Gene looping essay 2010

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Gene looping essay 2010

  1. 1. Feeling Loopy About Transcription:The Relationship of Gene Loops & the Nuclear Pore Complex to Transcriptional Regulation. Nova Syed 996167375 December 3, 2010 0
  2. 2. Introduction The subnuclear organization of DNA plays an important role in regulating geneexpression, and is often reflective of the transcriptional status of a cell (2). Many develop-mentally regulated genes in metazoan cells are translocated to the nuclear periphery to maintain arepressed state until differentiation is complete, when they relocalize to the nucleoplasm (3, 6).Similarly, the recruitment of telomeres to the nuclear envelope in yeast promotes the silencing ofsubtelomeric genes. Despite this association of the nuclear periphery with a transcriptionallyrepressive setting, recent studies have demonstrated that certain actively transcribed genes can beconditionally recruited to associate with the nuclear pore complex (NPC). These interactions areevolutionary conserved between S. cerevisiae and S. Pombe, while in D. melanogaster, thenuclear pore subunits are localized to active genes in the nucleoplasm (4, 8). Until recently, thefunctional relevance and mechanism of peripheral localization have been elusive. Presently it ispostulated that in the yeast S. cerevisiae, recruitment of certain inducible genes, such as GAL1,FMP27, HXK1, and INO1, upon activation confers a transcriptional “memory” that alters theirreinduction kinetics (2, 3, 8). Depending on the gene, this memory may be retained for one hourto a number of generations (2, 3). Following the discovery of looping interactions between thepromoter and the 3’ end of some activated genes in yeast, the nuclear basket myosin-like protein1 (Mlp1) has been implicated in the maintenance of short-term transcriptional memory bytethering ‘memory gene loops’ to allow a faster rate of initiation upon reinduction of some genes(2). However, the identification of unique sequences important for gene recruitment to thenuclear pore versus maintenance of transcriptional memory indicates that the process whichallows a gene to remember its previous transctiptionally active state is a two-step mechanism thatmay or may not involve a looped intermediate and Mlp1 (3, 8). In addition, the incorporation of 1
  3. 3. the non-canonical histone variant H2A.Z in the promoter nucleosomes of these inducible geneshas also been deemed essential for the persistent localization of the INO1 and GAL1 genes at thenuclear periphery immediately after repression (3, 6). The following is a discussion of recentstudies which investigated the molecular interactions involved in gene looping and subsequentrecruitment to the nuclear periphery, and their impact on the regulation of gene expression.Gene Looping in Yeast The discovery of gene loops resulted from investigating the enigmatic observation thatsome factors involved in transcriptional termination and 3’ end formation of pre-mRNA areapparently common to initiation as well, and can be found at the promoter of the same gene. In2004, O’Sullivan and colleagues demonstrated that GAL1::FMP27 and SEN1, two relativelylong genes in S. cerevisiae, primarily reside in a looped conformation by juxtaposing theirpromoters and terminators. In order to determine the density of RNA Polymerase II acrossFMP27 transcription/nuclear run-on (TRO) analysis was conducted. The TRO profile of FMP27showed an asymmetric accumulation of PolII was in the 5’ promoter region (due to promoterproximal pausing) and in the open reading frame, terminating beyond the PolyA signal. SinceFMP27 encodes an uncharacterized gene product, a heterologous reporter was created by placingthis ORF under the control of the GAL1 promoter, which is induced by galactose and repressedby glucose. Chromatin immunoprecipitation analysis on uninduced cells (grown in raffinose)using an antibody specific for RNAPII resulted in a significant asymmetrical PolII signalobtained specifically at the promoter and terminator regions, indicating the formation of a loop.To ensure the close proximity of the 5’ and 3’ regions in space, GAL1::FMP27 was placed underthe E. coli lac operator, in a strain expressing GFP tagged LacI. Repeating ChIP analysis usingan anti-GFP antibody, in both inducing and non-inducing conditions, resulted in detection of not 2
