ABSTRACTpl6ink4 has been implicated as a tumor suppressor that is lost from a variety of human tumors and human cell lines. pl6ink4 specifically binds and inhibits the cyclin-dependent kinases 4 and 6. In vitro, these kinases can phosphorylate the product of the retinoblastoma tumor suppressor gene. Thus, pl6ink4 could exert its function as tumor suppressor through inhibition of phosphorylation and functional inactivation of the retinoblastoma protein. Here we show that overexpression of pl6inlk4 in certain cell types will lead to an arrest in the G, phase of the cell cycle. In addition, we show that pl6ink4 can only suppress the growth of human cells that contain functional pRB. Moreover, we have compared the effect of pl6ink4 expression on embryo fibroblasts from wild-type and RB homozygous mutant mice. Wild-type embryo fibroblasts are inhibited by pl6ink4, whereas the RB nullizygous fibroblasts are not. These data not only show that the presence of pRB is crucial for growth suppression by p16ink4 but also indicate that the pRB is the critical target acted upon by cyclin D-dependent kinases in the G1 phase of the cell cycle. pl6ink4 was originally identified as a polypeptide bound to cyclin-dependent kinase 4 (cdk4) in human diploid fibroblasts transformed with the DNA tumor virus simian virus 40 (SV40)(1). The pl6ink4 gene was subsequently cloned using a twohybrid protein interaction screen to identify proteins that can associate with human cdk4 (2). Interestingly, the same gene was identified as a putative tumor suppressor through an extensive analysis of deletions and rearrangements of chromosome 9p21 present in human tumor lines (3, 4). The p16ink4 gene has since been found to be deleted or rearranged in a large number of human primary tumors (5). Binding of pl6ink4 to cdk4 prevents association of cdk4 with the D-type cyclins and results in an inhibition of the catalytic activity of the cyclin D/cdk4 enzymes (2). More recently, it was demonstrated that p16ink4 can also bind and inhibit cdk6, the alternative kinase partner of the D-type cyclins (6). Hence, pl6ink4 seems to target specifically the cyclin D-dependent kinase activity by competing with D cyclins for binding to their kinase partners.Complexes formed by cdk4/cdk6 and cyclin D have been strongly implicated in the control of cell proliferation during the G1 phase of the cell cycle (7,8). Three different human D-type cyclins have been identified, Dl, D2, and D3 (9, 10). In vitro, complexes of cyclin Dl, D2, or D3 and cdk4 or cdk6 can phosphorylate the product of the retinoblastoma tumor suppressor gene, pRB (11,12). The phosphorylation of pRB, occurring in mid/late G1, reverses its growth-inhibitory effect and enables cells to proceed from G1 to S phase. The timing of cyclin D-dependent kinase activity and the onset of pRB hyperphosphorylation appear to coincide in the cell cycle. Taken together, these findings suggest that cyclin D/cdk complexes play a critical role in pRB hyperphosphorylation in vivo. If so, pl6ink4 could negatively regulate cell ...

Chromatin influences gene expression by restricting access of DNA binding proteins to their cognate sites in the genome [1][2][3] . Large-scale characterization of nucleosome positioning in Saccharomyces cerevisiae has revealed a stereotyped promoter organization in which a nucleosome-free region (NFR) is present within several hundred base pairs upstream of the translation start site 4,5 . Many transcription factors bind within NFRs and nucleate chromatin remodelling events which then expose other cis-regulatory elements 6-9 . However, it is not clear how transcription-factor binding and chromatin influence quantitative attributes of gene expression. Here we show that nucleosomes function largely to decouple the threshold of induction from dynamic range. With a series of variants of one promoter, we establish that the affinity of exposed binding sites is a primary determinant of the level of physiological stimulus necessary for substantial gene activation, and sites located within nucleosomal regions serve to scale expression once chromatin is remodelled. Furthermore, we find that the S. cerevisiae phosphate response (PHO) pathway exploits these promoter designs to tailor gene expression to different environmental phosphate levels. Our results suggest that the interplay of chromatin and binding-site affinity provides a mechanism for finetuning responses to the same cellular state. Moreover, these findings may be a starting point for more detailed models of eukaryotic transcriptional control.When cells sense changes in environmental inorganic phosphate (P i ), the activity of the transcription factor Pho4 is modulated by phosphorylation 10 : Pho4 is phosphorylated on four sites, cytoplasmic and inactive when cells are grown in P i -rich medium; it is phosphorylated selectively on one site and localized to the nucleus in intermediate P i (about 100 mM) conditions; and it is unphosphorylated, nuclear and fully active in P i starvation 11,12 . The co-activator Pho2 interacts with unphosphorylated Pho4 and is required for induction of many PHO genes 13,14 ; however, it is not thought to be regulated in response to P i availability 12 . Despite being controlled by the same activators, the target gene PHO5 is expressed at a low level in intermediate P i conditions, whereas PHO84 is significantly induced 14 . Although both promoters contain a combination of highand low-affinity Pho4-binding sites [15][16][17] , more Pho4 is recruited to PHO84 in intermediate P i conditions than to PHO5 14 . We hypothesized that chromatin may influence gene expression by differentially regulating the accessibility of Pho4 sites in the PHO5 and PHO84 promoters.To test this hypothesis, we constructed variants of the PHO5 promoter controlling transcription of a green fluorescent protein (GFP) reporter gene (Fig. 1a). The PHO5 promoter contains five positioned nucleosomes (numbered 25 to 21), a low-affinity Pho4 site (CACGTTt) and a Pho2 site in the NFR, and a high-affinity Pho4 site (CACGTGg) and distal Pho2 sites occluded under nucleosome...

