Nucleosomes: regulators of transcription

/iEVIEWS 19 Jantzen, H-M., Admon, A., Bell, S.P. and Tjian, R. (I990) Nature 344, 830--836 20 Mishima, Y., Finanscek, I., Kominami, R., and Muramatsu, M. (1982) Nucleic Acids Res. 10, 6659--6669 21 Learned, R.M., Cordes, S. and Tjian, R. (1985) Mol. Cell. Biol. 5, 1358-1369 22 Schnapp, A. et al. (1990) Nucleic Acids Res. 18,

3 6 Henderson, S. L., Ryan, K. and Sollner-Webb, B. (1989) GenesDev. 3, 212-223 3 7 Bateman, E. and Paule, M.R. (1988) Cell 47, 985-992 3 8 Parker, K.A. and Steitz, J.A. (1987) Mol. Cell. Biol. 7,

28.99-2913 39 Hannon, G.J~et al. (1989) Mol. Cell. Biol. 9, 4422-4431 40 Tyc, K. and Steitz, J.A. (1989) EMBOJ. 8, 3113-3119 41 Hughes, J.M.X., Konings, D.A.M. and Cesareni, G. (1987) EMBOJ. 6, 2145-2155 42 Tolletx,ey, D. (1987) EMBOJ. 6, 4169-4175 43 Li, H.V., Zagorski, J. and Foumier, M.J. (1990) Mol. Cell. Biol. 10, 1145-1152 44 Henriquez, R., Blobel, G. and Aris, J.P. (1990) J. Biol. Chem. 265, 2209-2215 45 Schimmang, T. et al. (1989) EMBOJ. 8, 4015-4024 46 Caizergues-Ferrer, M. et al. (1989) Genes Dev. 3, 324-333 47 Borer, R.A., Lehner, C.E, Eppenberger, H.M. and Nigg, E.A. (1989) Cell 56, 379-390 48 Peter, M. et al. (1990) Cell 60, 791-801 49 Schmidt-Zachmann, M.S., Hugle-Dorr, B. and Franke, W.W. (1987) EMBOJ. 6, 1881-1890 5 0 Reddy, R. etal. (1983)J. Biol. Chem. 258, 1383-1386 51 Gold, H.A., Topper, J.N., Clayton, D.A. and Craft, J. (1989) Science 245, 1377-1380 52 Morgan, G.T., Reeder, R.H. and Bakken, A.H. (1983) Proc. Natl Acad. Sci. USA 80, 6490--6494

1385-1393 23 Tower, J., Culotta, V.C. and Sollner-Webb, B. (1986) Mol. Cell. Biol. 6, 3451-3462 24 Smith, S.D. etal. (1990) Mol. Cell. Biol. 10, 3105-3116 25 Bateman, E., Iida, C.T., Kownin, P. and Paule, M.R. (1985) Proc. Natl Aca#l. Sci. USA 82, 8004--8008 26 Brill, S.J., DiNardo, S., Voelkel-Meiman, K. and Stemglanz, R. (1987) Nature 326, 414-416 27 Paule, M.R. et aL (1984) Nucleic Acids Res. 12, 8161-8180 28 Buttgereit, D., Pflugfelder, G. and Grummt, I. (1985) Nucleic Acids Res. 13, 8165-8180 29 Tower, J. and Sollner-Webb, B. (1987) Cell 50, 873-883 3 0 Cavanaugh, A.H. and Thompson, E.A., Jr (1985) Nucleic Acids Res. 13, 3357-3369 31 Mahajan, P.B. and Thompson, E.A. (1990)J. Biol. Chem. 265, 16225-16233 32 Bartsch, I., Schoneberg, C. and Grummt, I. (1988) Mol. Cell. Biol. 8, 3891-3897 33 McStay, B. and Reeder, R.H. (1990) Mol. Cell. Biol. 10, 2793-2800 34 Connelly, S. and Manley, J. (1989) Mol. Cell. Biol. 9, 5254-5259 35 Kuhn, A., Bartsch, I. and Grummt, I. (1990) Na~.ure 344, ~59_5r.", •

..

