Chromatin and transcriptional activity in earlyXenopusdevelopment

seminars in

CELL BIOLOGY, Vol 6, 1995: pp 191–199

Chromatin and transcriptional activity in early Xenopus development Nicoletta Landsberger and Alan P. Wolffe

Developmental regulation of gene expression during Xenopus oogenesis and embryogenesis

Experiments with Xenopus oocytes and embryos have determined a direct biochemical relationship between chromatin structure and transcription. Nucleosomes within specific nucleoprotein architectures can either activate or repress transcription. Developmentally regulated changes in chromosomal composition direct the dominant repression of specific genes. Reconstruction of chromatin templates in vivo establishes that replication-coupled chromatin assembly both represses basal transcription and facilitates a full range of inducible gene activity. Chromatin structure emerges as a major contributory factor to the regulation of genes.

The genome of the Xenopus oocyte is vigorously transcribed. Synthesis of specific mRNAs, ribosomal RNA and tRNA occurs in a highly regulated manner over a differentiative period extending over several months. Only a fraction of the mRNAs synthesized during oogenesis are directly available for translation, most are sequestered in a translationally inert masked form. Following oocyte maturation into an egg, transcription ceases. The early cleavage divisions following fertilization rely on stores of proteins laid down during oogenesis and the recruitment of masked maternal mRNA to the translational machinery for all cellular assembly and differentiative processes. After 11 rapid cleavage divisions, the 4000 cell stage embryo activates zygotic transcription. This developmental process is known as the mid-blastula transition (MBT), it is also associated with a lengthening of the cell cycle and the acquisition of cell motility. At this time many genes are pleiotropically transcribed including both oocyte ‘specific’ genes that are normally repressed in somatic cells, and genes whose activity will later be restricted to specific cell lineages. Regulated gene transcription begins at the MBT. As gastrulation proceeds, cell lineage specific patterns of gene activity are established. Further global changes of gene activity during the gastrula-neurula transition (GNT) include extensive repression of transcription by RNA polymerase III. Maternal mRNA is degraded during gastrulation and subsequent development is directly substantially through transcriptional mechanisms (Figure 1).

Key words: Xenopus / linker histone / histone acetylation / HMGs / developmental control ©1995 Academic Press Ltd

Xenopus laevis OOGENESIS and embryogenesis provide outstanding systems for the biochemical analysis of chromosomal structure and function. The nuclei of the large oocytes are easily injected with DNA templates that are efficiently assembled into chromatin and potentially transcribed. DNA injected into Xenopus eggs is assembled into chromatin and nuclear structures. These pseudo-nuclei are competent for replication and can be made useful for the analysis of how chromosomal and nuclear structure influence transcription. Recent technical advances allow the chromosomal composition of both the pseudo-nuclei and endogenous chromosomes to be manipulated in vivo. The future offers the exciting prospect of understanding in molecular detail how DNA packaging and compartmentalization impact on the transcription process. Most importantly the Xenopus embryo allows the developmental consequences of this transcriptional control to be determined.

The developmental regulation of chromosomal composition in Xenopus — impact on nuclear processes

From the Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892-2710, USA ©1995 Academic Press Ltd 1043-4682/95/040191 + 09 $12.00/0

Core histones Large stores of core histones accumulate during 191

N. Landsberger and A. P. Wolffe Xenopus oogenesis.1,2 These core histones are enough to assemble a mass of DNA equivalent to 20,000 diploid nuclei into chromatin.3 They are stored in the oocyte nucleus together with the molecular chaperones, nucleoplasmin4 and N1/N2.5 Histones H2A and H2B associate with nucleoplasmin and histones H3 and H4 with N1/N2.6 The core histones are extensively modified by phosphorylation and acetylation. Histone H4 is both diacetylated and phosphorylated.7-9 Histone H2A is phosphorylated, as is a major variant histone H2A.X.9-10 The amount of translatable core histone mRNA is constant in the Xenopus oocyte, egg and cleavage embryo.11 Core histone transcripts are deadenylated at oocyte maturation yet this does not influence their overall translational competence. Translation of each histone mRNA amounts to a synthesis of 600pg of each protein per hour during early embryogenesis. This is enough to package a mass of DNA equivalent to more than 2,000 nuclei by the MBT. Core histone gene transcription is very active early in gastrulation, however unlike the situation in oocytes, transcription is now tightly linked to the S-phase of the cell cycle. Thus at all times during early development an excess of core histones is available from oogenesis through gastrulation to package DNA into chromatin. Chromatin assembly dependent on the core histones alone does not easily account for the differences in transcriptional activity between DNA injected into oocytes compared to that injected into fertilized eggs

