BBB cromosome Condensation 1s Caused by Condensins Nate be vesponsitte for either inactivating )Xist rset cerca * SHC pretons are ATPases that include cendensins and cohesis. '* Aheterodimer of SMC proteins associates with other subunits. > Condensins cause chramatin to be more tghtiy calles bby ntreducing positive supercnis inte DNA, * Candensins ar resporsibie for condensing chreme. Somes at mitosis. * Chreneseme-specfic condensins are responsible for Condensing inactive X chromosomes in elegans, CpG Istands Are Subject to Methylation '* Mast methyl groups in DNA ae feund on cytosine on both strands of the Cps doubt. * Replication converts a fully methylated site te a hemi- methylated site. '* Hemimethylated sites are converted te fully methyt- ated sites bya maintenance methyltransferase * TET proteins canver:5-mathyicytosine to 5:-Ay deny methylcytosine to lead to ONA demethyiatien DNA Methylation Is Responsible for Imprinting * Paternal and maternal alleles may have different pat- terns of methylation at fertilization. * Mthylation i usually asseciated with inactivation of the gene * When genes ae differentially imprinted, sual of the embryo may require that the functienal alee fs pro vided by the parent with the unmethylated al *» Suvival of heteezygotes for imprinted genes is fer ent, depending nthe gitection ef the cross Introduction GB sunmay Epigenetic effets can result from meditlestien ef ‘2 nucleic acid after it has been synthesized or by the perpetuatien ef protein structures. Epigenetic inheritance describes the ability of different states, which may have different phenotypic consequences, to be mherited without any change in the sequence of DNA | EDD Epigenetic Ffeets Can Be Inherited * Eolgenetcetfcs can rest from paitiaion ofa ‘mucieic acid after it has ‘been synthesized or by the Pe-petuation of protein stuctures. -* Epigenetic effects may be inherited through generations (RBBB Yeast Prions Show Unusual Inheritance * The Sup35 protein in fs witd-typeselubie form is termination factr for trans.atien. * Sup35 can also exist in an alternative farm of oisomeric aggregates, in which tis nat active in protein synthe, * The prasence ofthe eligomeric form causes nel syn thesized protein te acquire the inactive structure. * Cenversion between the two farsi influenced by chaperanes. * The wit-type form has the recessive genetic state psi~ and the mutant frm has the daminant genetic state PST, ns Cause Diseases in Mammals * The protein responsitie for scrapie exists in two forms: the wid-type noninfectious fem BrP, which Is sus- 2 ¥ chno- masemes, all but ene ae inactivated. ‘The Xie (K inactivation center) isa cis-acting ‘agian on the X chremeseme that is necessary and sufficient te ensure that only one ¥ chromosome * Hic includes the xist gene, which cedes foran RNA that is found oniy on inactive x chromosomes * Xistreeits Polycom complexes, which modify histones en the inactive X. + The mechanism that is respersible ter preventing {Xst RNA from accumulating an the ative chromo- seme is unkown, For species with chromosomal sex determina: tion, the sex of the individual presents an inter. esting problem for gene regulation, because of the variation in the number of X chromosomes, MX-linked genes were expressed equally well in each sex, females would have twice as much of each product as males, The importance of avoiding this situation is shown by the exis tence of dosage compensation, which equal izes the level of expression of X-linked genes in the two sexes. Mechanisms used in different species are summarized in FIGURE 29.10: + In mammals, one of the two female X chromosomes is inactivated com. pletely: The result is that females have only one active X chromosome, which is the same situation found in males, ‘The active X chromosome of females and the single X chromosome of males are expressed at the same level, + In Drosophila, the expression of the single male X chromosome is doubled relative to the expression of each female X chromosome + In Caenorhabditis elegans, the expression of each female (hermaphrodite) X chro: ‘mosome is halved relative to the expres. sion of the single male X chromosome 29.5 X Chromosomes Undergo Global Changes 867348, Mammals Flies Wore Inaet Dowie Have fone? x expraseion exprossion ox wo # x SS . => <> ° i> == o i—_— ao Y= = ‘tone FIGURE 29.10 Different means of sasage compensation 30 used to equalize X chremeseme expressian in males and Femates, ‘The common feature in all these mechanisms of dosage compensation is that the entirechromo. some is the target for regulation. A global change occurs that quantitatively affects almost all of the promoters on the chromosome. We know the mostabout heinactivation of the X chromo: some in mammalian females, where the entire chromosome becomes heterochromatic ‘The twin properties of heterochromatin arcits condensed state and associated inactivity (introducedin the Chromosomes chapter) Itcan be divided into two types: * Constitutive heterochromatin con. tains specific sequences that have no coding function. These include satel- Iite DNAs, which are often found at the centromeres. These regions are invari- ably heterochromatic because of their intrinsic nature. + Facultative heterochromatin takes the form of chromosome segments or entire chromosomes that are inac tive in one cell lineage, although they can be expressed in other lineages, ‘The example par excellence 1s the mara. malian X chromosome. The mactive X chromosome is perpetuated