R . H . REEDER IS IN THE FRED HUTC~IINSOI¢ CANCER RESEARCH

]c ~ m

n24 c o w ~ u s r ~ r , s~urr~ tra 9slo4, vs~

II

k.i.,

In the bacterial paradigm of gene regulation, control occurs by the recognition of DNA signals by specific transcription factors. One extension to this mode involves the changes in gene activity caused by altering DNA supercoiling, arguing that DNA topology is also an important contributor to gene regulation. The nucleosomal core particle is the building block of the eukaryotic chromosome, containing a histone octamer (two molecules each of histones H2A, H2B, H3 and H4) and 146 bp of DNA wrapped approximately 1.8 turns around the octamer. A single molecule of histone H1 interacts with the octamer and with linker DNA between core particles, sealing off approximately two full turns of DNA (164-166 bp) around the octamer. Nucleosomes contribute greatly to DNA topology, and have been shown to act as inhibitors of transcription in vitro. Despite this, much important work dealing with eukaryotic transcription has assumed histories to be 'invisible' to the regulatory factors that recognize their promoter elements. This review presents several lines of evidence that argue that the nucleosome is an integral component and regulator of transcriptional mechanisms. Experiments will be discussed that indicate that (1) histone modifications are important for the unfolding of chromatin domains even before transcription, (2) the nucleosome acts to repress transcription initiation in vivo and must be displaced to allow a certain level of basal transcription, and (3) individual histories may have unique functions; in particular a short segment of

Nucleosomes: regulators of transcription MICHAELGRUNSTEIN Histories and nucleosomes are involved in the folding of DNA in the eukaryottc cell Recent evidence suggests that they are also involved in a mulltstep process of DNA

unfo/d/ngasd &euercgu/at/og histone H4 is specifically involved in repression of the yeast silent mating loci.

Nudeosomes on repressed, inducible and active chromafin The work of Weintraub and his co-workers has shown that tissue-specific gene activity is accompanied by an unfolding (i.e. increased DNase I sensitivity) of the chromatin packaging that gene (reviewed in Ref. 1). Two important results have since emerged. First, chromatin unfolding appears to precede induction, and second, the region that is unfolded can be considerably greater than the transcribed gene itself. For example, Sippel's group has shown that the repressed chick lysozyme gene in an erythrocyte appears to be present in a folded nucleosomal array. However, in the oviduct the inducible (but inactive) and the active lysozyme gene have in common a number of 5'-specific

TIG DECEMBER1990 VOL.6 NO. 12 @1990 Elsevier Science Publishers lad (UK) 0168 - 9479/90/502.00

E]~]

BEVIEWS DNase-I-hypersensitive sites in the vicinity of the promoter. Upon induction, only one new hypersensitive site is evident (reviewed in Ref. 2). Similarly, there is a phased nucleosomal array along the entire length of the repressed ~-major globin gene in mouse fibroblasts. In erythroid cells, the [3-globin gene may be inactive but inducible, or alternatively it may be activated. In either case, the globin chromatin shows increased nuclease sensitivity and a stretch at the upstream regulatory region from which several nucleosomes have been altered or displaced3. These data argue that nuclease sensitivity and nucleosome displacement at the c/s-acting promoter precede induction in the appropriate cell type. The DNase-I-sensitive domain associated with inducibility or activity can be much greater than the gene and its immediate c~-acting promoter sequences. The chick lysozyme DNase-I-sensitive domain is approximately 24 kb long while the lysozyme transcript is only 4 kb z. S~ilarly, when human nonerythroid cells