of embryos. Duplex DNA is assembled with the core histones with comparable rates and efficiencies within oocytes and embryos.12 Although the large mass of core histones sequestered in the oocyte nucleus us theoretically enough to package 120ng of DNA.1,2 the rate and efficiency of chromatin assembly is such that much less duplex DNA ( ~ 3ng) can be efficiently packaged into chromatin even after a 24-hr incubation in the oocyte nucleus. This is because the physiological chromatin assembly process only normally occurs during replication, during which assembly is actively coupled to DNA synthesis.13,14 In fact due to inefficient chromatin assembly, most genes injected as double stranded DNA into oocytes are actively transcribed.15 In order to repress basal transcription in oocytes efficiently, chromatin assembly has to be coupled to replication through the injection of single-stranded templates that undergo second strand synthesis in the oocyte nucleus.16,17 Alternatively, chromatin assembly on duplex DNA has to be allowed to occur for extended periods of time ( > 4 hrs) before the trans-acting factors necessary to activate the basal transcriptional machinery are made available (see Figure 2).16-18 In contrast to the relative efficiency with which a wide variety of promoters are transcribed in the oocyte nucleus, the microinjection of duplex DNA into embryos leads to transcriptional quiescence prior to the mid-blastula transition.12,19-24 However, due to the toxicity of exogenous DNA for subsequent

Figure 1. Gene expression during early Xenopus development. Development is shown from left to right. Major regulatory mechanisms operating at a particular developmental stage are indicated.

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Chromatin and transcription in Xenopus embryogenesis, generally much less DNA ( ~ 300pg) is typically injected into fertilized eggs compared to oocytes ( ~ 3ng). This facilitates the assembly of templates into both chromatin and ‘pseudo-nuclei’. Some investigators have suggested that a simple titration of core histones regulates transcription during early embryogenesis and accounts for transcriptional quiescence.12 Almouzni and Wolffe14 have shown by titration and readdition of core histones within the developing embryo that in fact these proteins have a necessary, but far from sufficient role in generating a transcriptionally inactive state prior to the MBT (see later). An artificial increase in DNA

content within embryos prior to the MBT will lead to the detection of significant transcription from certain genes.12,23,24 However significant transcriptional activation of the microinjected c-myc promoter prior to the MBT occurs with core histones in considerable mass excess ( > 24 fold) over DNA.12 Thus it is possible that titration of another more limiting transcriptional repressor or some other independent process determines basal transcriptional activity. Several independent observations suggest that this is in fact the case. An important consideration concerning the simple histone titration model for transcriptional activation

Figure 2. Basal transcription and the induction of hsp70 promoter activity after heat shock are dependent on the pathway of chromatin assembly. Stage VI oocytes were injected with 3ng of double-stranded (lanes 1, 2) or single-stranded (lanes 3, 4) M13Hsp70CAT DNA and, as internal standard, 0.3ng of the CMV DNA. After 4 hrs at 18°C the oocytes were incubated 3 hrs at 18°C (lanes 1, 3) or 34°C (lanes 2, 4). Groups of 15 healthy oocytes were collected and processed both for RNA analysis by primer extension (A) and DNA analysis (B). In A the position of the correct primer extension products is indicated. In B the DNA was extracted, resolved on a 1% agarose gel, transferred and hybridized with radiolabeled M13Hsp70CAT DNA. The horizontal arrow indicates the position of the supercoiled CMVCAT DNA. Double-stranded form I (closed circular supercoiled), form II (nicked circular), form Ir (closed circular) and single-stranded DNA (ss) are indicated.