Ethanol toxicity in yeast Saccharomyces cerevisiae limits titer and productivity in the industrial production of transportation bioethanol. We show that strengthening the opposing potassium and proton electrochemical membrane gradients is a mechanism that enhances general resistance to multiple alcohols. Elevation of extracellular potassium and pH physically bolster these gradients, increasing tolerance to higher alcohols and ethanol fermentation in commercial and laboratory strains (including a xylose-fermenting strain) under industrial-like conditions. Production per cell remains largely unchanged with improvements deriving from heightened population viability. Likewise, up-regulation of the potassium and proton pumps in the laboratory strain enhances performance to levels exceeding industrial strains. Although genetically complex, alcohol tolerance can thus be dominated by a single cellular process, one controlled by a major physicochemical component but amenable to biological augmentation.

We describe a method of genome-wide analysis of quantitative growth phenotypes using insertional mutagenesis and DNA microarrays. We applied the method to assess the fitness contributions of Escherichia coli gene domains under specific growth conditions. A transposon library was subjected to competitive growth selection in Luria-Bertani (LB) and in glucose minimal media. Transposon-containing genomic DNA fragments from the selected libraries were compared with the initial unselected transposon insertion library on DNA microarrays to identify insertions that affect fitness. Genes involved in the biosynthesis of nutrients not provided in the growth medium were found to be significantly enriched in the set of genes containing negatively selected insertions. The data also identify fitness contributions of several uncharacterized genes, including putative transcriptional regulators and enzymes. The applicability of this high-resolution array selection in other species is discussed.

Microbial contamination is an obstacle to widespread production of advanced biofuels and chemicals. Current practices such as process sterilization or antibiotic dosage carry excess costs or encourage the development of antibiotic resistance. We engineered Escherichia coli to assimilate melamine, a xenobiotic compound containing nitrogen. After adaptive laboratory evolution to improve pathway efficiency, the engineered strain rapidly outcompeted a control strain when melamine was supplied as the nitrogen source. We additionally engineered the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica to assimilate nitrogen from cyanamide and phosphorus from potassium phosphite, and they outcompeted contaminating strains in several low-cost feedstocks. Supplying essential growth nutrients through xenobiotic or ecologically rare chemicals provides microbial competitive advantage with minimal external risks, given that engineered biocatalysts only have improved fitness within the customized fermentation environment.

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Lignocellulosic biomass remains unharnessed for the production of renewable fuels and chemicals due to challenges in deconstruction and the toxicity its hydrolysates pose to fermentation microorganisms. Here, we show in Saccharomyces cerevisiae that engineered aldehyde reduction and elevated extracellular potassium and pH are sufficient to enable near-parity production between inhibitor-laden and inhibitor-free feedstocks. By specifically targeting the universal hydrolysate inhibitors, a single strain is enhanced to tolerate a broad diversity of highly toxified genuine feedstocks and consistently achieve industrial-scale titers (cellulosic ethanol of >100 grams per liter when toxified). Furthermore, a functionally orthogonal, lightweight design enables seamless transferability to existing metabolically engineered chassis strains: We endow full, multifeedstock tolerance on a xylose-consuming strain and one producing the biodegradable plastics precursor lactic acid. The demonstration of “drop-in” hydrolysate competence enables the potential of cost-effective, at-scale biomass utilization for cellulosic fuel and nonfuel products alike.