Repressed

sP

are fused with murine erythroleukemia cells, approximately 80 kb of DNA surrounding the human ]]-globin gene are reorganized into a DNase-I-sensitive domain 4. Of special interest are the boundaries that help to define the unfolded domain. Sequence elements (A-elements) at the 5' and 3' boundaries of the chick lysozyme DNase-I-sensitive domain are important for position-independent high-level transcription after stable transfection. Also, Grosveld, Groudine and their colleagues have shown that position-independent high-level expression of the human ~-globin gene upon transformation requires both a nuclease-hypersensitive dominant control region (DCR) present approximately 50-60 kb upstream, and 3' sequences some 20 kb downstream of the ~-globin gene. Elements subcloned from the large (20 kb) DCR alone can direct elevated, position-independent expression of the [3-globin gene (reviewed in Ref. 2). Increased nuclease sensitivity of large, potentially active domains suggests that chromatin in these regions has unfolded. How these domains are targeted for unfoldk:g is unclear but it may involve specialized sequences at the boundaries of the DNase-I-sensitive domain. The chick lysozyme A-elements contain SAPS (nuclear scaffold-associated regions, otherwise known as nuclear matrix attachment regions or MARs) mediating the attachment of the chromatin domain to the chromosomal scaffold. SARs near other genes have been proposed to specify the base of chromosomal loops. SAPs are several hundred nucleotides long, rich in A.T base pairs and can bind topoisomerase II in vitroS. The globin DCR may be close to a SAR, but this association has not been well characterized. By interacting with specialized proteins, DCPs or even the SAPS themselves may provide signals at which histone modification enzymes initiate chromatin unfolding, as described below.

Histone modifications may unfold chromatin domains before transcription

H1 and core histone tail , modifications '

sP Chromatin unfolding FIGil Proposed stages in the unfolding of a developmentally regulated gene. A highly folded repressed chromatin domain is tethered to the nuclear scaffold proteins (SP) by topoisomerase II interaction with SARsequences. A dominant control region (DCR)or SARsequences themselves may serve as targets for histone modifications allowingchromatin unfolding. Only then may the activator protein recognize its binding site, setting up the inducible stage (Fig. 3).

Two histone modifications that can potentially alter the repressed chromatin domain and thereby generate the DNase-I-sensitive state are (1) an altered histone H1 interaction and (2) acetylation of lysine residues within the core histone amino-terminal tails. These are of special interest, since neither H1 nor the core histone amino-terminal tails are required for nucleosome assembly, yet both are required for the folding of the 10 nm 'beads on a string' chromatin fiber (associated with acti-'e chromatin) to the more compact 30 nm solenoidal fiber associated with inactive chromatin6. Other sources of chromatin variability, including histone H2B ubiquitination, the presence of certain histone H2A variants and HMG proteins, have all been associated with active chromatin. However, since little is known of possible mechanisms by which these interactions may cause chromatin unfolding, they will not be covered here. (1) An altered histone H1 interaction Histone HI contains a hydrophobic core (which interacts with the nucleosome dyad) and basic aminoand carboxy-terminal tails (which interact with linker DNA present between nucleosomes). Several examples

TIG DECEMBER1990 VOL.6 NO. 12

~9(~

[~EVIEWS point to an altered H1 interaction on active genes. Histones HI and H5 (an HI variant found in erythrocytes) are associated with both inactive (the viteUogenin gene) and inducible or active (globin genes) chromosomal regions in chicken erythrocytes 7. However, nuclease digestion studies suggest that H1 protects linker DNA of inactive genes more extensively. In other studies, H1 has been found to be depleted from purified chromatin of certain active or potentially active genes, including Xenopus oocyte 5S RNA genes 8 and the inducible or active mouse kappa immunoglobulin light chain gene locus9. Whether this depletion simply reflects selective H1 loss from active genes during experimental manipulations remains to be determined, especially as H1 is clearly present on the active Balbiani rings of Chironomus tentans as shown by immunoelectron microscopy ~0. How H1 interacts reversibly with linker DNA is unclear but this process is clearly a candidate for H1 tail phosphorylation H.

activation time in response to given conditions might dictate the stages involved. In the yeast Saccbaromyces cerevisiae, much of the genome appears to be continuously in an HI-depleted, hyperacetylated, unfolded, DNase-I-sensitive, inducible or active state 16,17. Given the evolutionary conservation of eukaryotic transcriptional mechanisms and the power of yeast genetics ~.n dissecting regulatory pathways, yeast is therefore well suited for the study of the transition from the inducible to the active state.

