Chromatin assembly factor 1 subunit


Protein interactions of the small subunit of CAF-1

Trapoxin is a microbially derived cyclotetrapeptide that inhibits histone deacetylation in vivo and causes mammalian cells to arrest in the cell cycle. A trapoxin affinity matrix isolated two nuclear proteins that copurify with histone deacetylase activity. After peptide microsequencing, one protein was cloned and found to be very similar to the yeast transcripitional regulator Rpd3p (see Drosophila Rpd3). The other protein has been identified as RbAp48. Both proteins are predominantly nuclear (Taunton, 1996).

The predominant cytoplasmic histone acetyltransferase activity was isolated from Saccharomyces cerevisiae. This enzyme acetylates the lysine at residue 12 of free histone H4 but does not modify histone H4 when packaged in chromatin. The activity contains two proteins, Hat1p and Hat2p. Hat1p is the catalytic subunit of the histone acetyltransferase and has an intrinsic substrate specificity that modifies lysine in the recognition sequence GXGKXG. The specificity of the enzyme in the yeast cytoplasm is restricted relative to recombinant Hat1p suggesting that it is negatively regulated in vivo. Hat2p, which is required for high affinity binding of the acetyltransferase to histone H4, is highly related to Rbap48, which is a subunit of the chromatin assembly factor, CAF-1, and copurifies with the human histone deacetylase HD1. It is proposed that the Hat2p/Rbap48 family members serve as escorts of histone metabolism enzymes to facilitate their physical interaction with histone H4 (Parthun, 1996).

A collection of yeast temperature-sensitive mutants was screened by an enzymatic assay to find a mutant defective in the acetylation of histone H4. The assay used a fractionated cell extract and measured acetylation of a peptide corresponding to amino acids 1-28 of H4. There are at least two activities in this fraction that acetylate the peptide. A mutation, hat1-1, that eliminates one of the activities was identified and mapped to a locus near the centromere of chromosome XVI. The HAT1 gene was cloned and found to encode a protein of 374 amino acids. Analysis of the peptide used in the assay demonstrates that the HAT1 enzyme acetylates lysine 12 of histone H4. hat1 mutants have no obvious growth defects or phenotypes other than the enzyme defect itself. The HAT1 protein expressed in Escherichia coli gives histone acetyltransferase activity in vitro, demonstrating that HAT1 is the structural gene for the enzyme (Kleff, 1995).

Chromatin assembly factor 1 (CAF-1) assembles nucleosomes in a replication-dependent manner. The small subunit of CAF-1 (p48) is a member of a highly conserved subfamily of WD-repeat proteins. There are at least two members of this subfamily in both human (p46 and p48) and yeast cells (Hat2p, a subunit of the B-type H4 acetyltransferase, and Msi1p). Human p48 can bind to histone H4 in the absence of CAF-1 p150 and p60. p48, also a known subunit of a histone deacetylase, copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150, p60, p48) and H3 and H4, and promotes DNA replication-dependent chromatin assembly. CAC histone H4 exhibits a novel pattern of lysine acetylation that overlaps with, but is distinct from, that reported for newly synthesized H4 isolated from nascent chromatin. Most of the p48 polypeptide present in a crude nuclear fraction is not actually associated with CAC. The bulk of the p48 protein sediments with an uncharacterized larger complex. These data suggest that CAC is a key intermediate of the de novo nucleosome assembly pathway and that the p48 subunit participates in other aspects of histone metabolism (Verreault, 1996).

