy used in many protein systems involved in different biological processes.

Performing Yeast One-Hybrid Library Screens.

Yeast one-hybrid (Y1H) assays are used to identify which transcription factor (TF) "prey" molecules can bind a DNA fragment of interest that is used as "bait". Y1H assays involve introducing plasmids that encode TFs into a yeast "bait strain" in which the DNA fragment of interest is integrated upstream of one or more reporters, and activation of these reporters indicates that a TF-DNA interaction has occurred. These plasmids express each TF as a hybrid protein (hence the "one-hybrid" name) fused to the activation domain (AD) of the yeast TF Gal4. The AD moiety activates reporter expression even if the TF to which it is fused typically functions as a repressor. Here, we describe how to perform a Y1H screen of a library of cDNA fragments cloned into a pPC86 plasmid expressing the protein encoded by the cDNA as an AD fusion. The method assumes availability of either commercially available libraries or libraries generated in house using mRNA extracted from a tissue of interest. We also assume that users have access to a yeast bait strain that possesses the DNA fragment of interest integrated upstream of two different reporters-HIS3, an auxotrophic marker, and LacZ, a colorimetric marker that changes colorless X-gal into a blue compound. Briefly, the screen involves transforming the AD-cDNA library into the yeast bait strain, identifying colonies that show activation of both reporters, retesting the interaction in a freshly grown bait strain, and sequencing the cDNA insert to identify the interacting TF.