  4. 4. only the 5’ promoter, but the terminator region as well (Figure 1). Since this could only resultfrom crosslinking between the two distant (linearly) regions, only a stable loop structure couldlead to this result. Furthermore, O’Sullivan et al. conducted a 3C chromosome conformationcapture technique under non-inducing, inducing and repressing growth conditions to illustrate theclose spatial proximity of the 5’ and 3’ regions of both GAL1::FMP27 and SEN1 (Figure 2). The3C technique captures bridging interactions between different sites on a gene, as the relativefrequencies with which two sites become crosslinked are determined by the amount ofintramolecular ligation product made, which is visualized by quantitative PCR (7). As shown inFigure 2, intramolecular ligation products detected by primers 1 and 4, 1 and 5, and a and d areall consistent with a bridging complex formed between the promoter and terminator regions inboth noninducing and inducing conditions. Notably, the absence of ligation products detectedduring repression conditions indicate that looping is not an artefact of DNA curvature, but isactually linked to a gene’s transcriptional status (1). To determine if this relationship is evident in the differentially CTD-phosphorylatedforms of promoter and terminator-specific RNAPII present, ChIP analyses were conducted onGAL1::FMP27 with antibodies specific for phosphorylated Ser5 and phosphorylated Ser2. Bothends of the gene under uninduced conditions contain an elongation-competent PolII, as bothpromoter and terminator-specific signals were significant for Ser5-phosphorylated PolII CTD.Thus, the kinase that phosphorylates PolII CTD at Ser5, Kin28p, was also found to reside in the5’ and 3’ regions of the gene. Inactivation of this kinase by a temperature sensitive mutation ledto the loss of Ser5 phosphorylated CTD and PolII in the promoter and terminator, and absence ofloop conformation as per intramolecular ligation. These data confirm that loop formation 3
  5. 5. requires initial transcriptional activation, as RNAPII that is elongation incompetent is notsufficient to juxtapose the promoter and terminator regions in a stable complex (1). Following the study by O’Sullivan et al, the molecular interactions necessary for theformation of gene loops have expanded to involve several factors in the transcription initiationand termination machineries. This is most likely due to the natural increase of transcriptionefficiency as a result of recycling of the same RNAPII, instead of reinitiating de novo (11). In2007, a study by Singh and Hampsey found the yeast TFIIB homolog Sua7p to be a necessaryfactor in gene loop formation, as it crosslinked to both the promoter and terminator regions ofPMA1 and BLM10 (12). An E62K mutant form of TFIIB loses its ability to specifically interactwith the terminator and is unable to support looping in all the tested genes (BLM10, SAC3,GAL10, SEN1, and HEM3), independent of TBP (12). mRNA processing factors like Ssu72 andPta1, and cis-acting mRNA processing elements have also been deemed essential in loopformation (13). Gene looping is dependent on the phosphatase activity of Ssu72, because itmediates the removal of phosphates from the CTD of RNAPII, facilitating its reinitiation (13).Recruitment of Genes to the Nuclear Periphery Although the nuclear envelope is generally characterized as being a transcriptionallyrepressive zone, recent evidence points to the possibility that for some inducible genes individualnuclear pore complex (NPC) components can promote activation and retaining of recenttranscription memory. Such an environment conducive to active transcription appears to belocated in the vicinity of NPC basket proteins like Mlp1, Mlp2 and Nup2 (12, 2). Thisphenomenon is specifically linked to inducible yeast genes, such as HXK1, INO1, GAL1,HSP104, SU2, and α-factor induced genes (12). Current literature focused on elucidating amechanism for the physical relocation of such genes implicates various factors responsible for 4