In eukaryotic cells, newly synthesized histone H4 is acetylated at lysines 5 and 12, a transient modification erased by deacetylases shortly after deposition of histones into chromosomes. Genetic studies in Saccharomyces cerevisiae have revealed that acetylation of newly synthesized histones H3 and H4 is likely to be important for maintaining cell viability; the precise biochemical function of this acetylation is not known, however. The identification of enzymes mediating site-specific acetylation of H4 at Lys5 and Lys12 may help explain the function of the acetylation of newly synthesized histones. A cDNA encoding the catalytic subunit of the human Hat1 acetyltransferase has been cloned and, using specific antibodies, the Hat1 holoenzyme was purified from human 293 cells. The human enzyme acetylates soluble (but not nucleosomal) H4 at Lys5 and Lys12, and acetylates histone H2A at Lys5. Unexpectedly, Hat1 was found in the nucleus of S-phase cells. Like its yeast counterpart, the human holoenzyme consists of two subunits: a catalytic subunit, Hat1, and a subunit, p46, that binds core histones. Binding of p46 to histones greatly stimulates the acetyltransferase activity of Hat1. Both p46 and the highly related p48 polypeptide (the small subunit of human chromatin assembly factor 1; CAF-1) bind directly to helix 1 of histone H4, a region that is not accessible when H4 is in chromatin. It is suggested that p46 and p48 are core-histone-binding subunits that target chromatin assembly factors, chromatin remodeling factors, histone acetyltransferases and histone deacetylases to their histone substrates in a manner that is regulated by nucleosomal DNA (Verreault, 1998).

Histone acetylation and deacetylation are catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. SAP30 has been identified as a novel component of the human histone deacetylase complex that includes Sin3, the histone deacetylases HDAC1 and HDAC2, histone binding proteins RbAp46 and RbAp48, as well as other polypeptides. A SAP30 homolog is described in yeast that is functionally related to Sin3 and the histone deacetylase Rpd3. The human SAP30 complex is active in deacetylating core histone octamers, but inactive in deacetylating nucleosomal histones due to the inability of the histone binding proteins RbAp46 and RbAp48 to gain access to nucleosomal histones. These results define SAP30 as a component of a histone deacetylase complex conserved among eukaryotic organisms (Zhang, 1998).

The protein associations and enzymatic requirements were investigated for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuclear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear. Xenopus RbAp48 and SIN3 have been cloned and it has been shown that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase activity and transcriptional repression of the TRbetaA promoter. This repression requires deacetylase activity and nuclear import of RPD3 mediated by a carboxy-terminal nuclear localization signal. Exogenous RPD3 is not incorporated into oocyte deacetylase and ATPase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus. RPD3 associates with RbAp48 through N- and C-terminal contacts and RbAp48 also interacts with SIN3. Xenopus RbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo. Exogenous RPD3 may be targeted to histones through interaction with endogenous RbAp48 to direct transcriptional repression of the Xenopus TRbetaA promoter in the oocyte nucleus. However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors (Vermaak, 1999).

MBD2 and MBD3 are two proteins that contain methyl-CpG binding domains and have a transcriptional repression function. Both proteins are components of a large CpG-methylated DNA binding complex named MeCP1, which consists of the nucleosome remodeling and histone deacetylase complex Mi2-NuRD and MBD2. MBD3L2 (methyl-CpG-binding protein 3-like 2) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG-binding domain. Unlike MBD3L1, which is specifically expressed in haploid male germ cells, MBD3L2 expression is more widespread. MBD3L2 interacts with MBD3 in vitro and in vivo, co-localizes with MBD3 but not MBD2, and does not localize to methyl-CpG-rich regions in the nucleus. In glutathione S-transferase pull-down assays, MBD3L2 is found associated with several known components of the Mi2-NuRD complex, including HDAC1, HDAC2, MTA1, MBD3, p66, RbAp46, and RbAp48. Gel shift experiments with nuclear extracts and a CpG-methylated DNA probe indicate that recombinant MBD3L2 can displace a form of the MeCP1 complex from methylated DNA. MBD3L2 acts as a transcriptional repressor when tethered to a GAL4-DNA binding domain. Repression by GAL4-MBD3L2 is relieved by MBD2 and vice versa, and repression by MBD2 from a methylated promoter is relieved by MBD3L2. The data are consistent with a role of MBD3L2 as a transcriptional modulator that can interchange with MBD2 as an MBD3-interacting component of the NuRD complex. Thus, MBD3L2 has the potential to recruit the MeCP1 complex away from methylated DNA and reactivate transcription (Jin, 2005).

Histone methylation by PRC2 is inhibited by active chromatin marks

The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). This study examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin by providing the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. It was found that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains (Schmitges, 2011).

