InteractiveFly: GeneBrief

XNP: Biological Overview | References


Gene name - XNP

Synonyms - dATRX, dXNP

Cytological map position - 96E1-96E2

Function - chromatin modification enzyme

Keywords - nucleosome assembly, regulation of gene expression, formation of pericentric heterochromatin, heterochromatic gene silencing

Symbol - XNP

FlyBase ID: FBgn0039338

Genetic map position - chr3R:21303590-21308549

Classification - DEAD-like helicases superfamily

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Chavez, J., Murillo-Maldonado, J. M., Bahena, V., Cruz, A. K., Castaneda-Sortibran, A., Rodriguez-Arnaiz, R., Zurita, M. and Valadez-Graham, V. (2017). dAdd1 and dXNP prevent genome instability by maintaining HP1a localization at Drosophila telomeres. Chromosoma [Epub ahead of print]. PubMed ID: 28688038
Summary:
An important component of the telomeres is the heterochromatin protein 1a (HP1a). Mutations in Su(var)205, the gene encoding HP1a in Drosophila, result in telomeric fusions, retrotransposon regulation loss and larger telomeres, leading to chromosome instability. Previously, it was found that several proteins physically interact with HP1a, including dXNP and dAdd1 (orthologues to the mammalian ATRX gene). This study found that mutations in the genes encoding the dXNP and dAdd1 proteins affect chromosome stability, causing chromosomal aberrations, including telomeric defects, similar to those observed in Su(var)205 mutants. In somatic cells, dXNP and dAdd1 participate were shown to participate in the silencing of the telomeric HTT array of retrotransposons, preventing anomalous retrotransposon transcription and integration. Furthermore, the lack of dAdd1 results in the loss of HP1a from the telomeric regions without affecting other chromosomal HP1a binding sites; mutations in dxnp also affected HP1a localization but not at all telomeres, suggesting a specialized role for dAdd1 and dXNP proteins in locating HP1a at the tips of the chromosomes. These results place dAdd1 as an essential regulator of HP1a localization and function in the telomere heterochromatic domain.
BIOLOGICAL OVERVIEW

Most nucleosomes that package eukaryotic DNA are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. Replication-independent nucleosome replacement using the H3.3 histone variant efficiently repackages these regions, but how histones are recruited to these sites is unknown. This study used an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to define steps in the mechanism of nucleosome replacement. Xnp chromatin remodeler and the Hira histone chaperone were found to independently bind nucleosome-depleted chromatin. Surprisingly, these two factors are only displaced when new nucleosomes are assembled. H3.3 deposition assays reveal that Xnp and Hira are required for efficient nucleosome replacement, and double-mutants are lethal. It is proposed that Xnp and Hira recognize exposed DNA and serve as a binding platform for the efficient recruitment of H3.3 predeposition complexes to chromatin gaps. These results uncover the mechanisms by which eukaryotic cells actively prevent the exposure of DNA in the nucleus (Schneiderman, 2012).

DNA in the eukaryotic nucleus is associated with histone proteins to form nucleosomes, the fundamental units of chromatin. Most nucleosomes are assembled during DNA replication, but chromatin structure is routinely disrupted in active regions of the genome. These regions are repackaged by replication-independent (RI) nucleosome replacement using the H3.3 histone variant. This process results in the enrichment of the H3.3 histone variant at all sites where nucleosomes are unstable or disrupted (Mito, 2005; Goldberg, 2010; Schneiderman, 2012 and references therein).