Chromatin structure and gene regulation in yeast Yeast genes, like those of higher eukaryotes, contain activator binding sites (upstream activator sequences or UASs). They also contain TATA elements just upstream of the initiation site, at which basal factors and RNA polymerase II (i.e. the preinitiation complex) bind to start transcription. TFIID, the basal protein binding the TATA element, is especially important in that its binding then allows the rest of the preinitiation (2) Histone acetylation complex to recognize the promoter. One of the best The core histone amino-terminal ends all contain studied examples of yeast chromatin is that for the acid lysine residues whose epsilon amino groups are acety- phosphatase PH05 gene. The uninduced PH05 prolated and their positive charges neutralized reversibly moter, in repressive high phosphate media, has one of during the cell cycle. The laboratories of Allfrey, its weaker UAS elements (UASp-1) in a nucleaseGorovsky and Bradbury have shown that highly acety- sensitive site. Under these conditions the TATA element lated levels of H3 and H4 are strongly correlated with is folded in a nucleosomal structure TM. Upon activation chromatin unfolding and elevated levels of in low phosphate media, four precisely positioned transcription (reviewed in Ref. 1). Also, partial histone nucleosomes bracketing this UAS element are dishyperacetylation (induced by sodium butyrate treat- placed, liberating a stronger UASp-2 element and the ment of HeLa cells) leads to partial unfolding of inter- TATA promoter element. This argues for the presence phase chromatin as viewed by electron microscopy12. of the PH05 gene in two possible states only Finally, antibodies specific for acetylated lysine residues (inducible and active). The divergently transcribed GALl and GALIO genes of histone H4 preferentially interact with the active ¢x-D globin chromatin but not the inactive ovalbumin chro- of yeast are repressed in media containing glucose and matin 1-~. Significantly, indirect immunofluorescence are fully complexed in nucleosomes under these conusing similar antibodies in Tetrahymena has shown that ditions. In media containing glycerol or ethanol, the acetylation of histone H4 precedes transcription t't. It is genes are more rapidly inducible (but not yet active). possible that histone acetylation may not only be nec- In galactose-containing media the genes are fully essary for generating inducible chromatin but also for active. Activation is accompanied by nucleosome disthe efficient recognition by RNA polymerase of a pro- placement from the promoter region, including the moter folded in a nucleosome. This suggestion is sup- region around the TATA element. While the UASc ported by the finding that histone acetylation can alter element is in a DNase-I-hypersensitive site under all DNA topology (i.e. cause negative supercoiling) in a growth conditions, the activator GAL4 protein is bound manner that would be expected to stimulate initiation to the UASG element in the inducible and active states onlyl9. Therefore, the GAL1-GALIO genes may exhibit of transcription by RNA polymerase 15. all three chromosomal states of activation, although it is not known whether these genes are folded in a Conclusion It may be postulated that certain potentially active defined domain in the repressed state. The PH05 and GAL1-GALIO chromatin structures DNase-I-sensitive domains are specified at their boundaries by SAR (MAR) sequence elements. These may be ai'gue that yeast gene activation involves, first, the tethered by topoisomerase II to the nuclear scaffold. presence of a factor at the UAS that makes the UAS Chromatin unfolding before transcription, to create the nuclease sensitive in the absence of induction. In the inducible state, may then be initiated by the recogni- case of GAL1-GALIO this may be a function of the tion of a particular DNase-I-hypersensitive sequence GRF2 protein, which has the ability to prohibit a (perhaps the SAR or even DCR-like sequences for nucleosome from occupying the UASG (Ref. 20). In the other genes) by histone modification enzymes mediat- inducible state, GAL4 recognizes the UASG. Upon ing displacement of H1 and the amino-terminal tails of induction, GAL4 activates initiation of transcription by core histones (Fig. 1). Only after chromatin is unfolded 'communicating' with the preinitiation complex, allowin this manner might the activator proteins bind and ing binding at the TATA promoter and initiation. Unclear in each of these examples is the role of the stimulate transcription initiation. Not all genes in a eukaryote should be expected to nuc!eosome in gene regulation. How do changes in undergo the transition from the repressed to the nucleosome structure mediate the transition from inducible state before activation. Differences in inducible to active chromatin? TIG DECEMBER1990 VOL.6 NO. 12 39,