Understanding how histone modification patterns are propagated during cell division is essential for understanding the molecular basis of epigenetic inheritance. Trimethylation of H3K27 by PRC2 has emerged as a key step in generating transcriptionally repressed chromatin in animals and plants. This study investigates how PRC2 recognizes the H3 tail and responds to H3-associated marks of active chromatin. Crystallographic analyses reveal the molecular basis for H31-14 recognition by the Nurf55-Su(z)12 module of PRC2 and demonstrate that H3 tails carrying K4me3 are no longer recognized by Nurf55-Su(z)12. In the context of the whole PRC2 complex, H3K4me3 triggers allosteric inhibition of PRC2, a process that requires H3K4me3 to be present on the same histone molecule containing the substrate Lys27. PRC2 inhibition by H3K36me2/3 was also observed. PRC2 inhibition by active chromatin marks (H3K4me3 and H3K36me2/3) is conserved in PRC2 complexes reconstituted from humans, mouse, flies, and plants (Schmitges, 2011).

Minimal PRC2 complexes lacking Nurf55 retain partial catalytic activity and are inhibited by H3K4me3. H3K4me3, once free of Nurf55, is thereby able to trigger PRC2 inhibition. A model is favored where Nurf55-Su(z)12 serves in sequestration and release of histone H3. It is proposed that the release of the H3 tail from Nurf55-Su(z)12 is required, but not sufficient, to induce H3K4me3 inhibition as it needs to trigger allosteric inhibition in conjunction with Su(z)12 and the E(z) SET domain. Unmodified H3, H3K9me3, or H3R2me-modified tails, on the other hand, remain sequestered and are shielded from other chromatin factors. These sequestered marks are also not expected to interfere with PRC2 regulation. In line with this prediction, it is observed that H3K9me3, which remained bound to Nurf55-Su(z)12, also did not interfere with PRC2 activity in vitro. In vitro, binding of Nurf55 to the N terminus of H3 was not critical for the overall nucleosome binding affinity of PRC2 under the assay conditions. However, small differences in PRC2 affinity amplified by large chromatin arrays could skew PRC2 recruitment toward sites of unmodified H3K4. Additionally, the Nurf55 interaction might play a more subtle role in positioning the complex correctly on nucleosomes (Schmitges, 2011).

The in vitro findings suggest that active chromatin mark inhibition by PRC2 is largely governed through allosteric inhibition of the PRC2 HMTase activity thereby limiting processivity of the enzyme. A minimal trimeric PRC2 subcomplex that retains both activity and H3K4me3/H3K36me2/3 inhibition was defined. This minimal complex consists of ESC, an E(z) fragment that comprises the ESC binding region at the N terminus, the Su(z)12 binding domain in the middle, and the C-terminal catalytic domain, and the Su(z)12 C terminus harboring the VEFS domain. The importance of Su(z)12 is underlined by findings on the Arabidopsis PRC2 complexes that revealed that active mark inhibition is determined by the choice of Su(z)12 subunit (i.e., inhibition with EMF2, but not with VRN2). As the extent of methylation inhibition and the domains required for inhibition were similar for H3K4me3 and H3K36me2/3, it is hypothesized that both peptides function through a related mechanism allosterically affecting E(z) SET domain processivity with the help of Su(z)12. Further structural studies are required to reveal how these active marks are recognized and how this recognition is linked to inhibition of the E(z) SET domain (Schmitges, 2011).

H3K27me3 recognition by PRC2 has been reported to recruit and stimulate PRC2, a mechanism implicated in creating and maintaining the extended H3K27me3 domains at target genes in vivo. Such positive feedback, however, necessitates a boundary element curtailing the expansion of H3K27me3. The results suggest that actively transcribed genes (i.e., marked with H3K4me3 and H3K36me2/3) that flank domains of H3K27me3 chromatin may represent such boundary elements. In conjunction with H3K27me3-mediated stimulation, this provides a model how PRC2 could template domains of H3K27me3 chromatin during replication without expanding H3K27me3 domains into the chromatin of active genes. The inhibitory circuitry present in PRC2, however, does not function as a binary ON/OFF switch. PRC2 is able to integrate opposing H3K4me3 and H3K27me3 modifications into an intermediary H3K27 methylation activity (Schmitges, 2011).