in a het erochromatic state, whereas the active X chromosome is euchromatic, Thus, identical DNA sequences are involved in both states. Once the inactive state hhas been established, itis inherited by descendant cells. Thisis an example of epigenetic inheritance, because it does not depend on the DNA sequence. ‘Ourbasicview ofthe situation ofthe female ‘mammalian X chromosomes was formed by the single X hypothesis in 196], Female mice that are heterozygous for X-linked coat color muta: tions have a variegated phenotype in which some areas of the coat are wild type but others are mutant, FIGURE 29.11 shows that this can be explained if one of the two X chromosomes is. CHAPTER 29 Epigenetic Effects Are Inherited inactivated at randomin each cellof a small pre- cuusor population. Cellsin which the X chromo- some carrying the wild-type genes inactivated ive rise to progeny that expressonly the mutant allcle on the active chromosome. Cells derived from a precursor where the other chromosome ‘was inactivated have an active wild-type gene, In the case of coat color, cells descended from @ particular precursor stay together and thus form a patch of the same color, creating the Pattern of visible variegation, In other cases, individual cellsin a population will express one. or the other of X-linked alleles; for example, in heterozygotes for the X-linked locus G&D, any particular red blood cell will express only one of the two allelic forms. (Random inactivation of one X chromosome occurs in etithervan mam: ‘mals. In marsupials, the choice is directed: Itis always the X chromosome inherited from the father thatis inactivated) Inactivation of the X chromosome in femalesis governedby the n=l rule: Regardless of how mony X chromosomes are present, all but one wall be inactivated In normal females there are of course two X chromosomes, butin rare cases where nondisjunction has generated 3X or greater genotype, only one X chromo: some remains active. This suggests a general model in which aspecificeventis limited to one X chromosome and protectsit from an inactiva tion mechanism that applies to all the others. oth X otvomasomes are active in precurser vot Wiksyps coat color SED. Mutant coat coer gene mE ‘One X sxomoeome ‘nactivatedin each cel S25 ative allele ome ve ste ar Mutant sler ‘Exprossion ot wie ype oat color Linked variegation i caused ty the ran- ‘dam inactivation of ene X chromosome in each precursor call Celis in which the allele fs on the active chromo. same have wild phenetype; cals in which the 2 allele 's ‘nthe active chramasere have mutant phenetype.A single locus on the X chromosome is sufficient for inactivation. When a transloca tion occurs between the X chromosome and an autosome, this locus is present on only ‘one of the reciprocal products, and only that product can be mactivated, By comparing dit ferent translocations, itis possible to map this locus, which is called the ic (X-Inactivation center}. A cloned region of450 kb contains all the properties of the ic. When this sequence is inserted as a transgene onto an autosome, the autosome becomes subject to inactivation (atleastin a cellculture system) Pairing of Xic loci on the two X chromosomes hasbeen impli catedin the mechanism for the random choice of X inactivation. Moreover, differencesin the sister chromatid cohesion correlates with the outcome of the choice of the X chromosome to be inactivated, indicating that alternate states resent before the inactivation process may: direct the choice of which X chromosome will become inactivated icis a as-acting locus that contains the information necessary to count X chromosomes and inactivate all copies but one, Inactivation spreads from Xic along the entire X chromo some. When Xicis presenton an X chromosome autosome translocation, inactivation spreads into the autosomal regions (although the effect is not always complete). icisa complex geneticlocus that expresses several long noncoding RNAs (ncRNAS). The most important of these ia gene called Xist (X inactive specific transcrip), which is stably expressed only on the inactive X chromosome. The behavior of this gene is etfectively the opposite ofall other loci on the chromosome, Which are turned off Deletion of Xist prevents an X chromosome from being inactivated Iedoes not, however, interfere with the count. ingmechanism (because other X chromosomes «an be inactivated). Thus, we can distinguish ‘uvo features of ic an unidentified elements) required for counting, and the 2ist gene requized for inactivation, The n-I rule suggests that stabilization of Xist RNA is the “default,” and that some blocking mechanism prevents stabilization at ‘one X chromosome (which will be the active X). ‘This means that, athough Xicis necessary and sufficient for a chromosome to be iinictivated, the products of other loci are necessary for the establishment of an active X chromosome ‘The Aisttranscriptis regulated in a negative manner by Thay, its antisense partner. Loss of ‘Nix expression on the future inactive X chro. mosome permits Xist to become upregulated and stabilized, and persistence of Tsix on the future active X chromosome prevents Xist upregulation. Toit is regulated by Xite, which hasa Tyix-specific enhancer andis located 10 kb upstream of Tsix FIGURE 29.12illustrates the role of Xist RNA in X-inactivation. Aist codes foran ncRNA that Jacks open reading frames. The Ait RNA “coats” the X chromosome from which it is synthe. sized, which suggests that it has @ structural