i

EVIEWS both viral and yeast promoters in vivo is generally much lower than that seen in vitro in nuclear extracts lacking nucleosomes24, 25. Also, nucleosome assembly conditions in vitro lead to a much greater decrease in basal transcription than in activated transcription mediated by mammalian transcriptional activators and GAL4-VP16 (a fusion of the yeast GAL4 DNA-binding site with the activation domain of the viral activator VP16) (Ref. 26; J.L. Workman, I. Taylor and R.E. Kingston, unpublished). These data also argue that the activator must directly or indirectly mediate nucleosome displacement from the TATA element. This could occur through an interaction between the activator and TFIID27 that increases the rate or stability of TFIID binding26; in fact there are data showing that an activator can alter the footprint produced by a partially pure mammalian TFIID fraction near the TATA elemen#S. The altered TFIID interaction might be sufficient to displace an already bound nucleosome from the TATAelement. Alternatively, activators may interact directly with histones, thereby displacing them from DNA. If nucleosomes prohibit basal transcription in vivo, then nucleosomal displacement in the absence of the UAS mechanism should activate initiation of otherwise inactive genes.

Unfolded (inducible) chromatin structures may allow activator binding Replication has been proposed to allow the clearing of nucleosomes to allow factor binding. However replication is unnecessary for the activation of many eukaryotic genes, implicating trans-acting factors in nucleosome displacement21. Once repressed chromatin has been unfolded by the histone modification enzymes, the activator or a post-translationally modified activator may recognize its binding site. In the case of the globin or lysozyme structures, an activator protein may first bind to one of the unfolded repeated enhancer elements (some of which are probably not folded in a nucleosome). In certain yeast genes, a GRF2-1ike protein may allow more efficient activator binding by preventing a nucleosome from occupying the UAS. The activator, once bound, may cause further nucleosome displacement at the promoter. This is supported by the finding of the Hager, Wrange and Beato laboratories that the mouse mammary tumor virus glucocorticoid receptor protein can form a complex with its upstream regulatory sequence when the sequence is folded in a nucleosome in vitro. Hormone action in vivo leads to displacement of a nucleosome at this position. Only then can the basal transcription factor NF1 bind, leading to initiation 2.

UKY412

Physiological function of

repression of initiation by nucleosomes

GAL (G)

In contrast to the situation at the activator site, there is considerable evidence that nucleosomes will repress the TATA promoter, probably by preventing access of TFIID. Nucleosomes present at a certain critical density or positioned on a promoter sequence will prevent initiation of transcription in vitro],Zz. However, prior incubation of a promoter with proteins of the transcription complex allows initiation23. The physiological purpose for the repression of initiation by nucleosomes may be to keep basal transcription (defined as transcription caused by recognition of the TATA element by the preinitiation complex) repressed in the absence of activation. Evidence for this comes from experiments showing that basal transcription of

GLU (D)

Nucleosome loss in yeast

G

or

D

+

+

G

D

UKY403 .,~

/,/

-

-

GAL (G) or G GLU(D)

. . . . . . .