The crosstalk between H3K4me3 and H3K36me2/3 versus H3K27me3 has been extensively studied in vivo. Specifically, HOX genes in developing Drosophila larvae, or in mouse embryos, show mutually exclusive H3K27me3 and H3K4me3 domains that correlate with transcriptional OFF and ON states, respectively. In Drosophila, maintenance of HOX genes in the ON state critically depends on the trxG regulators Trx and Ash1, which methylate H3K4 and H3K36, respectively. At the Ultrabithorax (Ubx) gene, lack of Ash1 results in PRC2-dependent H3K27me3 deposition in the coding region of the normally active gene and the concomitant loss of Ubx transcription. Similarly, in the Arabidopsis Flowering Locus C (FLC), CLF-dependent deposition of H3K27me3 reduces H3K4me3 levels, while deletion of the H3K4me3 demethylase FLD increases H3K4me3 levels and concomitantly diminishes H3K27me3 levels. The results provide a simple mechanistic explanation for these observations in plants and flies. It is proposed that H3K4 and H3K36 modifications in the coding region of active PcG target genes function as barriers that limit H3K27me3 deposition by PRC2 (Schmitges, 2011).

It is noted that a number of HMTase complexes contain histone mark recognition domains that bind the very same mark that is deposited by their catalytic domain. While this positive feedback loop guarantees the processivity of histone mark deposition, it also requires a control mechanism that avoids excessive spreading of marks. The direct inhibition of HMTases by histone marks, as seen for PRC2, may offer a paradigm of how excessive processivity can be counteracted in other HMTases (Schmitges, 2011).

Arabidopsis VRN2 is implicated in the control of the FLC locus after cold shock. FLC is a bivalent locus containing both repressive H3K27me3 and active H3K4me3 marks. In a VRN2-dependent fashion, H3K27me3 levels increase at FLC during vernalization. This study found that while EMF2-containing PRC2 complexes are sensitive to H3K4me3 and H3K36me3, their VRN2-containing counterparts are not. In response to environmental stimuli plant PRC2 H3K4me3/H3K36me3 inhibition can thus be switched OFF (or ON). This offers the possibility that inhibition in animal PRC2 could also be modulated either by posttranslational modification of SUZ12 or by association with accessory factors (Schmitges, 2011).

Quantitative mass spectrometry analyses of posttranslational modifications on the H3 N terminus in HeLa cells found no evidence for significant coexistence of H3K27me3 with H3K4me3 on the same H3 molecule. Similarly, the fraction of H3 carrying both H3K27me3 and H3K36me3 was reported to be extremely low (~0.078%), while H3K27me3 and H3K36me2 coexist on ~1.315% of H3 molecules. However, H3K27me3/H3K4me3 and H3K27me3/H3K36me2/3 bivalent domains have been reported to exist in embryonic stem cells, and they have been implicated to exist on the same nucleosome. Given that PRC2 is inhibited by active methylation marks, how then could such bivalent domains be generated? Two main possibilities are envisioned. First, PRC2 inhibition in vivo could be alleviated by specific posttranslational modifications on PRC2 in embryonic stem cells (see in plants, VRN2). Second, H3K27me3 could be deposited prior to modification of H3K4 or H3K36. According to this view, one would have to postulate that the HMTases depositing H3K4me3 or H3K36me2/3 can work on nucleosomes containing H3K27me3. In support of this view, it was found that H3K36 methylation by an NSD2 catalytic fragment is not inhibited by H3K27me3 marks on a peptide substrate (Schmitges, 2011).

In summary, this study found that mammalian and fly PRC2 complexes are not only activated by H3K27me3, but they are also inhibited by H3K4me3 and H3K36me2/3. PRC2, as a single biochemical entity, can thus integrate the information provided by histone modifications with antagonistic roles in gene regulation. While the biological network overseeing crosstalk between active and repressive chromatin marks in vivo probably extends beyond PRC2, including other chromatin modifiers such as histone demethylases, this identified a regulatory logic switch in PRC2 that intrinsically separates active and repressive chromatin domains. Given the dynamic nature of the nucleosome template that makes up eukaryotic chromosomes, this circuitry probably equips PRC2 with the necessary precision to heritably propagate a repressed chromatin state (Schmitges, 2011).