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N. Landsberger and A. P. Wolffe expression of genes in vertebrates (see article by Richard Schultz in this issue, also ref 28).

during embryogenesis is whether the transcription obtained in the presence of exogenous DNA prior to the MBT reflects basal or regulated transcription. Under normal developmental conditions only certain class II genes are activated at the MBT, others remain repressed.22 In a detailed study Almouzni and Wolffe14 found that a simple titration of the core histones by the addition of a mass of DNA equivalent to that found at the MBT (25ng) would activate exogenous class III gene transcription, but not exogenous class II promoters, including the CMV immediate early promoter or the adenovirus E4 promoter. This result is consistent with that of Lund and Dahlberg25 who found that the transcriptional activation of exogenous Xenopus U1 genes prior to the MBT would not be achieved by a simple increase in the DNA content of the embryo. Taken together these results demonstrate that the activation of c-myc transcription prior to the MBT12 is a promoter specific phenomenon. The key step for transcription of the CMV and E4 promoters following injection into developing Xenopus embryos prior to the MBT is the recruitment of the basal transcriptional machinery to the TATA homology. This can be accomplished either by titration of chromatin and addition of exogenous TBP, or by expression of a transcriptional activator that can penetrate chromatin, with binding sites next to the promoter.14 Presumably such activators are either not required for c-myc transcription or c-myc promoter specific activators are present in excess within the preMBT embryo.12 The capacity of transcriptional activators to overcome repressive influences on transcription prior to the MBT demonstrates that the class II gene basal transcriptional machinery including endogenous TBP is fully competent in the cleavage embryo. Therefore, the basal transcriptional machinery must be prevented from stably associating with promoter elements by inhibitory proteins. Since exogenous TBP alone does not overcome this inhibition, the inhibitory proteins must be dominant over the basal class II transcriptional machinery (Figure 3) (see also refs 13, 26, 27). Transcriptional activators can be dominant over inhibitory chromatin structures and can recruit the basal transcriptional machinery to activate transcription. Thus one component of transcriptional quiescence in the Xenopus embryo prior to the MBT must be the absence or functional constraint of transcriptional activators. Selective utilization of particular transcription factors during early embryogenesis may have important consequences for the regulated

Core histone acetylation The only modification of the core histones that has been extensively studied with respect to the functional consequences for transcription during Xenopus development is acetylation. This post-translational modification occurs in the highly basic amino (N)-terminal tail domains of the histones that lie on the outside of the nucleosome.29 When practically all of the acetylatable lysines in the N-terminal tails are modified, the core histones are described as being hyperacetylated. This type of modification is usually associated with transcriptionally active chromatin.30 Extensive acetylation of the core histones may facilitate the access of transcription factors and RNA polymerase to DNA in the nucleosome.31 Reversible acetylation of the N-terminal tails is one potential site at which signal transduction mechanisms might impact on chromatin structure and gene expression (see article by Bryan Turner in this issue). Histone acetylation is developmentally regulated.8,32,33 In both the sea-urchin and Xenopus, histone H4 is stored within unfertilized eggs in the diacetylated form. During the rapid cleavage divisions following fertilization, histone H4 is gradually deacetylated.8,33 Extensive accumulation of deacetylated histone H4 correlates with a lengthening of the cell

Figure 3. A model for transcriptional control in the cleavage Xenopus embryo (see ref 14). The default state in the cleavage Xenopus embryo is for the core histones and other chromatin components (Histones) to repress transcription, either non specific DNA (NS DNA) in the presence of exogenous TATA-binding protein (TBP) or transcriptional activators (GAL4-VP16) can overcome this repression.

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Chromatin and transcription in Xenopus cycle. Experiments with the inhibitors of histone deacetylase, sodium butyrate and Trichostatin A demonstrate that the capacity to stably accumulate hyperacetylated core histones within embryonic chromatin is developmentally regulated. This developmental regulation of histone hyperacetylation correlates with the capacity to activate transcription of the histone H1° promoter.8,34,35 The deacetylase inhibitors do not cause histone hyperacetylation or activation of histone H1° gene transcription in Xenopus oocytes. High concentrations of sodium butyrate (40mM) have been found to cause the appearance of vestigial lampbrush loops in mature Xenopus oocytes36 and to stimulate transcription of microinjected human globin genes.37

However experiments with sodium butyrate alone have to be treated with caution, since it has been shown to have pleiotropic effects on enzymes and other macromolecular structures (reviewed in ref 38). The induction of histone hyperacetylation with sodium butyrate and Trichostatin A first appears during embryogenesis early in gastrulation (Figure 4).8 This could reflect the release from inhibition, or the de novo synthesis of histone acetyltransferases. The induction of histone H1° gene expression in developing embryos by deacetylase inhibitors correlates with the capacity to hyperacetylate the core histones even on exogenous DNA assembled into

Figure 4. Changes in chromatin composition during early development in the presence of sodium butyrate. Audioradiographs of chromatin-associated proteins synthesized during early development (A, morula; B,blastula; C, gastrula; and D, neurula), or silver-stained gel (E, F, and G) are shown. The positions of the core and linker histones is indicated together with the acetylation status of histone H4. See Dimitrov et al (ref 8) for details.

