Step 1 Sie Sire Recruitment of St | Sir wih bound \l Sir, and of i Sir3 to TG repeat ey I {¥ bound Rap1 \ \ and yKu apt binding sites Subtelomeric regions Step2 Sir2-mediated [ fs r deacetylation of histone | | H4K16 in nearby Sy [ Step3 Spreading of the Sir complex: along nearby nucleosomes Step 4 TG, repeats )} ici wl DR zi yw pn Dy Dh wD) Folding of a silent telomere into a higher- order structure Figure 6. Model for stepwise assembly of heterochromatin in yeast. (Step 1) At telomeres, Rap] and yKu recruit Sir even in the absence of Sir2 or Sir3. Only Sir4 can be recruited in the absence of the other Sir proteins, and its binding is antagonized by Rif] and Rif? (Mishra and Shore 1999). (Step 2) Sir4-Sir2 and Sir4-Sir3 interact strongly creating Sir complexes along the ‘TG repeats. Sir? NAD-dependent histone deacetylase activity is stimulated by complex formation and Sir2 deacetylates the acetylated histone H4K 16 residue in nearby nucleosomes. (Step 3) Sit complexes sptead along the nucleosomes, perhaps making use of the O-acety!-ADP-ribose intermediate produced by NAD hydrolysis (Liou et al. 2005). Sir3 and Sird bind the deacetylated histone H4 tails. Although the deacetylated histone H3 amino-terminal tail also binds Sir3 and Sir4 proteins, it is not shown here. (Step 4) The silent chromatin “matures” at the end of M phase to create an inaccessible structure. This may entail higher-order folding and sequestering at the nuclear envelope. ——Gg, @ H3K9me2/3 @ Hak4mes ® H3S10ph Figure 4. Cell-cycle regulation of centromere heterochromatin assembly. (A) Heterochromatin located at chr somal centromeres becomes differentially methylated and phosphorylated on histones throughout the indicated. These modifications control the binding of the heterochromatin protein Swi6. During mitosis Sw displaced by H3S10 phosphorylation. Swi6 binding is reestablished during subsequent DNA replication ( when a more accessible chromatin structure permits RNA Pol II to transcribe centromeric DNA. This, | recruits the RNAi machinery to direct H3K9me methylation. (B) Replication-coupled RNAi model (Li et: ‘This figure illustrates an alternative model for how RNAi works at centromeres. Here, RNAi serves to releas II from chromatin to avoid collision with DNA replication machinery during S phase. See text for furtherd Adapted from Djupedal and Ekwall 2008.)Diffusion omnes Heterochromatin Euchromatin Inverted repeat barrier - RNA Fol Il dependent —- r B-box barrier ubiquitination by Cul4-Dabt AINA barrier - RNA Pol Il dependent Heterochromatin ‘Centromeric chromati UTR barrier Phe ‘Subtelomeric chromatin” ‘Eucnromatin Euchromatin Heterochromatin Euchromatin Figure 5. Chromatin boundaries and the boundary mechanism involving Epel (for enhancement of position effect) in S. pombe. (A) A schematic representation of different types of boundary elements in S, pontbe. (B) The jechanism, ofboundary function by Epe!. Epel associates with Swié, however, when the antisilencing factor is ubiquitinated by the Culd-Ddb1 ligase and degraded in the heterochromatin region to allow for heterochromatin assembly, However, at boundaries, Epel is somehow protected from degradation, thus restricting the spreading of heterochromatin, Phosphorylation of Swi6 contributes to the dissociation of Epe! at heterochromatin, while promoting the associ- ation with the HDAC complex SHREC in maintaining histone hypoacetylation, (A, Adapted from Scott et al. 2007.) S. pombe: diffuse boundaries, RNA Pol IT inverted repeat (IR) barriers, B-box barriers, RNA Pol III-dependent tRNA barriers, and RNA Pol III-independent LTR barriers (long terminal repeat sequences derived from retrotransposons) elements (Fig. 5A) (Scott et al. 2007; Stralfors et al. 2011). Several tRNA genes reside between the outer repeats and the central kinetochore domain (double arrowheads in Fig. 2A), These have been shown to act as a barrier preventing heterochromatin from encroaching into the central do- main (Scott et al. 2006) One of the best-studied boundary factors, Epel, was first identified through a mutation that enhances hetero- chromatin formation (Ayoub et al. 2003), Epel interacts with Swi6 and somehow restricts H3K9 methylation to within heterochromatic regions. It also reduces siRNA pro- duction and promotes RNA Pol II occupancy in het- erochromatin (Zofall and Grewal 2006; Isaac et al. 2007; Trewick et al. 2007), thereby limiting heterochromatin as- sembly beyond (IR) boundaries in the mat2-mat3 region and pericentric regions of