Dithizone Staining of Intracellular Zinc: An Unexpected and Versatile Counterscreen for Auxotrophic Marker Genes in Saccharomyces cerevisiae

Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle

Features of Cryptic Promoters and Their Varied Reliance on Bromodomain-Containing Factors

The Set2-Rpd3S pathway is important for the control of transcription memory. Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicate that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters

In Vitro Recombination of Non-Homologous Genes Can Result in Gene Fusions that Confer a Switching Phenotype to Cells

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which β-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to β-lactam even though they do not encode allosteric enzymes. These ‘phenotypic switch’ genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo

An Ash2L/RbBP5 Heterodimer Stimulates the MLL1 Methyltransferase Activity through Coordinated Substrate Interactions with the MLL1 SET Domain

Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with transcription activation and is mainly catalyzed by the MLL/SET1 family histone methyltransferases. A common feature of the mammalian MLL/SET1 complexes is the presence of three core components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Unlike most other histone lysine methyltransferases, all four proteins are required for efficient H3 K4 methylation. Despite extensive efforts, mechanisms for how three core components regulate MLL/SET1 methyltransferase activity remain elusive. Here we show that a heterodimer of Ash2L and RbBP5 has intrinsic histone methyltransferase activity. This activity requires the highly conserved SPRY domain of Ash2L and a short peptide of RbBP5. We demonstrate that both Ash2L and the MLL1 SET domain are capable of binding to S-adenosyl-L- [methyl-3H] methionine in the MLL1 core complex. Mutations in the MLL1 SET domain that fail to support overall H3 K4 methylation also compromise SAM binding by Ash2L. Taken together, our results show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall catalysis of MLL1 mediated H3 K4 methylation. The results we describe here provide mechanistic insights for unique regulation of the MLL1 methyltransferase activity. It suggests that both Ash2L/RbBP5 and the MLL1 SET domain make direct contacts with the substrates and contribute to the formation of a joint catalytic center. Given the shared core configuration among all MLL/SET1 family HMTs, it will be interesting to test whether the mechanism we describe here can be generalized to other MLL/SET1 family members in the future

Phosphorylation of Not4p Functions Parallel to BUR2 to Regulate Resistance to Cellular Stresses in Saccharomyces cerevisiae

Background The evolutionarily conserved Ccr4-Not and Bur1/2 kinase complexes are functionally related in Saccharomyces cerevisiae. In this study, we further explore the relationship between the subunits Not4p and Bur2p. Methodology/Principal Findings First, we investigated the presence of post-translational modifications on the Ccr4-Not complex. Using mass spectrometry analyses we identified several SP/TP phosphorylation sites on its Not4p, Not1p and Caf1p subunits. Secondly, the influence of Not4p phosphorylation on global H3K4 tri-methylation status was examined by immunoblotting. This histone mark is severely diminished in the absence of Not4p or of Bur2p, but did not require the five identified Not4p phosphorylation sites. Thirdly, we found that Not4p phosphorylation is not affected by the kinase-defective bur1-23 mutant. Finally, phenotypic analyses of the Not4p phosphomutant (not4S/T5A) and bur2Δ strains showed overlapping sensitivities to drugs that abolish cellular stress responses. The double-mutant not4S/T5A and bur2Δ strain even revealed enhanced phenotypes, indicating that phosphorylation of Not4p and BUR2 are active in parallel pathways for drug tolerance. Conclusions Not4p is a phospho-protein with five identified phosphorylation sites that are likely targets of a cyclin-dependent kinase(s) other than the Bur1/2p complex. Not4p phosphorylation on the five Not4 S/T sites is not required for global H3K4 tri-methylation. In contrast, Not4p phosphorylation is involved in tolerance to cellular stresses and acts in pathways parallel to BUR2 to affect stress responses in Saccharomyces cerevisiae

An Increase in Mitochondrial DNA Promotes Nuclear DNA Replication in Yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication

Growth Trajectories of Preterm Infants: Birth to 12 Years

Introduction: Birth weight often is used to predict how preterm infants will grow, but scant attention has been paid to the effect of neonatal morbidities on growth trajectories. We investigated birth weight and neonatal morbidity in preterm infants’ growth to age 12 years. Method: A five-group, prospective, longitudinal study was conducted with 194 infants: 46 full term; 29 healthy preterm without morbidity; 56 preterm with medical illness (MPT); 34 preterm with neurologic illness; and 29 preterm small for gestational age (SGA). Height, weight, and body mass index were measured at six ages. Results: The full-term group had greater height than the preterm groups to age 8 years, when healthy preterm and MPT groups caught up. Only the SGA group had smaller height at age 12 years. The MPT, preterm with neurologic illness, and SGA groups had lower weight through age 12 years. Body mass index was appropriate for preterm groups by age 4 years. Across time, neonatal morbidity had a significant effect on height and weight trajectories. Birth weight was significant for weight trajectories only. Discussion: With variation in growth trajectories, details of neonatal morbidity in health history interviews will inform child health assessments