The large subunit of CAF-1

In the yeast Saccharomyces cerevisiae, telomere repeat DNA is assembled into a specialized heterochromatin-like complex that silences the transcription of adjacent genes. The general DNA-binding protein Rap1p binds telomere DNA repeats, contributes to telomere length control and to telomeric silencing, and is a major component of telomeric chromatin. Rap1p localization factor 2 (RLF2) was identified in a screen for genes that alleviate antagonism between telomere and centromere sequences on plasmids. In rlf2 mutants, telomeric chromatin is perturbed: Telomeric silencing is reduced and Rap1p localization is altered. In wild-type cells, Rap1p and telomeres localize to bright perinuclear foci. In rlf2 strains, the number of Rap1p foci is increased, Rap1p staining is more diffuse throughout the nucleus, Rap1p foci are distributed in a much broader perinuclear domain, and nuclear volume is 50% larger. Despite the altered distribution of Rap1p in rlf2 mutant cells, fluorescence in situ hybridization to subtelomeric repeats shows that the distribution of telomeric DNA is similar in wild-type and mutant cells. Thus in rlf2 mutant cells, the distribution of Rap1p does not reflect the distribution of telomeric DNA. RLF2 encodes a highly charged coiled-coil protein that has significant similarity to the p150 subunit of human chromatin assembly factor-I(hCAF-I), a complex that is required for the DNA replication-dependent assembly of nucleosomes from newly synthesized histones in vitro. Furthermore, RLF2 is identical to CAC1, a subunit of yeast chromatin assembly factor-I (yCAF-I) which assembles nucleosomes in vitro. In wild-type cells, epitope-tagged Rlf2p expressed from the GAL10 promoter localizes to the nucleus with a pattern distinct from that of Rap1p, suggesting that Rlf2p is not a component of telomeric chromatin. This study provides evidence that yCAF-I is required for the function and organization of telomeric chromatin in vivo. It is proposed that Rlf2p facilitates the efficient and timely assembly of histones into telomeric chromatin (Enomoto, 1997).

Mechanisms contributing to the maintenance of heterochromatin in proliferating cells are poorly understood. Chromatin assembly factor 1 (CAF-1) binds to mouse HP1 proteins via an N-terminal domain of its p150 subunit, a domain dispensable for nucleosome assembly during DNA replication. Mutations in p150 prevent association with HP1 in heterochromatin in cells that are not in S phase and the formation of CAF-1-HP1 complexes in nascent chromatin during DNA replication in vitro. It is suggested that CAF-1 p150 has a heterochromatin-specific function distinct from its nucleosome assembly function during S phase. Just before mitosis, CAF-1 p150 and some HP1 progressively dissociate from heterochromatin concomitant with histone H3 phosphorylation. The HP1 proteins reassociate with chromatin at the end of mitosis, at the time histone H3 is dephosphorylated (Murzina, 1999).

Enhancer of zeste [E(z)] is a Polycomb-group transcriptional repressor and one of the founding members of the family of SET domain-containing proteins. Several SET-domain proteins possess intrinsic histone methyltransferase (HMT) activity. However, recombinant E(z) protein was found to be inactive in a HMT assay. A multiprotein E(z) complex has been isolated from humans that contains extra sex combs, suppressor of zeste-12 [Su(z)12], and the histone binding proteins RbAp46/RbAp48. This complex, which has been termed Polycomb repressive complex (PRC) 2, possesses HMT activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3. The HMT activity of PRC2 is dependent on an intact SET domain in the E(z) protein. It is hypothesized that transcriptional repression by the E(z) protein involves methylation-dependent recruitment of PRC1. The presence of Su(z)12, a strong suppressor of position effect variegation, in PRC2 suggests that PRC2 may play a widespread role in heterochromatin-mediated silencing (Kuzmichev, 2002).

The polypeptide composition of the PRC2, specifically the presence of Su(z)12, suggests that PRC2 plays a more general role in transcriptional silencing outside of the repression of HOX genes. Su(z)12 is a protein with dual PcG and Su(var) functions, and this, therefore, suggests that PRC2 has functions other than homeotic gene repression and, in fact, may play a more general role in heterochromatin-mediated silencing. The observation that human E(z) can function as an inducer of silencing in yeast and as an enhancer of PEV in Drosophila supports this notion. It is speculated that the requirement for E(z) and ESC during early embryonic development reflects its function in general transcriptional silencing. The multifunctional nature of both the E(z) and Su(z)12 proteins suggests that they may also display biochemical heterogeneity. For example, the heterogeneous elution profile of E(z) on various columns suggests that E(z) exists in several distinct complexes (Kuzmichev, 2002).