  6. 6. recruitment, depending on the loci under study. These include transcriptional activators, mRNAprocessing and export factors, and distinct NPC subunits (8). In 2009, Tan-Wong et al. identifiedMlp1 as a requirement to maintain a tether to the nuclear periphery, by the gene HXK1 (2). Asshown in Figure 3, they used GFP-Nup49 to label the nuclear envelope, LacI-GFP, and placed256 LacOP repeats upstream of HXK1, to visualize the LacI-GFP-HXK1 locus as a green dot inthe nuclear periphery under inducing conditions. This regulated localization was abolished in astrain deleted for Mlp1. Given that Mlp1 was also demonstrated to bind both 5’ and 3’ ends ofHXK1, this underlined an essential role for Mlp1 in tethering the looped gene to the NPC (2).However, because Mlp1 localization in the nucleoplasm was not investigated, this protein cannotbe confidently declared as actively involved in the recruitment process that directs HXK1 to theNPC. Indeed, Ahmed et al recently discovered two redundant gene recruitment sequences in theS. cerevisiae INO1 promoter that serve as “zip codes”, directing it to the nuclear peripheryfollowing transcriptional activation (4). Despite the fact that these GRSs are not present in allgenes recruited to the NPC, this discovery opens up the possibility of similar zip codes thatmediate translocation of other loci. Each gene recruitment sequence was found to beindependently sufficient in promoting a physical interaction between a GRS containing locus andcomponents of the nuclear pore complex (4). In addition, GRSI was found to maintain zip codefunction in S. pombe, a very distant relative of S. cerevisiae, suggesting at least 1 billion years inevolutionary conservation of this mechanism for gene targeting to the NPC (4, 8). 5
  7. 7. The link between gene looping and the NPC: Transcriptional Memory Based on the evidence discussed, the act of gene looping alone does not necessarilyconfer recruitment to the nuclear periphery. This is because gene looping has been attributed toall transcriptionally active genes tested thus far, by 3C technique, but all active genes are notfound at the nuclear periphery (15). Instead, gene looping is indispensable to rapid reactivationof all inducible genes at the NPC that have experienced a short period of repression (2). Thus,such an alteration of gene architecture (looping) is more likely to be a mechanism for increasingtranscription efficiency by recycling RNAPII and other important transcription factors (11). As aresult, the main role of recruiting active induced genes to the nuclear periphery may just be tohold the gene in a primed state for reinduction. This is done by facilitating/maintaining loopformation after a short period of repression, which is marked by the non-canonical histoneH2A.Z (3). Evidence from recent literature supporting this hypothesis is discussed below. Transcriptional memory describes the retention of information regarding a gene’s recenttranscriptional activity in order to facilitate robust reinduction after a temporary repressionperiod. It is associated with localization of the gene to the nuclear periphery, and chromatinarchitecture and state. In the 2009 study conducted by Tang-Wong et al, transcription dependentgene loop structures of HXK1 and GAL1::FMP27 are demonstrated to be maintained as memorygene loops (MGLs) at the nuclear periphery for up to an hour of repression. Retention of theseMGLs were found to be highly dependent on the NPC basket protein Mlp1, which was illustratedto have a 5’/3’ bimodal distribution on HXK1. Transcriptional memory of HXK1 andGAL::FMP27 was tested by RT-qPCR, Chromatin immunoprecipitation using an antibodyspecific for PolII, and 3C analysis in a time course assay of galactose induction and reinduction.As shown in figure 4, a lack of MGL presence (determined by 3C) at the beginning reinduction 6