Read-through Activation of Transcription in a Cellular Genomic Context

Read-through transcription from the adjacent E1a gene region is required for wild-type (wt) activity of the downstream adenovirus E1b promoter early after infection (read-through activation). However, whether a cellular chromosomal template can support read-through activation is not known. To address this issue, read-through activation was evaluated in the context of stably expressed templates in transfected cells. Inhibition of read-through transcription by insertion of a transcription termination sequence between the E1a and E1b promoters reduced downstream gene expression from stably integrated templates. The results indicate that the mechanism of read-through activation does not depend on the structure of early adenovirus nucleoprotein complexes, a structure that is likely to be different from that of cellular chromatin. Accordingly, this regulatory interaction could participate in the coordinated control of the expression of closely linked cellular genes

Short Telomeres Compromise β-Cell Signaling and Survival

The genetic factors that underlie the increasing incidence of diabetes with age are poorly understood. We examined whether telomere length, which is inherited and known to shorten with age, plays a role in the age-dependent increased incidence of diabetes. We show that in mice with short telomeres, insulin secretion is impaired and leads to glucose intolerance despite the presence of an intact β-cell mass. In ex vivo studies, short telomeres induced cell-autonomous defects in β-cells including reduced mitochondrial membrane hyperpolarization and Ca2+ influx which limited insulin release. To examine the mechanism, we looked for evidence of apoptosis but found no baseline increase in β-cells with short telomeres. However, there was evidence of all the hallmarks of senescence including slower proliferation of β-cells and accumulation of p16INK4a. Specifically, we identified gene expression changes in pathways which are essential for Ca2+-mediated exocytosis. We also show that telomere length is additive to the damaging effect of endoplasmic reticulum stress which occurs in the late stages of type 2 diabetes. This additive effect manifests as more severe hyperglycemia in Akita mice with short telomeres which had a profound loss of β-cell mass and increased β-cell apoptosis. Our data indicate that short telomeres can affect β-cell metabolism even in the presence of intact β-cell number, thus identifying a novel mechanism of telomere-mediated disease. They implicate telomere length as a determinant of β-cell function and diabetes pathogenesis

Human RNA Polymerase II-Association Factor 1 (hPaf1/PD2) Regulates Histone Methylation and Chromatin Remodeling in Pancreatic Cancer

Change in gene expression associated with pancreatic cancer could be attributed to the variation in histone posttranslational modifications leading to subsequent remodeling of the chromatin template during transcription. However, the interconnected network of molecules involved in regulating such processes remains elusive. hPaf1/PD2, a subunit of the human PAF-complex, involved in the regulation of transcriptional elongation has oncogenic potential. Our study explores the possibility that regulation of histone methylation by hPaf1 can contribute towards alteration in gene expression by nucleosomal rearrangement. Here, we show that knockdown of hPaf1/PD2 leads to decreased di- and tri-methylation at histone H3 lysine 4 residues in pancreatic cancer cells. Interestingly, hPaf1/PD2 colocalizes with MLL1 (Mixed Lineage Leukemia 1), a histone methyltransferase that methylates H3K4 residues. Also, a reduction in hPaf1 level resulted in reduced MLL1 expression and a corresponding decrease in the level of CHD1 (Chromohelicase DNA-binding protein 1), an ATPase dependent chromatin remodeling enzyme that specifically binds to H3K4 di and trimethyl marks. hPaf1/PD2 was also found to interact and colocalize with CHD1 in both cytoplasmic and nuclear extracts of pancreatic cancer cells. Further, reduced level of CHD1 localization in the nucleus in hPaf1/PD2 Knockdown cells could be rescued by ectopic expression of hPaf1/PD2. Micrococcal nuclease digestion showed an altered chromatin structure in hPaf1/PD2-KD cells. Overall, our results suggest that hPaf1/PD2 in association with MLL1 regulates methylation of H3K4 residues, as well as interacts and regulates nuclear shuttling of chromatin remodeling protein CHD1, facilitating its function in pancreatic cancer cells