How H3.3 is delivered to dynamic chromatin sites is unknown. However, biochemical isolation of predeposition complexes has identified shared and distinctive assembly factors that associate with the H3 and H3.3 histones and mediate the replication-coupled or RI assembly of nucleosomes, respectively. These factors include histone chaperones and chromatin remodelers that are important for new nucleosome assembly, and might potentially target histones to active chromatin regions. However, mutants in some of these factors have surprisingly limited phenotypes. The Hira chaperone promotes H3.3 deposition at genes (Goldberg, 2010) but is only essential for H3.3 deposition on sperm chromatin during fertilization (Loppin, 2005; Bonnefoy, 2007). In Drosophila the ATRX/XNP remodeler homolog Xnp colocalizes with H3.3 in somatic cells, but is not essential (Schneiderman, 2009). In mammals, ATRX/XNP promotes H3.3 deposition only at telomeres and some heterochromatic sequences (Goldberg, 2010; Drané, 2010; Wong, 2010). These results have raised the possibilities that H3.3 assembly factors are redundant or that additional factors involved in the deposition of this histone variant exist. Loss of H3.3 itself can be compensated in somatic cells by the major H3 histone, suggesting that assembly of any nucleosome suffices (Sakai, 2009; Schneiderman, 2012 and references therein).

This work used an inducible system that produces nucleosome-depleted chromatin at the Hsp70 genes in Drosophila to study the mechanism of nucleosome replacement. Evidence is provided that H3.3 predeposition factors mediate two separable steps in RI nucleosome assembly. The Xnp and Hira factors bind genomic sites when nucleosomes are disassembled, thereby marking sites for RI assembly. Strikingly, it was also demonstrated that Hira and Xnp are redundant for RI nucleosome assembly in somatic nuclei. The results further reveal that RI nucleosome replacement is essential for chromatin structure and viability, and uncover a cellular system that surveys chromatin for defects and promotes its repair (Schneiderman, 2012).

This study used the inducible Hsp70 genes as a controlled in vivo system to deplete nucleosomes from chromatin. Heat-shock activates transcription of Hsp70, displacing nucleosomes and increasing the sensitivity of the locus to digestion by micrococcal nuclease (MNase). After heat-shock, H3.3-containing nucleosomes repackage the locus (Schwartz, 2005). To generate persistently nucleosome-depleted chromatin, the Hsp70 genes in H3.3-deficient cells. ChIP experiments using an anti-H3 antibody show similar amounts of histone H3 at the Hsp70 genes in wild-type and H3.3-deficient salivary glands, and the genes show similar protection from MNase digestion. These data demonstrate that Hsp70 sequences are fully protected by nucleosomes before induction in wild-type and H3.3-deficient salivary glands. In both genotypes induced Hsp70 genes become hypersensitive to MNase as nucleosomes are lost. In wild-type cells after heat-shock nuclease protection is restored, but is not restored in H3.3-deficient glands. This finding demonstrates that nucleosomes are not replaced after Hsp70 induction in the absence of H3.3. This system allows the teasing apart of cause-and-effect in analyzing the effects of nucleosome assembly factors (Schneiderman, 2012).

Previous work has described that the Xnp chromatin remodeler colocalizes with H3.3 in chromatin (Schneiderman, 2009). Mammalian data has suggested that the Xnp homolog ATRX mediates H3.3 deposition at telomeres and at transcribed heterochromatic repeat sequences (Goldberg, 2010; Drané, 2010), although its nuclear distribution is much broader (McDowell 1999). The Hira histone chaperone has been implicated in global nucleosome replacement after the removal of protamines from the sperm nucleus during fertilization and near genes in mammalian cells. Purification of nuclear soluble H3.3-containing complexes showed that ATRX and Hira are in two separate complexes that mediate H3.3 deposition at distinct target sites. ASF1 is a general histone chaperone that complexes with new histone dimers in the cytoplasm and escorts them into the nucleu. This study used antibodies to Drosophila Xnp, Hira, and ASF1 to track their localization during Hsp70 induction and subsequent RI nucleosome assembly in polytene chromosomes of larval salivary glands (Schneiderman, 2012).