+ +

D

G

D

F/G! Derepression of PH05 transcription upon nucleosome loss. Northern blot illustrating the synthesis of PH05 mRNA in UKY412 and UKY403 yeast strains. The two strains, constructed by Ung-Jin Kim in our laboratory, are isogonic except that UKY412contains its sole histone H4 gone attached to its own promoter while UKY403 contains an H4 gene under the control of the GALl promoter. Low levels of inorganic phosphate (Pi-) activate, while high levels (Pt +) repress PH05. Note that nucleosome loss obtained by repressing the H4 gene in glucose (D) in high Pi + activates the PHO5gene. TIG DECEMBER

1990 VOL. 6 L

NO. 1 2

Changes in histone gene dosage have been shown to affect transcription originating from certain yeast genes located next to transposable elements 29. In our laboratory, we have asked whether nucleosome depletion fn vivo will activate initiation of otherwise inactive genes. By creating yeast strains in which histone H2B or H4 synthesis may be repressed, we have obtained loss of approximately half the nucleosomes in G2-arrested cells. Nucleosome loss caused by depleting histone H4 had little obvious effect on constitutive transcription of rRNA, 5S RNA, tRNA and mRNA genes. An examination of uninduced mRNA levels produced by regulated genes initially yielded conflicting results. For example, the uninduced steady-state level of P H 0 5 mRNA was increased considerably (Fig. 2), while that of CUP1 was not noticeably increased after nucleosome loss-~.n.

EVIEWS However, we now have evidence to suggest that activation by nucleosome loss can be obscured by post-transcriptional degradation of (CUP1) mRNAs. In some cases it rnwy potentially also be reduced by competing catabolite repression acting through UAS elements of glucose-repressible genes. When UAS elements were deleted from different regulated promoters (PH05, CYC1, GALl) that were then fused to the E. coli ~galactosidase (lacZ) gene, nucleosome loss activated each of these promoters to approximately 5--15% of the normal fully induced level, depending on the promoter. Even inactivated HIS3 and CUP1 promoters attached to lacZ were activated to high levels by nucleosome loss (Ref. 31; L. Durrin and M. Grunstein, unpublished). These data show that nucleosome loss activates genes in the absence of the UAS mechanism and that the preinitiation complex must be at least partially active in vivo in the absence of a functional UAS mechanism.

Conclusion Our data suggest two levels of activation of these regulated yeast genes (Fig. 3). In the inducible state, the activator is bound to the UAS, possibly with the help of an additional (GRF-2-1ike) protein. Upon induction, the activator may mediate nucleosome loss from the TATA element by altering the binding of TFIID or interacting directly with the nucleosome. Nucleosome displacement may allow' access of the partially active preinitiaticn complex to the TATA element, allowing a basal level of transcription. A second level of activation may then occur in which TFIID or the preinitiation complex is modified by the activator (possibly by an intermediate adaptor molecule 25) allowing the fully active state. This latter step is likely to be independent of chromatin structure. The relative contributions of these two steps could differ for different UAS and TATA elements.

Histone H4 and repression of the silent

mUnglocl

(a) Inducible

TFIID~

activator

TATA

0

i ,,_ ww

basal transcription (chromatin dependent)

U mRNA .

active transcription (chromatin independent)

I

~

mRNA •

?

o

,--,

MMMM FIGI~ Proposed stages in the activationof an inducible yeast gene. (a) The inducible stage in which the activatoris bound to the unfolded potentially active gene. Co) Chromatin-dependentactivation. The activatormediates nucleosome displacement from the TATAelement. ,'ITfismay occur by its affecting the rate or extent of TFI!Dblndmg to the TATAelement or by interacting directlywith histones. Nucleosome displacement would cause a paoAall'yactive preinitiationcomplex to initiate a basal level of transcription. (c) Chromatin-independentactivation. The activator interacts with or modifies TFIID, possibly through aa adaptor proteinz~, to allow full induction.