  8. 8. after short-term repression (1hr) in the mlp1∆ strain matches a lack of fast RNAPII binding, andrapid mRNA production. For these particular genes, short-term memory of past transcriptionalactivity was only retained for one hour, corresponding to the time point at which memory geneloops were lost and had to be reformed upon reinduction (2). However, Mlp1 is not the only NPC protein involved in tethering MGLs. The yeast geneINO1, encoding Inositol 1-phosphate synthase, was identified by to differentially interact withthe specific NPC proteins Nup2 and Nup100 depending on the length of repression theyunderwent. Following the discovery of gene recruitment sequences, Brickner et al. determinedthat these elements were not sufficient to retain localization of recently repressed INO1 at thenuclear pore complex. Through deletion analysis of the INO1 promoter, they identified a secondcis-acting element, called ‘memory recruiting sequence’ that was required for peripherallocalization during a short period of repression (after initial induction). This MRS zip code wasalso found to sufficiently induce H2A.Z incorporation into the INO1 promoter by the chromatinremodeler Swi/Snf. The H2A.Z histone variant has a boundary function that involves physicalinteraction with the NPC to separate transcriptionally silent loci from non-silent regions. It isoften specifically found in the promoters of genes belonging to a variety of species. In 2007,Brickner et al determined H2A.Z to have an essential part in transcriptional memory. The loss ofthis variant histone did not impact the recruitment of INO1 to the nuclear periphery in eitherpermanently induced or repressed conditions. However, mutants unable to express H2A.Z wereincapable of remaining at the NPC during a short period of repression that followed initialinduction, and subsequently demonstrated a strong defect in transcriptional memory for bothINO1 and GAL1 (measured by RT-qPCR of the respective mRNA in reinduction conditions) (6).Adding to the development of a model for transcriptional memory, Light et al determined that 7
  9. 9. deletion of genes encoding Nup100 and histone H2A.Z, and mutation of MRS all result in theloss of transcription memory of INO1 (gauged by RT-qPCR and ChIP with anti Rbp1 shown inFigure 5). Since all of these components are postulated to be involved in one specific aspect oftranscriptional memory, this suggests a possible two step mechanism that consists of an initialrecruitment of an induced gene to the nuclear periphery directed by a GRS and in a loopedconformation due to active transcription (Figure 6) (3,8). During temporary repression,maintenance of the localization of the specific gene occurs through H2A.Z incorporation via anMRS and tethering to Nup100 (3). In the case of HXK1 and GAL1::FMP27 genes studied byTang-Wong et al, only one tethering step is required by Mlp1, as this protein was found tomaintain MGLs. Notably, INO1 may not exist as an MGL while waiting for reinduction at thenuclear periphery, as it was observed to lose its gene loop conformation (2, 8). This may alsoaccount for this gene’s slower reinduction kinetics in comparison to GAL1::FMP27 and HXK1(2, 8). Since INO1 was still observed to maintain its localization at the nuclear periphery byLight et al, it is very likely to have switched from interacting with nuclear pore complex basketproteins during transcription to binding an internal NPC core protein after H2A.Z incorporationand loss of loop conformation due to repression. Thus, despite having slow reactivation kinetics,INO1 cannot retain any transcription memory if it is not localised at the NPC or does not haveH2A.Z incorporated into its promoter (3). In a broader context, the studies discussed here fittogether in elucidating that the mechanism of transcriptional memory involves at least two majorsteps, and that the slight alteration of these steps in different inducible genes contributes to theirrelative ability to ‘remember’ the cell’s previous expression status. This type of ‘adaptive’memory renders eukaryotes like S. cerevisiae an ability to utilize the immediate environment as asource of information and thereby learn from recent experience. 8
  10. 10. Figure 1: lacO-GAL1::FMP27 cells expressing HIS3:lacI-GFP were induced with3-aminotriazole and then subjected to GAL1::FMP27. Galactose was used as the growth mediafor induction, glucose for repression, and raffinose for non-induction conditions. ChIP analysiswas conducted with an antibody against GFP (1).Figure 2: Maps of the restriction enzyme cut sites and primers used in 3C analysis are shown forboth FMP27 and SEN1. 3C analysis of GAL1::FMP27 in induced (galactose), non-induced(raffinose) and repressed (glucose) conditions. Con denotes the genomic DNA negative control.For SEN1, expression conditions were limited to growth in raffinose (1). 9