Heat-shock rapidly activates transcription of Hsp70, and elongating RNA polymerase II becomes strongly localized in puffs at the transcribing genes. After cessation of a heat-shock, the puffs regress and RNA polymerase II leaves the locus within 30 min. Neither Xnp, Hira, nor ASF1 were found to be enriched at the Hsp70 loci before induction. However, upon induction all three proteins are rapidly recruited to the Hsp70 loci. Xnp, Hira, and ASF1 remain associated with chromatin and the genes are transcribed, but then leave the Hsp70 genes after heat-shock. Their dynamic recruitment implicates these assembly factors in cotranscriptional nucleosome dynamics (Schneiderman, 2012).

The induction of Hsp70 was followed in H3.3-deficient salivary glands, where transcription produces nucleosome-depleted chromatin. Both Northern analysis and cytological observations of RNA polymerase II showed that Hsp70 was induced in wild-type and H3.3-deficient salivary glands, although H3.3-deficient glands produce ~50% less mRNA. Two effects on the recruitment of RI assembly factors were observed . First, Xnp, Hira, and ASF1 are all rapidly recruited to the induced Hsp70 genes at moderately reduced levels, indicating that the Hsp70 genes are less efficiently induced in this genotype. Second, both Xnp and Hira - but not ASF1 - accumulate and persist at the Hsp70 genes after heat-shock. The correspondence between persistent nucleosome depletion and the persistent binding of these factors is striking. This correspondence is a distinctive property of these two RI assembly factors, because the histone chaperone ASF1 is also cotranscriptionally recruited but dissociates both in wild-type and in H3.3-deficient cells. Finally, retention of Xnp and Hira is not a result of ongoing transcription, because RNA polymerase II rapidly leaves the Hsp70 loci after heat-shock and transcript production ceases. It is concluded that once Xnp and Hira bind nucleosome-depleted chromatin, they are only displaced when new nucleosomes assemble (Schneiderman, 2012).

How are Xnp and Hira recruited to nucleosome-depleted chromatin? These factors may be directly recruited by transcriptional machinery to active genes. Alternatively, Xnp and Hira may bind a structural feature common to chromatin gaps, or may simply bind exposed DNA. These factors might be complexed with DNA-binding factors or may bind DNA themselves. The homologous ATRX remodeler contains an ADD (ATRX-DNMT3-DNMT3L) domain that can bind DNA or histone tails. Indeed, ATRX is recruited to the genomes of DNA viruses as they enter the nucleus, suggesting that it may directly bind exposed DNA. A recent study has shown that the mammalian Hira chaperone may also directly bind exposed DNA at chromatin gaps (Ray-Gallet, 2011). This study found that Xnp and Hira bind independently at induced Hsp70 genes in null mutants of the other factor, implying that there may be multiple ways that RI assembly factors recognize exposed DNA (Schneiderman, 2012).

Although both Xnp and Hira have been implicated in RI nucleosome assembly, mutants in these factors have surprisingly limited phenotypes. These results have raised the possibility that H3.3 assembly factors are redundant. To test if these factors promote nucleosome replacement at these sites, deposition of GFP-tagged truncated H3.3 histone into chromatin was assayed in wild-type and mutant genotypes. H3.3core-GFP can only be incorporated by RI nucleosome assembly, and the histone labels active genes.A pulse of H3.3core-GFP was produced in salivary glands and chromosome spreads were prepared 2 h later to assess the efficiency of RI nucleosome assembly. In wild-type cells, H3.3core-GFP strongly labels chromosome arms and active genes. In contrast, the H3.3core-GFP protein is efficiently produced in xnp-null mutant cells, but only a fraction of the protein deposits onto chromosomes; instead, most of the protein accumulates within the nucleolus. This protein does not coincide with DNA in the nucleolus, and may be predeposition or aggregated histones. Hira mutants have a similar reduction in H3.3 deposition: H3.3core-GFP protein is produced, but most protein accumulates in the nucleolus. These results demonstrate that the rate of RI nucleosome assembly is reduced in both xnp and Hira mutants, although some assembly can still occur. Indeed, longer expression of tagged histones in xnp or Hira mutants does achieve apparently normal levels (Schneiderman, 2012).