chromosomes 1 and 3 (‘Itewick et al. 2007; Braun et al. 2011). Based on its function in restricting heterochromatin domains from spreading into euchromatin, Epel has been termed an antisilencing factor. How, then, is the function of Epe! restricted to chromatin boundary regions? It is thought to uniformly associate with heterochromatin through association with Swi6; however, the ubiquitinating action of the cullin-dependent ligase Cul4-Ddb1 degrades Epel in heterochromatin so that it becomes restricted to the heterochromatin/euchromatin boundary regions (Fig, 5B) (Braun et al. 2011). What pre- vents the ubiquitin ligase from acting on Epel at bound- aries is currently an open question.A Wildtype x wild type B IES* x wildtype c IES* x wild type (no genetic exchange) 2 Figure 6, Inhibition of IES excision by the parental macronucleus is mediated by factors transmitted through the cytoplasm, (A) During macronuclear differentiation following mating of wild-type cells, IFSs (eg, the red bar) are excised efficiently; however, (B) the presence of an IES (IES") in the maternal macronucleus of one partner can signal its presence (depicted by radio tower emissions) to the developing macronuclei within both partners, biting excision of the homologous IESs in all four resulting progeny. (C) The IES* maternal macronucleus can inhibit IES excision in its wild-type mating, partner even when genetic exchange is blocked. parental macronuclear genome is transmitted through fac- tors able to freely move through the cytoplasm. 63. “Spontaneous” Elimination of Foreign Sequences Introduced into the Micronuclear Genome In Tetrahymena, when the neo gene is integrated into a mictonuclear chromosome, it may be deleted from the genome of new macronuclei during successive rounds of conjugation (Yao et al. 2003; Liu et al. 2005; Howard-Till and Yao 2007). The neo gene is derived from the bacteria transposon Tn, and thus is unlikely to contain by chance some specific signal to spontaneously induce its own elim- ination. Therefore, this result presents the possibility that any “foreign” sequence introduced into the micronuclear genome of Tetrahymena may be recognized as an IES. Deletion of the neo gene is generally inefficient relative to the nearly 100% elimination observed for naturally oc- curring IESs. Furthermore, the efficiency of neo elimination from different micronuclear loci varied significantly (Liu et al. 2005; Howard-Till and Yao 2007). Not only did the "genomic environment surrounding the neo gene influence elimination of this transgene, but multiple neo insertions into the micronuclear genome enhanced its DNA elimina- tion (Liu et al. 2005; Howard-Till and Yao 2007). Thus, both the presence of a foreign sequence in the micronucleus as well as its repetitiveness in the genome affected DNA elim- ination. These position and copy number effects on trans- gene elimination will be discussed in Section 8 in the light of small RNA-directed regulation of DNA elimination mech- anism. More generally, neo gene elimination suggests that IESs are recognized by more than sequence alone. 7 DNA ELIMINATION IS GUIDED BY SMALL RNA-DIRECTED TRANS-NUCLEAR COMPARISON OF WHOLE GENOMES The homology-dependent effects described in Section 6 show that a cross talk occurs between the genomes of the parental macronucleus and the new macronucleus during nuclear differentiation, which can profoundly alter DNA elimination patterns and cellular phenotypes. The obser- vations that only highly homologous sequences are affected suggest strongly that this cross talk is mediated by nucleic acids. As discussed in Sections 7.2 and 7.3, it is likely that the interplay between parental macronuclear transcriptsox ‘Transcriptional + 4 Promoter-proximal @-— pausing RNA (25-50 base pairs) Active Inactive . dt P-TEFb complex P-TEFb complex Recruitment of P-TEFD Pause release and transcriptional elongation Directional forces: ‘on Pol I ? Fulklength mRNA Figure 11. Coupling of transcriptional initiation to elongation. The key biochemical events that control the tran= sition from transcriptional initiation to elongation are shown. The arrows shown to the right illustrate the combi- natorial nature of forces acting on the RNA polymerase II (Pol II) at various phases of RNA expression. SITE signal induced transcription factor; GT, general transcription factors; P-S5, phosphorylation of serine 5 in the carboxy= terminal domait nation between transcriptional initiation and elongation at primary and secondary gene loci. In