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N. Landsberger and A. P. Wolffe nuclear structures.35 This regulated expression of exogenous DNA provides a useful system to dissect the regulatory elements within the H1° gene that confer induction by inhibitors of histone deacetylase. Extensive mutational analysis demonstrates that no single element within over 500bp of the H1° promoter is necessary for transcriptional induction, this is suggestive of a requirement for the modification of preexisting chromatin structure to facilitate general access by transcription factors and RNA polymerase. It should not noted that histone hyperacetylation does not significantly influence the sequestration of linker histones into chromatin,8 a result in agreement with in-vitro reconstruction experiments.39 Concentrations of the histone deacetylase inhibitor, Trichostatin A, sufficient to induce histone hyperacetylation in Xenopus embryos, delay gastrulation and cause diminished midtrunk and posterior formation, suggesting defects in mesoderm formation.35 Thus although constitutive hyperacetylation of the histones does not prevent either the cell division or differentiation sufficient for early morphogenesis, it has a role in establishing stable states of differential gene activity during gastrulation. In this respect it will be of interest to examine the regulation of specific genes whose expression might be enhanced or inhibited by histone acetylation during early gastrulation.

efficiently,47 however B4 mRNA is degraded following gastrulation.48 Xenopus eggs contain stores of histone B4 adequate for the assembly of 2,000 nuclei.8,9,49 Although histone B4 has approximately 30% sequence identity with normal histone H1, the protein is considerably less basic (B4 has 74 basic amino acids, but also has 27 acidic amino acids, H1 has 65 basic amino acids, but only six acidic amino acids). Histone B4 has all of the characteristics of a linker histone: it interacts preferentially with DNA wrapped around an octamer of core histones rather than with naked DNA, it protects additional DNA flanking the nucleosome core from digestion with micrococcal nuclease and it requires linker DNA to form a stable complex with the nucleosome core.49,50 Nevertheless it might be expected on the basis of the overall differences in basicity compared to normal somatic histone H1, that histone B4 would interact with DNA in the nucleosome less tightly than normal somatic histone H1. This ‘looser’ interaction might be expected to result in a less condensed chromatin fiber and both a greater accessibility to trans-acting factors and a chromatin structure that provides less impediment to processive enzyme complexes like those involved in DNA replication. Histone B4 is incorporated into pronuclei in the fertilized Xenopus egg.9 Depletion of histone B4 eliminates the accumulation of chromatosome length DNA ( ~ 168bp) during micrococcal nuclease digestion of pronuclear chromatin. This further confirms that histone B4 associates with DNA rather like histone H1 in the nucleosome.9 Nevertheless histone B4 is not essential either for nuclear assembly or mitotic chromosome condensation.51,52 In contrast to the maternal mRNA encoding histone B4 which is translated, maternal mRNA encoding histone H1 is synthesized during oogenesis but is masked away from the translational machinery.53 This masking process depends on the transcription process itself and the packaging of mRNA with the Y-box proteins.54 Recruitment of histone H1 mRNA to the translational machinery only occurs following fertilization.53,55 Thus somatic histone H1 synthesis only becomes significant at the blastula stage of development. By the MBT, histone B4 and normal somatic histone H1 are presented in approximately equal amounts. However by the time of neurulation, histone B4 has substantially disappeared from chromatin. Major changes in the accessibility of class III genes to transacting factors and RNA polymerase III between gastrulation and neurulation correlate with the sub-