  11. 11. Figure 3: Activation allows for Mlp1-dependent HXK1 localization to the NPC. (B)Fluorescence microscopy shows a LacI-GFP-HXK1 dot in subnuclear positions of thenucleoplasm (centre) or periphery (outer zone). (C) Comparing the percentage of cells seen witha fluorescing LacI-GFP-HXK1 signal tethered to the NPC due to galactose activation andglucose repression in wild type and mlp1∆ strains (2). 10
  12. 12. Figure 4: Gene loops (shown by 3C analysis as the presence of a PCR band) are facilitated byMlp1, and required for transcriptional memory in HXK1. (A) Flow-chart of galactoseinduction/reinduction time course. (B) HXK1 RT–qPCR (top panel), Pol II ChIP (middle panel),and 3C (bottom panel) showing correlation in loop formation, PolII recruitment and mRNAproduction in wildtype versus mlp1∆ strains (2). 11
  13. 13. Figure 5: Figure 5. The memory recruitment sequence and H2A.Z are both required frotranscription memory of INO1 in S. cerevisiae. Repressing medium contained 100 mM inositol,whereas medium without inositol served as the inducing condition. RT-qPCR was conducted toquantify the relative INO1 mRNA levels to ACT1 (which should not have been affected by thechanging conditions). Recruitment of RNA PolII was determined by ChIP, using an antibodyagainst Rbp1 (3). 12
  14. 14. Figure 6: Hypothesized model of transcription memory, incorporating gene looping, recruitmentto the NPC (by GRS or other presently unknown sequences in the promoter of the targeted gene),initial binding to specific basket proteins during active transcription and reinduction. H2A.Zincorporation would theoretically occur after recent repression. Binding of INO1 to a NPC coreprotein resulting in the loss of loop structure may be responsible for lack of as robust areactivation as GAL1 (8). 13
  15. 15. References:1. OSullivan JM, Tan-Wong SM, Morillon A, Lee B, Coles J, Mellor J, Proudfoot NJ. Gene loops juxtapose promoters and terminators in yeast. Nat Genet. 2004 Sep;36(9):1014-8.2. Tan-Wong SM, Wijayatilake HD, Proudfoot NJ. Gene loops function to maintain transcriptional memory through interaction with the nuclear pore complex. Genes Dev. 2009 Nov 15;23(22):2610-24.3. Light WH, Brickner DG, Brand VR, Brickner JH. Interaction of a DNA Zip code with the nuclear pore complex promotes H2A.Z incorporation and INO1 transcriptional memory. Mol Cell. 2010 Oct 8;40(1):112-25.4. Ahmed S, Brickner DG, Light WH, Cajigas I, McDonough M, Froyshteter AB, Volpe T, Brickner JH. DNA zip codes control an ancient mechanism for gene targeting to the nuclear periphery. Nat Cell Biol. 2010 Feb;12(2):111-8. Epub 2010 Jan 24.5. Birse CE, Lee BA, Hansen K, Proudfoot NJ. Transcriptional termination signals for RNA polymerase II in fission yeast. EMBO J. 1997 Jun 16;16(12):3633-43.6. Brickner DG, Cajigas I, Fondufe-Mittendorf Y, Ahmed S, Lee PC, Widom J, Brickner JH. H2A.Z-mediated localization of genes at the nuclear periphery confers epigenetic memory of previous transcriptional state. PLoS Biol. 2007 Apr;5(4):e81.7. Dekker J, Rippe K, Dekker M, Kleckner N. Capturing chromosome conformation. Science. 2002 Feb 15;295(5558):1306-11.8. Kerr SC, Corbett AH. Should INO stay or should INO Go: a DNA "zip code" mediates gene retention at the nuclear pore. Mol Cell. 2010 Oct 8;40(1):3-5.9. El Kaderi B, Medler S, Raghunayakula S, Ansari A. Gene looping is conferred by activator -dependent interaction of transcription initiation and termination machineries. J Biol Chem. 2009 Sep 11;284(37):25015-25. Epub 2009 Jul 14.10. Singh BN, Hampsey M. A transcription-independent role for TFIIB in gene looping. Mol Cell. 2007 Sep 7;27(5):806-16.11. Ansari A, Hampsey M. A role for the CPF 3-end processing machinery in RNAP II dependent gene looping. Genes Dev. 2005 Dec 15;19(24):2969-78. Epub 2005 Nov 30.12. Strambio-De-Castillia C, Niepel M, Rout MP. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat Rev Mol Cell Biol. 2010 Jul;11(7):490- 501.13. Brickner JH. Transcriptional memory: staying in the loop. Curr Biol. 2010 Jan 12;20(1):R20-1. 14

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