Purified PRC2 displayed specificity for K9 and K27 of the histone H3 tail. The complex, under the conditions of the assays used, displayed a strong preference for K27. However, when the H3-tail was used as a GST-fusion protein, PRC2 displayed apparently equal specificity for K9 and K27. Analyses of the amino acid sequence in which these lysines are embedded shows a great deal of conservation. K9 is present within the sequence QTARK9STG, whereas K27 is present within the sequence KAARK27SAP. Therefore, at least two different possibilities can be postulated to account for the specificity observed. In one case, the specificity of PRC2 is relaxed in vitro, under the assay conditions used, and the methylation of K9 is nonspecific because of the sequence similarity of the residues within which K9 resides. An apparently similar situation was observed in studies analyzing the specificity of the histone methyltransferase G9a, which biochemically behaves as a H3-histone methyltransferase that preferentially targets K9 and, to much lower levels, K27. In vivo, however, G9a clearly targets H3-K9: whether or not the extent of H3-K27 methylation is decreased in G9a-null cells is unknown. A second possibility is that E(z) targets both K9 and K27, but that this is a regulated process such that methylation of K9 and/or K27 is modulated by factors that associate with E(z) and/or by other modifications existing in the nucleosome. This second possibility is favored based on the following observations. First, the E(z) protein can be considered to be a PcG as well as a TrxG. Not surprisingly, the analyses demonstrate that E(z) is present in distinct complexes. One of the complexes containing E(z) is PRC2; however; this complex also includes Su(z)12. Su(z)12 is a polypeptide that has been found in genetic analyses to regulate the expression of the HOX genes, but loss of function of Su(z)12 suppresses PEV. Therefore, the presence of Su(z)12 in PRC2 may regulate the methylation sites within the histone H3 tail (Kuzmichev, 2002).

Methylation of histone H3-K9 was shown to be an essential step in the establishment of inactive X chromosome. H3-Lys 9 methylation of the inactive X chromosome is not mediated by Suv39 or by G9a. Studies have demonstrated that the imprinted inactivation of the X chromosome in females is lost in mutant mice lacking eed (the mammalian homolog of ESC). Moreover, studies have also demonstrated that during imprinted X inactivation, the mammalian ESC-E(z) complex is localized to the inactive X chromosome in a mitotically stable manner. It is speculated, in light of the accumulated data, that H3-K9 methylation of the inactive X chromosome might be mediated by E(z) within PRC2 or a PRC2-like complex. Importantly, however, the function of methylation of histone H3 at K27 has not been analyzed in the establishment and/or maintenance of the inactive X chromosome. In light of the results discussed above, it is postulated that methylation of H3-K27 may also be important in the process of X inactivation (Kuzmichev, 2002).

It is proposed that the role of E(z) HMT activity in the repression of homeotic gene expression is to establish a binding site for other PcG proteins. It is suggested that PRC2 is recruited to the HOX gene cluster by a transiently acting repressor, for example, through an EED-YY1/Pho interaction or an RbAp46/48-HDAC/dMi2/Hb interaction. Once recruited, PRC2 methylates K27 on histone H3, and this mark recruits PC1. The PC1 protein can convert this mark into a permanently repressed state through methylation of K9 through the recruitment of the Su(var)3-9 H3-K9-specific histone methyltransferase and/or the recruitment of PRC1. Alternatively and/or additionally, PC1 may stimulate the H3-K9 HMT activity of PRC2. This hypothesis is supported by studies demonstrating that trimethylation of K27 is necessary for binding of PC1 to an H3 tail peptide. These findings are in full agreement with studies demonstrating loss of chromosome binding for several PRC1 components upon inactivation of E(z). Interestingly, immunolocalization experiments using antibodies specific for methylated H3-K9 suggest that almost all of the H3-K9 methylation is concentrated in the chromocenter of Drosophila polytene chromosomes, with almost no staining detectable on the chromosomal arms. In contrast, E(z) and other PcG proteins, with subnuclear localization that is regulated by E(z), bind only to discrete bands along the arms of polytene chromosomes. These observations suggest that methylation at K27, rather than methylation at K9, is more likely to establish a binding site for the PC1 protein. This may explain why methylation of K9 alone was not sufficient to allow PC1 to recognize specifically the H3 tail in vitro. The observed PC1 binding was independent of DNA. However, repression of HOX genes in vivo is dependent on PRE. From these results, it must be concluded that although methylation of the H3 tail is important in creating a recognition site for PC1 binding, stable and specific binding must require additional factors and or modifications. A likely candidate is a nucleosome on the PRE with the histone H3-tail methylated at position 27 (Kuzmichev, 2002).