The data described above deal with the histone octamer unit as a repressor of transcription. However, each of the ,histories has a different pO2,aw sequence and is likely to have distinct functions in the regulation of folding and activity of DNA. A most dramatic example of a separately definable histone function occurs with respect to yeast mating. In S. cerevisiae, there are three loci on chromosome III [HM//x, MAT (a or a) and HMRa] that encode information that determines mating type. Despite the presence of identical promoter sequences at the silent loci and MAT(e.g. ~ and MATa or HMRa and MATa), only the info__rmation located at the MAT locus is

expressed and determines the mating type of the cell (a, a or, in the case of a diploid cell, a/a). The production of both a and a information in the same cell acts to shut clown haploid functions and prevent further mating. Therefore, the expression of haploid functions depends on the efficient repression of r-/M/a and HMRa. Repression occurs through four nonessential repressor proteins SIR1, SIR2, SIR3 and SIR4 that act

TIG DECEMBER1990 rot. 6-No. 12

~gtJ

EVIEWS

References 1 van Holde, K.E. (1988) in Chromatin (Rich, A., ed.), Springer-Verlag 2 Elgin, S.C.R. (1990) Curr. Opin. Cell Biol. 2, 437-445 2) Benezra, R., Cantor, C.R. and Axel, R. (1986) Cell 44, 697-704 4 Fon'ester, W.C. et al. (1987) Nucleic Acids Res. 15, 10159-10177 5 Adachi, Y., Kas, E. and Laemmli, U.K. (i989) EMBOJ. 8, : i n 3997-4006 i I ~.~..J , ; ! == 6 Allan, J., Harborne, N., Rau, D.C. and Gould, It. (1.982) J. Cell Biol. 93, 285-297 E I 7 Weintraub, H. (1984) Cell 38, 17-27 HML 8 Wolffe, A.P. (1990) NewBioL 2, 211-218 9 Garrard, W.T. (1989) in Tissue Specific Gene Expression FIGll (Renkawitz, R., ed.), pp. 1-31, VCH Publishers The histone H4 amino-terminal residues 16-19 (the four plus symbols, representing the highly conserved amino acid 10 Ericsson, C., Grossbach, U., Bjorkroth, B. and Daneholt, B. (1990) Cell60, 73-83 sequence Eys-gfg-His-Arg)are involved in the repression of the yeast silent mating loci HML(shown here, open box) and HMR 11 Hill, C.S., Packman, L.C. and Thomas, J.O. (1990) EMBO J. 9, 805--813 by a direct or indirect interaction with the repressor protein SIR3. E and I are the silencer DNA elements, necessary for 12 Annunziato, A.T., Frado, L-L.Y., Seale, R.L. and Woodcock, C.L.E (1988) Chromosoma 96, 132-138 effective repression of HMLand HMR.Since SIR3does not appear to recognize the silencer elements themselves, it is 13 Hebbes, T.R., Thome, A.W. and Crane-Robinson, C. (1988) EMBOJ. 7, 1395-1402 proposed that another protein(s) (X) mediates the interaction between SIR3and E (or I ) DNA, thereby providing the 14 Pfeffer, U., Ferrari, N., Tosetti, E and Vidali, G. (1989)J. Cell. Biol. 109, 1007-1014 specificity for this interaction at the silent mating loci. 15 Norton, V.G., Imai, B.S., Yau, P. and Bradbury, E.M. (1989) Cell 57, 449-457 through the E and I silencer sequences which border 16 Lohr, D. and Hereford, L. (1979) Proc. NatlAcad. Sci. on both silent mating loci. Surprisingly, while two USA 76, 4285--4288 additional proteins, encoded by RAP1 and ABF1, have 17 Nelson, D. (1982)J. Biol. Chem. 257, 1565-1568 been shown to bind to these sites, there is no evi- 18 Almer, A., Rudolph, H., Hinnen, A. and Horz, W. (1986) EMBOJ. 