To test if Xnp and Hira have redundant roles in nucleosome replacement, Hira;xnp double-mutant animals were generated. Single-mutants are fully viable, but double-mutant larvae grow slower than wild-type siblings and die during larval development. Therefore H3.3core-GFP deposition was measured in these double-mutants. Strikingly, high levels of H3.3core-GFP were produced in Hira;xnp animals, but all of the protein accumulates in the nucleolus, with no detectable staining of chromosomes. It is concluded that both Hira and Xnp contribute to the efficiency of H3.3 RI nucleosome assembly, but this fails when both factors are eliminated (Schneiderman, 2012).

The results lead to the suggestion that Xnp and Hira identify nucleosome-depleted chromatin and promote new nucleosome assembly through a stepwise process (see Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant). In the first step, Xnp and Hira bind exposed DNA at chromatin gaps, thereby marking sites where a nucleosome has been displaced. In the second step, delivery factors carrying new histones are recruited by binding to Xnp and Hira at chromatin gaps. In the final step, these factors assist in the transfer of histones from delivery chaperones to DNA, and Xnp and Hira are released when nucleosome assembly is complete (Schneiderman, 2012).

A previous study showned that the Xnp remodeler is found at all sites where H3.3 is enriched, including active genes (Schneiderman, 2009 ). The Hira chaperone also localizes at active genes. However, there are additional sites in the genome where the two assembly factors do not coincide. First, the major site for Xnp binding is at a nucleosome-depleted satellite block, where Hira is not found. Second, most Hira is localized to the repeated ribosomal DNA (rDNA) within the nucleolus, where Xnp is not found. This finding implies that the two factors are not redundant at all sites where H3.3 is deposited (Schneiderman, 2012).

The rDNA genes are repressed in late-stage salivary glands. Therefore, deposition of H3.3 was assayed in the somatic follicle cells of ovaries, where rDNA is highly transcribed. In this cell type, Xnp localizes broadly in the nucleus but not within the nucleolus, but most Hira protein forms foci within the nucleolus. A pulse of epitope-tagged H3.3 produced in follicle cells broadly labels the nucleus and foci within the nucleolus, corresponding to transcriptionally active sites in this cell type. Follicle cells from xnp mutant ovaries also show nucleolar labeling with H3.3-GFP, demonstrating that Xnp is not required for nucleolar RI assembly. In contrast, a pulse of epitope-tagged H3.3 in Hira mutant follicle cells does not deposit in the nucleolus. Thus, rDNA chromatin must rely on the Hira chaperone for H3.3 deposition. It is concluded that Xnp and Hira are redundant at many sites within the genome, but some sites rely on individual factors for replacement nucleosome assembly. Although neither Hira nor Xnp are individually required for viability, there may be more subtle phenotypes that occur in repetitive sites in the genome (Schneiderman, 2012).

If Xnp and Hira are generally involved in recognizing chromatin gaps, the localization of these factors should be affected in H3.3-deficient cells. Indeed, new binding sites for Xnp appear in rDNA chromatin after H3.3 knock-down. Hira is also recruited to the nucleosome-depleted satellite block after H3.3 knock-down. Strikingly, the area of the nucleosome-depleted satellite block is ∼2.5-times larger after H3.3 knock-down, and the Xnp signal at this site is elevated. This finding implies that RI assembly is normally required for compaction of the satellite. The redundancy of Hira and Xnp implies that their importance for H3.3 deposition is underestimated in single-mutants. Both the relocalization and increased binding of Xnp and Hira after H3.3 knock-down support the idea that these assembly factors are recruited to aberrant, nucleosome-depleted chromatin. As persistent exposure of DNA may disrupt transcriptional regulation or allow DNA damage, surveying chromatin for gaps with RI assembly factors may be critical for genome stability and function (Schneiderman, 2012).