The presence of the RbAp46 and RbAp48 proteins in the ESC-E(z) complex may be important for several reasons. First, these histone-binding proteins are often found in complexes with enzymes involved in the covalent modification of histones. For example, RbAp46 is essential for substrate recognition by, and enzymatic activity of, the histone acetyltransferase enzyme Hat1. Therefore, it is speculated that the inability to detect HMT activity in preparations of recombinant E(z) protein is owing, in part, to the lack of the RbAp46/RbAp48. Another implication of the presence of RbAp proteins in the E(z) complex is that they might facilitate interaction with HDACs. During development, GAP proteins facilitate repression of the HOX genes. GAP proteins, such as Hunchback, are short-lived. Hunchback represses HOX genes by recruiting the Drosophila homolog of human Mi-2 protein, a constituent of the NuRD complex which also contains HDACs 1 and 2 and RbAp46/RpAp48. An interesting possibility is that HDACs or RbAp proteins initially recruited by Hunchback can later recruit PRC2 containing HMT activity via interaction with E(z). This may constitute a switch from short-term to long-term repression (Kuzmichev, 2002).

In support of this hypothesis, PRC2 contains E(z) and RbAp proteins. In addition, there is strong experimental evidence for an interaction between HDACs and E(z). One function of the E(z)-HDAC interaction is to deacetylate histones so that the E(z)-containing complex can methylate them. A similar mechanism was found to operate in yeast, in which methylation of H3-K9 by Clr4 requires deacetylation of H3-K9 and Lys 14 (K14) by Clr6 and Clr3, respectively. A similar mechanism is likely to operate in higher eukaryotes because acetylation and methylation are mutually exclusive marks, and methylation of H3-K9 by Suv39h1 requires deacetylation of this residue. The findings demonstrating two distinct ESC-E(z) complexes, one of which coelutes with HDAC1, raises the possibility that the PRC2 can transiently associate with an HDAC complex. This observation raises the possibility that PRC2 HDAC1 may be a highly-specialized complex dedicated to the methylation of H3-K27, which apparently is not acetylated in vivo in higher eukaryotes. Therefore, it is possible that these two different ESC-E(z) multiprotein complexes establish different marks on the histone H3 tail (Kuzmichev, 2002).

Cellular localization of CAF-1

In the S-phase of the eukaryotic cell cycle, newly replicated DNA is assembled into chromatin. Indirect immunofluorescence microscopy was used to localize the sites of chromatin assembly with respect to DNA replication. Replication foci in the nuclei of permeabilized HeLa cells were labeled by incorporation of biotin-16-dUTP and detected by fluorescent streptavidin. Prelabeling of replication foci in vivo with bromodeoxyuridine shows that replication in permeabilized cells proceeds at preexisting replication forks. The localization of chromatin assembly factor 1 (CAF-1) was determined with subunit-specific monoclonal antibodies. CAF-1 is not detectable in mitotic cells and is detectable only at background levels in about 60% of all interphase nuclei. The other interphase nuclei show an intense punctate immunostaining of CAF-1. These sites of CAF-1 colocalize with replication foci during all stages of the S-phase. No other discrete sites of CAF-1 are observed. Human replication protein A (RPA) colocalizes with these replication/chromatin assembly sites. In addition, extra nuclear sites of RPA are observed that probably represent prereplication foci, poised for initiation of DNA replication (Krude, 1995).