role. Prior to X-inactivation, it is synthesized by both female X chromosomes. Following inactivation, the RNA is found only on the inactive X chromosome. The transcription rate remains the same before and afterinactivation, so the transition depends on posttranscriptional events. Prior to X-inactivation, Xise RNA decays with a half-life of ~2 hours, X-inactivation is mediated by stabilizing the Xist RNA on the inactive X chromosome. The Xist RNA shows a Punctate distribution along the X chromosome, which suggests that association with proteins 10 form particulate structures may be the means of stabilization, We do not know yet what other factors may be involved in this reaction and how the ist RNA is limited to spreading in as along the chromosome. = FF \ 7 t t NN IN Sa Sa tants TaXPNA eet ono he cio f / \ ‘Aclvex active, FIGURE 29.12 X-inactivation involves stailization of ist RNA, which coats the inactive chremeseme. Tx pre- vents xst expression on the future active X- 29.5 X Chromosomes Undergo Global Changes 349850 Por Gone ee PCRS I silencing Macro — p— FEL Hypoaceryiation athietone is x XisPNA HRAKiHOub! Late x Poli'exc. H9K27me3' —ropignion “aK 20m} ‘Xist-eperdent tation of sienoing Nistindependent on inaetvation FIGURE 29.13 2st RNA preduced from the 2ic Locus accumulates on the future inactive X (4). this excludes transcription machivery, such as RNA. Dolyrierase I (Pol 1), Polycomb croup cempiexes ate recruited to the Xt, ‘covered chramesome and establish chromesome- ide histone maificatens. Histone macroH2A becomes enriched an the Ki and preneters of genes on the Xi ate methylated, n this phase X-inativatien is ineversile and Xt is ‘not required far maintenarce of the silent state. Adapted from A. Mute and 3. Gritnau, Cur. Opin. Genet. Dey. 17 (2007): 387-393. Accumulation of Xie on the future inae- tive X results in exclusion of transcription machinery (stich as RNA polymerase II), and. leads to the recruitment of Polycomb repressor complexes (PRC1 and PRC2), which trigger a Series of chromosome-wide histone modi- fications (H2AK119 ubiquitination, H3K27 methylation, H4K20 methylation, and H4. deacetylation). Late in the process, an inac. tive X-specific histone variant, macroH2A, is incorporated into the chromatin, and pro. moter DNA is methylated. These changes are shown in FIGURE 29.13. (The repressive effects of promoter methylation are discussed in the {following sections.) At this point, the hetero- chromatic state of the inactive X is stable, and Aistis not required to maintain the silent state of the chromosome Despite these findings, none of the chro: matin components or modifications found have been shown on their own to be essential for X chromosome silencing, indicating potential redundancy among them or the existence of pathways that are yet to be identified. Global changes also occur in other types of dosage compensation. In Drosophila, a large ribonucleoprotem complex, MSL, is found only in males, where it localizes on the X chromosome. This complex contains two noncoding RNAs, which appear to be needed for localization to the male X (per haps analogous to the localization of Xist to the inactive mammalian X), and a histone acetyltransterase that acetylates histone H4 on K 16 throughout the male X. The net result of the action of this complex is the twofold CHAPTER 29 Epigenetic Effects Are Inherited increase in transcription of all genes on the male X. In the next section, we discuss a third mechanism for dosage compensation, a global reduction in X-linked gene expression in XX (hermaphrodite) nematodes GB Chromosome Condensation Is Caused by Condensins SC proteins are ATPases that include cendensins and catering. + heterodimer of SMC pret ‘ther subunits, * Condensins cause chromatin to be more tightly coiled by introducing positive supercoks into DNA. * Cendensins are response fer condensing cire- miesames at mitosis. ‘+ Chromoseme-speciticcendonsins ae respensibe far condensing imctive X chromosomes in ¢. elegans. in asocintes with ‘The structures of entire chromosomes are influ enced by interactions with proteins of the struc: tural maintenance of chromosome (SMC) family. They are ATPases that fallinto two func tional groups. Condensins are mvolvedin the controlof overall structure and are responsible for the condensation into compact chromo. somes at mitosis. Cohesins are concerned with connections between sister chromatids that concatenate through a cohesion ring, which must be released at mitosis. Both consist of dimers formed by SMC proteins. Condensins form complexes that have a core of the het crodimer SMC2-SMC4 associated with other (non-SMC) proteins. Cohesins have a similar organization but consist of SMCI and SMC3, and also interact with smaller non-SMC sub- units termed Secl/Rad2I and Sec3/SA, FIGURE 29.16 shows that an SMC protein hhas a coiled-coil structure in its center that is interrupted by a flexible hinge region. Both the amino and carboxyl termini have ATP- and DNA-binding motifs. The ATP-binding motif is also known as a “Walker module” SMC monomers fold at the hinge region, forming an antiparallelinteraction between the two halves of each coiled coil. This allows the amino and carboxyl termini to interact to form a “head” domain. Different models have been proposed for the actions of these proteins depending on whether they dimerize by intra- or intermo- lecular interactions, Folded SMC proteins form dimers via several different interactions, The most stable