Faculty Bibliography

Poly(A) binding proteins (PABPs) specifically bind the polyadenosine tail of mRNA and have been shown to be important for RNA polyadenylation, translation initiation, and mRNA stability. Using a modified L1 retrotransposition vector, we examined the effects of two PABPs (encoded by PABPN1 and PABPC1) on the retrotransposition activity of the L1 non-long-terminal-repeat (non-LTR) retrotransposon in both HeLa and HEK293T cells. We demonstrated that knockdown of these two genes by RNA interference (RNAi) effectively reduced L1 retrotransposition by 70 to 80% without significantly changing L1 transcription or translation or the status of the poly(A) tail. We identified that both poly(A) binding proteins were associated with the L1 ribonucleoprotein complex, presumably through L1 mRNA. Depletion of PABPC1 caused a defect in L1 RNP formation. Knockdown of the PABPC1 inhibitor PAIP2 increased L1 retrotransposition up to 2-fold. Low levels of exogenous overexpression of PABPN1 and PABPC1 increased L1 retrotransposition, whereas unregulated overexpression of these two proteins caused pleiotropic effects, such as hypersensitivity to puromycin and decreased L1 activity. Our data suggest that PABPC1 is essential for the formation of L1 RNA-protein complexes and may play a role in L1 RNP translocation in the host cell.

Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the epsilon-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

Recent studies employing genome-wide approaches have provided an unprecedented view of the scope of L1 activities on structural variations in the human genome, and further reinforced the role of L1s as one of the major driving forces behind human genome evolution. The rapid identification of novel L1 elements by these high-throughput approaches demands improved L1 functional assays. However, the existing assays use antibiotic selection markers or fluorescent proteins as reporters; neither is amenable to miniaturization. To increase assay sensitivity and throughput, we have developed a third generation assay by using dual-luciferase reporters, in which firefly luciferase is used as the retrotransposition indicator and Renilla luciferase is encoded on the same or separate plasmid for normalization. This novel assay is highly sensitive and has a broad dynamic range. Quantitative data with high signal-to-noise ratios can be obtained from 24- up to 96-well plates in 2-4 days after transfection. Using the dual-luciferase assays, we have characterized profiles of retrotransposition by various human and mouse L1 elements, and detailed the kinetics of L1 retrotransposition in cultured cells. Its high-throughput and short assay timeframe make it well suited for routine tests as well as large-scale screening efforts.

Transcriptional silencing in Saccharomyces cerevisiae is mediated by heterochromatin. There is a plethora of information regarding the roles of histone residues in transcriptional silencing, but exactly how histone residues contribute to heterochromatin structure is not resolved. We address this question by testing the effects of a series of histone H3 and H4 mutations involving residues in their aminoterminal tails, on the solvent-accessible and lateral surfaces of the nucleosome, and at the interface of the H3/H4 tetramer and H2A/H2B dimer on heterochromatin structure and transcriptional silencing. The general state, stability, and conformational heterogeneity of chromatin are examined with a DNA topology-based assay, and the primary chromatin structure is probed by micrococcal nuclease. We demonstrate that the histone mutations differentially affect heterochromatin. Mutations of lysine 16 of histone H4 (H4-K16) and residues in the LRS (loss of rDNA silencing) domain of nucleosome surface markedly alter heterochromatin structure, supporting the notion that H4-K16 and LRS play key roles in heterochromatin formation. Deletion of the aminoterminal tail of H3 moderately alters heterochromatin structure. Interestingly, a group of mutations in the globular domains of H3 and H4 that abrogate or greatly reduce transcriptional silencing increase the conformational heterogeneity and/or reduce the stability of heterochromatin without affecting its overall structure. Surprisingly, yet another series of mutations abolish or reduce silencing without significantly affecting the structure, stability, or conformational heterogeneity of heterochromatin. Therefore, histone residues may contribute to the structure, stability, conformational heterogeneity, or other yet-to-be-characterized features of heterochromatin important for transcriptional silencing.

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.

Our results suggest the possible benefit of manipulating an intrinsic aging pathway that is independent of nutrition availability, a potential therapeutic route that might be able to bypass shortcomings of calorie restriction.

The DNA entry and exit points on the nucleosome core regulate the initial invasion of the nucleosome by factors requiring access to the underlying DNA. Here we describe in vivo consequences of eliminating a single protein-DNA interaction at this position through mutagenesis of histone H3 Lys 42 to alanine. This substitution has a dramatic effect on the Saccharomyces cerevisiae transcriptome in both the transcriptional output and landscape of mRNA species produced. We attribute this in part to decreased histone H3 occupancy at transcriptionally active loci, leading to enhanced elongation. Additionally we show that this lysine is methylated in vivo, and genetic studies of methyl-lysine mimics suggest that this modification may be crucial in attenuating gene expression. Interestingly, this site of methylation is unique to Ascomycota, suggesting a recent evolutionary innovation that highlights the evolvability of post-translational modifications of chromatin.

Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory beta subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast.

Long interspersed elements, type 1(LINE-1, L1) are the most abundant and only active autonomous retrotransposons in the human genome. Native L1 elements are inefficiently expressed because of a transcription elongation defect thought to be caused by high adenosine content in L1 sequences. Previously, we constructed a highly active synthetic mouse L1 element (ORFeus-Mm), partially by reducing the nucleotide composition bias. As a result, the transcript abundance of ORFeus-Mm was greatly increased, and its retrotransposition frequency was > 200-fold higher than its native counterpart. In this paper, we report a synthetic human L1 element (ORFeus-Hs) synthesized using a similar strategy. The adenosine content of the L1 open reading frames (ORFs) was reduced from 40% to 27% by changing 25% of the bases in the ORFs, without altering the amino acid sequence. By studying a series of native/synthetic chimeric elements, we observed increased levels of full-length L1 RNA and ORF1 protein and retrotransposition frequency, mostly proportional to increased fraction of synthetic sequence. Overall, the fully synthetic ORFeus-Hs has > 40-fold more RNA but is at most only ~threefold more active than its native counterpart (L1RP); however, its absolute retrotransposition activity is similar to ORFeus-Mm. Owing to the elevated expression of the L1 RNA/protein and its high retrotransposition ability, ORFeus-Hs and its chimeric derivatives will be useful tools for mechanistic L1 studies and mammalian genome manipulation.