This study characterized changes of nucleosome assembly activity, intracellular localization, and reversible phosphorylation of the human chromatin assembly factor CAF-1 during the somatic cell division cycle. HeLa cells were synchronized in the G1, S, G2, and M phases of the cell cycle. All three subunits of human CAF-1 (p150, p60, and p48) are present during the entire cell cycle. In interphase, p150 and p60 are bound to the nucleus, but they predominantly dissociate from chromatin during mitosis. During S phase, p150 and p60 are concentrated at sites of intranuclear DNA replication. Only a fraction of total p48 is associated with p150 and p60, and the majority is present in other high molecular weight complexes. The other nucleosome assembly protein, NAP-1, is predominantly cytosolic throughout the cell cycle. Human CAF-1 efficiently mediates nucleosome assembly during complementary DNA strand synthesis in G1, S, and G2 phase cytosolic extracts. Active CAF-1 can be isolated as a 6.5 S complex from G1, S, and G2 phase nuclei. In contrast, CAF-1 isolated from mitotic cytosol does not support nucleosome assembly during DNA synthesis. In mitosis, the p60 subunit of inactive CAF-1 is hyperphosphorylated, whereas active CAF-1 in interphase contains hypophosphorylated and/or phosphorylated forms of p60 (Marheineke, 1998).

Direct conversion of C. elegans germ cells into specific neuron types

The ability of transcription factors to directly reprogram the identity of cell types is usually restricted and is defined by cellular context. Through the ectopic expression of single Caenorhabditis elegans transcription factors, this study found that the identity of mitotic germ cells can be directly converted into that of specific neuron types: glutamatergic, cholinergic, or GABAergic. This reprogramming event requires the removal of the histone chaperone LIN-53 (RbAp46/48 in humans), a component of several histone remodeling and modifying complexes, and this removal can be mimicked by chemical inhibition of histone deacetylases. These findings illustrate the ability of germ cells to be directly converted into individual, terminally differentiated neuron types and demonstrate that a specific chromatin factor provides a barrier for cellular reprogramming (Tursun, 2011).

CAF-1 and repair

DNA repair in the eukaryotic cell disrupts local chromatin organization. To investigate whether the resetting of nucleosomal arrays can be linked to the repair process assays were developed with both Xenopus egg extract and human cell extracts, to follow repair and chromatin assembly in parallel on circular DNA templates. Both systems are able to carry out nucleotide excision repair of DNA lesions. UV-dependent DNA synthesis occurs simultaneously with chromatin assembly, strongly indicating a mechanistic coupling between the two processes. A complementation assay established that chromatin assembly factor I (CAF1) is necessary for this repair associated chromatin formation (Gaillard, 1996).

Caf-1 complexes and differentiation

Chromatin-modifying complexes are important for transcriptional control, but their roles in the regulation of development are poorly understood. Components of the nucleosome remodelling and histone deacetylase (NURD) complex antagonize vulval development, which is induced by the Ras signal transduction pathway. In three of the six equivalent vulval precursor cells, the Ras pathway is active, leading to the production of vulval fates; in the remaining three, the Ras pathway is inhibited and vulval fates repressed. Inhibition of Ras signaling occurs in part through the action of the synthetic multivulval (synMuv) genes, which comprise two functionally redundant pathways (synMuvA and synMuvB). Five C. elegans members of the NURD chromatin remodelling complex inhibit vulval development through both the synMuvA and synMuvB pathways [hda-1, rba-1 (a Caf-1 homolog), lin-53 (another Caf-1 homolog), chd-3 (an Mi-2 homolog) and chd-4]; another two members, the MTA1-related genes egr-1 and egl-27, act only in the synMuvA pathway. It is proposed that the synMuvA and synMuvB pathways function redundantly to recruit or activate a core NURD complex, which then represses vulval developmental target genes by local histone deacetylation. These results emphasise the importance of chromatin regulation in developmental decisions. Furthermore, inhibition of Ras signaling suggests a possible link between NURD function and cancer (Solari, 2000).

Other histone acetyltransferases

Chromatin assembly factor 1 subunit Evolutionary Homologs part 3/3 | back to part 1/3

Chromatin assembly factor 1 subunit: Biological Overview | Regulation | Developmental Biology | Effects of RNAi | References

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