5, 2689-2696 dence that SIR proteins bind silencer sequences 19 Selleck, S.B. and Majors, J.E. (1987) Mol. Cell. Biol. 7, (reviewed in Ref. 32). 3260-3267 Deletion or nonconservative amino acid substi20 Chasman, D.I. et al. (1990) Genes Dev. 4, tution in an extremely conserved sequence (residues 503-514 16--19; Lys-Arg-His-Arg) at the H4 amino terminus 21 Chiu, C-P. and Blau, H.M. (1984) Cell 37, specifically activates the silent mating loci32-34. The 879-887 involvement of a ubiquitous protein such as histone 22 Lorch, Y., LaPointe, J.W. and Kornberg, R.D. (1987) Cell H4 in this highly specific function of silencing seems 49, 203-210 paradoxical. However, mutations at the N-terminus of 23 Workman, J.L. and Roeder, R.G. (1987) Cell 51, SIR3, a repressor specific to the silent mating loci, will 1613-1622 ,~upp:'ess point mutations in residues 16-19 but not a 24 Berk, A.j. (i986) Annu. Rev. Genet. 20, 45-79 viable deletion of the H4 amino terminus33. Therefore, 25 Lewin, B. (1990) Cell61, 1161-1164 SIR3 requires the presence of the H4 amino terminus 26 Workman, J.L., Roeder, R.G. and Kingston, R.E. (1990) EMBOJ. 9, 1299-1308 for suppression, and silencing is likely to occur through a direct or indirect interaction between SIR3 27 Stringer, K.F., Ingles, C.J. and Greenblatt, J. (1990) Nature 345, 783-786 and histone H4 (Fig. 4). It is interesting to note that 28 Horikoshi, M. et al. (1988) Cell 54, 1033-1042 the deletion that affects silencing (H4 del 4-19 but not 29 Clark-Adams, C.D. et al. (1988) Genes Dev. 2, H4 del 4-14) also affects the ability of the yeast (:x2 150-159 operator/repressor (involved in suppressing a-mating 30 Han, M., Kim, U-J., Kayne, P. and Grunstein, M. (1988) type-specific genes in haploid a and diploid a/0t cells) EMBOJ. 7, 2221-2228 to position a nucleosome directly adjacent to the oper- 31 Han, M. and Grunstein, M. (1988) Cell 55, ator (Ref. 35, S. Roth and R. Simpson, unpublished). 1137-1145 Undoubtedly, further investigation will uncover new 32 Kayne, P.S. et al. (1988) Cell 55, 27-39 mechanisms by which histones interact with regulatory 33 Johnson, L.M., Kayne, P.S., Kahn, E.S. and Grunstein, M. Proc. Naa Acad. Sci. USA (in press) proteins and therefore regulate transcription in yeast 34 Megee, P.C., Morgan, B.A., Mittman, B.A. and Smith, and in other eukaryotes. M.M. (1990) Science 247, 841--845 35 Roth, S?lr., Dean, A. and Simpson, R.T. (1990) Mol. Cell. Acknowledgements Biol. 10, 2247-2260 I thank many colleagues who sent me their published and unpublished papers. I am especially than'ld'ul to Arnold 13erk for his thoughtful criticisms of this manuscript. I also apologize to those colleagues whose work was quoted in the reviews of others as a result of space considerations. The M. GRUNSTEINIS IN THE MOLECULARBIOLOGYI N S T ~ AND work from our own !~boratory described here was supported TIlE DEPARTMENTOF BIOLOGY, UNIVERSITYOF CALIFORNIA,LOS by Public Health Service grants from the NIH (GM23674; ANGELES, CA 90077, US~L GM42421). "rIGDECEMBER1990 VOW.6 NO. 12 t-0(1