Trimethylation of Lys36 on H3 restricts gene expression change during aging and impacts life span.

Functional data indicate that specific histone modification enzymes can be key to longevity in Caenorhabditis elegans, but the molecular basis of how chromatin structure modulates longevity is not well understood. In this study, we profiled the genome-wide pattern of trimethylation of Lys36 on histone 3 (H3K36me3) in the somatic cells of young and old Caenorhabditis elegans. We revealed a new role of H3K36me3 in maintaining gene expression stability through aging with important consequences on longevity. We found that genes with dramatic expression change during aging are marked with low or even undetectable levels of H3K36me3 in their gene bodies irrespective of their corresponding mRNA abundance. Interestingly, 3' untranslated region (UTR) length strongly correlates with H3K36me3 levels and age-dependent mRNA expression stability. A similar negative correlation between H3K36me3 marking and mRNA expression change during aging was also observed in Drosophila melanogaster, suggesting a conserved mechanism for H3K36me3 in suppressing age-dependent mRNA expression change. Importantly, inactivation of the methyltransferase met-1 resulted in a decrease in global H3K36me3 marks, an increase in mRNA expression change with age, and a shortened life span, suggesting a causative role of the H3K36me3 marking in modulating age-dependent gene expression stability and longevity.

Two SET domain containing genes link epigenetic changes and aging in Caenorhabditis elegans.

Changes in epigenetic status and chromatin structure have been shown to associate with aging in many organisms. Here, we report an RNAi screen of putative histone methyltransferases and demethylases in wild-type Caenorhabditis elegans using reproduction inhibitor. We identified six genes that when inactivated by RNAi, consistently extend lifespan. Five of these genes do not require germline proliferation to affect lifespan. We further characterized two of these genes, the highly homologous SET domain containing genes, set-9 and set-26. They share redundant functions in maintaining normal lifespan, while exhibiting differential tissue expression patterns. Furthermore, we found that set-9 and set-26 partially act through the Forkhead box O (FOXO) transcription factor, DAF-16, to modulate lifespan. Interestingly, inactivation of somatic SET-26 alone results in a robust lifespan extension and alters the levels of histone H3 protein and the repressive histone marks, H3K9me3 and H3K27me3, in an age-dependent manner. We hypothesize that inactivation of SET-26 triggers compensation mechanisms to restore repressive chromatin structure and hence affects chromatin stability to promote longevity.

RNAi screens to identify components of gene networks that modulate aging in Caenorhabditis elegans.

Our understanding of the genetic mechanisms of organismal aging has advanced dramatically during the past two decades. With the development of large-scale RNAi screens, the last few years saw the remarkable identifications of hundreds of new longevity genes in the roundworm Caenorhabditis elegans. The various RNAi screens revealed many biological pathways previously unknown to be related to aging. In this review, we focus on findings from the recent large-scale RNAi longevity screens, and discuss insights they have provided into the complex biological process of aging and considerations of the RNAi technology will continue to have on the future development of the aging field.

P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo.

Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.

Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS.

Uninduced heat shock genes are poised for rapid activation, with RNA polymerase II (Pol II) transcriptionally engaged, but paused or stalled, within the promoter-proximal region. Upon heat shock, this Pol II is promptly released from the promoter region and additional Pol II and transcription factors are robustly recruited to the gene. Regulation of the heat shock response relies upon factors that modify the efficiency of elongation through the initially transcribed sequence. Here, we report that Pol II is susceptible to transcription arrest within the promoter-proximal region of Drosophila hsp70 and that transcript cleavage factor TFIIS is essential for rapid induction of hsp70 RNA. Moreover, using a tandem RNAi-ChIP assay, we discovered that TFIIS is not required to establish the stalled Pol II, but that TFIIS is critical for efficient release of Pol II from the hsp70 promoter region and the subsequent recruitment of additional Pol II upon heat induction.

Coordination of transcription, RNA processing, and surveillance by P-TEFb kinase on heat shock genes.

Positive transcription elongation factor b (P-TEFb) is a kinase that phosphorylates the carboxyl-terminal domain (CTD) of RNA Polymerase II (Pol II). Here, we show that flavopiridol, a highly specific P-TEFb kinase inhibitor, dramatically reduces the global levels of Ser2--but not Ser5--phosphorylated CTD at actively transcribed loci on Drosophila polytene chromosomes under both normal and heat shocked conditions. Brief treatment of Drosophila cells with flavopiridol leads to a reduction in the accumulation of induced hsp70 and hsp26 RNAs. Surprisingly, the density of transcribing Pol II and Pol II progression through hsp70 in vivo are nearly normal in flavopiridol-treated cells. The major defect in expression is at the level of 3' end processing. A similar but more modest 3' processing defect was also observed for hsp26. We propose that P-TEFb phosphorylation of Pol II CTD coordinates transcription elongation with 3' end processing, and failure to do so leads to rapid RNA degradation.

Evolutionary dynamics and population control during in vitro selection and amplification with multiple targets.

Iterative cycles of in vitro selection and amplification allow rare functional nucleic acid molecules, aptamers, to be isolated from large sequence pools. Here we present an analysis of the progression of a selection experiment that simultaneously yielded two families of RNA aptamers against two disparate targets: the intended target protein (B52/SRp55) and the partitioning matrix. We tracked the sequence abundance and binding activity to reveal the enrichment of the aptamers through successive generations of selected pools. The two aptamer families showed distinct trajectories of evolution, as did members within a single family. We also developed a method to control the relative abundance of an aptamer family in selected pools. This method, involving specific ribonuclease digestion, can be used to reduce the background selection for aptamers that bind the matrix. Additionally, it can be used to isolate a full spectrum of aptamers in a sequential and exhaustive manner for all the different targets in a mixture.

High Expression of a cDNA Coding for Human Lymphotoxin alpha Derivative in E. coli and Purification of Its Protein Product.

Using PCR, a cDNA coding for human lymphotoxin alpha derivative (hLTalphaDa) lacking 27 amino acids at N-terminal of natural hLT was constructed. The expression construct was expressed in E. coli BL21 (DE3). The product of expression was in the form of inclusion bodies, and accounted for 60%-80% of total bacterial proteins. The product protein was purified to over 95% by treatments of inclusion bodies. The specific activity of hLTalphaDa was above 2x10(8) IU(per mg protein). Its cytotoxic activity can be neutralized by monoclone antibody against hLT. The anti-tumor effects of hLTalphaDa were also tested in vitro and in vivo.