Quantitative and functional interrogation of parent-of-origin allelic expression biases in the brain.

The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased--rather than monoallelic--expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.

Combinatorial control of diverse metabolic and physiological functions by transcriptional regulators of the yeast sulfur assimilation pathway.

Methionine abundance affects diverse cellular functions, including cell division, redox homeostasis, survival under starvation, and oxidative stress response. Regulation of the methionine biosynthetic pathway involves three DNA-binding proteins-Met31p, Met32p, and Cbf1p. We hypothesized that there exists a "division of labor" among these proteins that facilitates coordination of methionine biosynthesis with diverse biological processes. To explore combinatorial control in this regulatory circuit, we deleted CBF1, MET31, and MET32 individually and in combination in a strain lacking methionine synthase. We followed genome-wide gene expression as these strains were starved for methionine. Using a combination of bioinformatic methods, we found that these regulators control genes involved in biological processes downstream of sulfur assimilation; many of these processes had not previously been documented as methionine dependent. We also found that the different factors have overlapping but distinct functions. In particular, Met31p and Met32p are important in regulating methionine metabolism, whereas p functions as a "generalist" transcription factor that is not specific to methionine metabolism. In addition, Met31p and Met32p appear to regulate iron-sulfur cluster biogenesis through direct and indirect mechanisms and have distinguishable target specificities. Finally, CBF1 deletion sometimes has the opposite effect on gene expression from MET31 and MET32 deletion.

Assembly of standardized DNA parts using BioBrick ends in E. coli.

Synthetic biologists have adopted the engineering principle of standardization of parts and assembly in the construction of a variety of genetic circuits that program living cells to perform useful tasks. In this chapter, we describe the BioBrick standard as a widely used method. We present methods by which new BioBrick parts can be designed and produced, starting with existing clones, naturally occurring DNA, or de novo. We detail the procedures by which BioBrick parts can be assembled into construction intermediates and into biological devices. These protocols are based on our experience in conducting synthetic biology research with undergraduate students in the context of the iGEM competition.

Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast.

A yeast strain lacking Met4p, the primary transcriptional regulator of the sulfur assimilation pathway, cannot synthesize methionine. This apparently simple auxotroph did not grow well in rich media containing excess methionine, forming small colonies on yeast extract/peptone/dextrose plates. Faster-growing large colonies were abundant when overnight cultures were plated, suggesting that spontaneous suppressors of the growth defect arise with high frequency. To identify the suppressor mutations, we used genome-wide single-nucleotide polymorphism and standard genetic analyses. The most common suppressors were loss-of-function mutations in OPI1, encoding a transcriptional repressor of phospholipid metabolism. Using a new system that allows rapid and specific degradation of Met4p, we could study the dynamic expression of all genes following loss of Met4p. Experiments using this system with and without Opi1p showed that Met4 activates and Opi1p represses genes that maintain levels of S-adenosylmethionine (SAM), the substrate for most methyltransferase reactions. Cells lacking Met4p grow normally when either SAM is added to the media or one of the SAM synthetase genes is overexpressed. SAM is used as a methyl donor in three Opi1p-regulated reactions to create the abundant membrane phospholipid, phosphatidylcholine. Our results show that rapidly growing cells require significant methylation, likely for the biosynthesis of phospholipids.