both types of genes, elongation requires the activity of factors that enable RNA. Pol II processivity. These factors could be schematically assigned to a few key regulatory hubs that determine the efficiency of elongation (Fig. 11). The first of these regula- tory hubs is at gene promoters where RNA Pol II comes under the control of factors that significantly slow or halt elongation (Zhou et al. 2012). Two negative elongation factors, the 5,6-dichloro-1-B-p-ribofuranosylbenzimida- TD) of Pol IIs NELE negative elongation factor; DSIE, DRB sensitivity-inducing factor; P-TEFb, positive transcription-elongation factor-b; BRD4, bromodom: bis-acetimide inducible 1; P-S2, phosphorylation of serine 2 in the CTD of Pol I. containing protein 4; HEXIM, hexamethylene zole (DRB) sensitivity-inducing factor (DSIF) and nk elongation factor (NELF), associate with RNA PollId initiation, leading to the generation of poised pol (Adelman and Lis 2012). The amount of promoter] mal RNA Pol II determines the size of the polymeras potentially available for clongation The release of Pol 11 into elongation depends on activity and/or amount of gene-bound cyclin-deper kinase-pausing machinery (i.e., P-TEFb and associat tors), which enables RNA Poll II processivity (PeterliESC NSC Neurogenic genes (e.g., 042) Neurogenic genes (2.3, 3 3 ACTIVE REPRESSED “8 Key developmental genes: H " SILENT (bivalent poised) ‘ mane Neuronal genes: SILENT (bivalent primed) 38 oe — BB Ea eS Binoy ca Pluripotency genes: REPRESSED QB HK27me2 GR H3KOme3 ~— AB H9K4mes © Unmethrylated cytosine @ SMethylcytosine © S-Hydroxymethyleyto Figure 2. Epigenetic mechanisms involved in the regulation of gene expression during neurogenesis. Typically, CpG island containing genes are mostly housekeeping genes and, consequently, are ubiquitously active. In early devel- opment, just postimplantation, these genes may not yet be active, but are marked by a bivalent chromatin signature, consisting of H3K4me3 and H3K27me3. Most of these genes will be activated during development via H3K27 demethylation (top line). A subset of these genes that are tissue-specific neurogenic genes may, subsequently, become silenced in nonneuronal or fully differentiated neuronal cells, through H3K9 methylated heterochromatin forma~ tion (sop right). Other neuronal-specific genes may delay activation, but remain bivalently primed for expression until neuronal differentiation ensues (middle line). Pluripotency genes, largely, do not contain CpG islands in their promoters. They are active during early development and become repressed following differentiation by conven tional H3K9 methylation and DNA methylation mechanisms. ESC, embryonic stem cell; NSC, neuronal stem cells CGI, CpG island. An explanation for this finding may be provided by the discovery that 5-hydroxymethylcytosine (ShmC) is present in the mammalian genome, and is approximately 10-fold more abundant in neurons than in peripheral tissues or embryonic stem (ES) cells (Kriaucionis and Heintz 2009; Munzel et al. 2010; Szulwach et al. 2011). Until recently, it was not possible to distinguish between 5mC and 5hmC. We now know that the distribution of 5amC varies between distinct regions of the brain and between the brain and ES cells (Szulwach et al. 2011). For example, ShmC is enriched in gene bodies and depleted from TSS in neurons, whereas the opposite is the case in ES cells. More recent studies revealed that the relationship between the distribution of shmC, 5mC, and gene expression is brain cell specific, and the methyl-CpG-binding protein 2 (MeCP2) binds to ShmC in the brain (Mellen et al. 2012). Additional studies led to the proposal that when MeCP2 binds to 5hmC, it facilitates transcription in neural cell types, but can. repressor when bound to SmC-containing DNA ( This proposal was based on the observation that is enriched in euchromatin, whereas 5mC is en heterochromatin (Mellen et al. 2012). Remarkably, proteins bearing a Rett syndrome mutation R133C di altered binding to ShmC DNA, suggesting that mutations alter the chromatin distribution of the p1 The investigators of this work speculated that neuronal ck specific dynamic gene regulation is the consequence of t three-dimensional (3D) chromatin structure, which termined by the levels of SmC, ShmC, and MeCP2 [I et al. 2012). Dnmt3a-dependent de novo DNA methylation been shown to inhibit the binding of the Polycomb repr sive complex PRC2 (Wu et al. 2010). The neurogenic: that require Dnmt3a are known targets of PRC2 in non