Linker histones Linker histone gene expression is dramatically regulated during early vertebrate development.40 Only in Xenopus have the functional consequences of this regulation been extensively explored. Normally there is one molecule of linker histone per nucleosome. The linker histone binds to DNA at the periphery of the DNA wrapped around the core histones.41 In addition linker histones are believed to help nucleosomal arrays fold into stable chromatin fibers (for review see ref 29). Normal somatic histone H1 in Xenopus oocytes and eggs has not been found.6,42-44 Although Destr´ee and colleagues reported stores of normal somatic histone H1 in oocytes,45 this result appears to have been due to the presence of residual somatic follicle cells on the oocytes used to extract histones. A solution to the apparent deficiency in linker histone in oocytes and eggs came from the discovery of the expression of the histone H1-like protein, B4 that is restricted to early Xenopus development.46 Xenopus oocytes synthesize histone B4 from translationally active maternal mRNA. Following oocyte maturation, B4 mRNA is translated a little more 196

Chromatin and transcription in Xenopus stitution of histone B4 with histone H1.8,56 The accumulation of histone H1 in chromatin has been proposed to have a causal role in the repression of class III genes44,57-59 This hypothesis has been directly tested by manipulation of the levels of histone B4 and histone H1 during early Xenopus embryogenesis. Precocious expression of histone H1 through the injection of mRNA encoding the protein into fertilized Xenopus eggs directs the specific repression of the Xenopus oocyte 5S rRNA genes.60 An increase in histone H1 content specifically restricts transcription factor TFIIIA-activated transcription of the oocyte 5S rRNA genes, and a decrease in histone H1 within chromatin facilitates the activation of the oocyte 5S rRNA genes by TFIIIA. These effects are very selective since the transcription of the U1 and U2 genes by RNA polymerase II or that of the somatic 5S RNA genes by RNA polymerase III is not affected by replacement of histone B4 by histone H1. Thus, the regulated expression of histone H1 during Xenopus development has a specific and dominant role in mediating the differential expression of the oocyte and somatic 5S rRNA genes. This provides an important example of how histones can exert dominant repressive effects on the transcription of a gene in vivo in spite of an abundance of transcription factors for that gene. The final major developmental transition concerning linker histones in Xenopus is the accumulation of the differentiation specific histone H1°. Histone H1° has a structured domain41 that is more arginine rich that that of normal somatic histone H1. This might stabilize the interaction of H1° with DNA in the nucleosome.40 Histone H1° protein is absent during Xenopus embryogenesis up to stage 42, a swimming tadpole. After this stage, H1° accumulates with a highly specific pattern of expression, in particular it is associated with cells that are advanced in their differentiation program such as neurons in the peripheral part of the brain.61 Expression patterns remain unchanged through stage 46, after which histone H1° accumulates in early all cell types. Recently it has been shown that mRNA encoding H1° starts to accumulate dramatically after stage 35.35 Whole amount in-situ hybridization again detects H1° expression preferentially in relatively differentiated cells such as those of the cement gland, nervous system and somites (Grunwald and Khochbin, unpublished data). Thus there is a correlation between the expression of histone H1°, the arrest of cells in the G0/G1 phase of the cell cycle and terminal differ-

entiation. Future studies will explore a causal role for histone H1° in directing these effects.

HMG proteins HMG1- and 2 are stored abundantly in X. laevis oocytes.62,63 It has been suggested that HMG1 might be capable of replacing histone H1 in chromatin.64 Normally HMG1 or 2 are associated with a relatively minor fraction of chromatin ( < 5 %), however within pronuclei the stoichiometry of these proteins relative to the core histones, implies that one HMG1- or 2 molecule in present within chromatin for every other nucleosome.9 Replacement of linker histone H1 by HMG1- or 2 might also facilitate rapid replication of chromatin, for the same reasons as we have proposed for histone B4. The HMG1- or 2 content of chromatin progressively declines through gastrulation until less than 5% of the nucleosomes in the neurula contain HMG1- or 2. Future experiments will explore the role of HMG1 and 2 in nuclear function. HMG14/17-like molecules are not detected in oocytes or eggs.63,65 These proteins appear to accumulate late in Xenopus development rather like histone H1°. They may also be involved in maintaining chromatin structures in differentiated cells over a long period of time.

Conclusion Developmentally regulated changes in chromosomal composition during Xenopus embryogenesis have a determinative influence on gene activity. A combination of in-vivo footprinting, coupled to manipulation of transcription factor and histone abundance leads to models of transcriptional regulation in which nonhistone and histone proteins play equally important roles. Understanding the developmental regulation of differential gene activity will require the role of transcription factors to be understood in both a chromatin and chromosomal context.

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