The Drosophila DAXX like protein (DLP) cooperates with ASF1 for H3.3 deposition and heterochromatin formation

Histone variants are non-allelic isoforms of canonical histones and they are deposited, in contrast to canonical histones, in a replication-independent (RI) manner. RI deposition of H3.3, a histone variant from the H3.3 family, is mediated in mammals by distinct pathways involving either the histone regulator A (HIRA) complex or the death-associated protein (DAXX)/alpha-thalassemia X-linked mental retardation protein (ATRX) complex. This study investigated the function of Drosophila DAXX Like Protein (DLP) by using both fly genetics approaches and protein biochemistry. DLP specifically interacts with H3.3 and shows a prominent localization on the base of the X chromosome, where it appears to act in concert with XNP the Drosophila homolog of ATRX, in heterochromatin assembly and maintenance. The functional association between DLP and XNP is further supported by a series of experiments, which illustrate genetic interactions and DLP-XNP-dependent localization of specific chromosomal proteins. In addition, DLP both participates in RI deposition of H3.3 and associates with the anti-silencing factor-1 (ASF1). It is suggested, in agreement with a recently proposed model, that DLP and ASF1 are part of a pre-deposition complex, which is recruited by XNP and is necessary to prevent DNA exposure in the nucleus (Fromental-Ramain, 2017).

This study has identified DLP as the Drosophila homolog of DAXX. DLP is involved, likely in concert with XNP/dATRX, in the formation of pericentric heterochomatin of the X chromosome. Moreover, DLP is implicated in RI deposition of the histone variant H3.3 and may constitute with ASF1 the central core of a pre-deposition complex, recruited to chromatin gaps by XNP. The existence of such complex was recently suggested (Fromental-Ramain, 2017 and references therein).

In spite of the fact that both proteins do not molecularly associate as their mammal homologs do, this study provides evidence that DLP and XNP functions are closely linked. DLP and XNP are located on the base of the X chromosome and analysis of animals simultaneously mutant for both dlp and xnp revealed that DLP and XNP likely act together during heterochromatin formation. In addition, both DLP and XNP are located next to distal heterochromatic marker HP1 on the X chromosome of larvae carrying the ln(1)wm4h rearrangement. Functional interactions between DLP and XNP 55 were also supported by the similar behavior of DLP and XNP in H3.3 deficient cells. In wild-type cells, in addition to the base of the X chromosome, expression of XNP is detected at many sites across the chromosome arms where DLP is not observed. In H3.3 knock-down-cells, DLP and XNP are present at many euchromatic sites of the chromosomes and are simultaneously associated with nucleolar chromatin of the rDNA. Finally, overexpressed DLP binds to many interbands on the polytene chromosomes, suggesting that DLP may also be involved in chromatin organization at euchromatic sites in addition to the pericentric heterochromatin. However, this latter observation should be viewed with caution since it cannot be ruled out that over-expressed DLP is not present in its usual complex and is consequently mis-targeted. Additional support for functional interactions between XNP and DLP is provided by genetic interactions between xnp and dlp. Indeed, loss of xnp function is characterized by reduced viability, which is further aggravated when dlp function is simultaneously reduced, strongly indicating that xnp and dlp may functionally cooperate during regulation of common targets. How XNP is recruited to nucleosome-depleted chromatin remains an important issue. XNP may be recruited by transcriptional machinery to active genes. Alternatively, XNP may bind structural motifs common to chromatin gaps, or may simply bind exposed DNA. The homologous ATRX contains a PHD domain that can bind DNA or histones tails. Recent work demonstrates that mammalian Hira may bind exposed DNA at chromatin gaps. Moreover, Hira and XNP bind active regions independently of one another. Hence, there may be multiple ways that RI assembly factors recognize exposed DNA (Fromental-Ramain, 2017).

In Drosophila, loss of H3.3 has a large impact on viability and fertility of both males and females. The Drosophila genome encompasses two single copy genes, H3.3A and H3.3B, which code for the same protein. H3.3 is highly expressed in mitotic, meiotic and post-meiotic male germ cells, probably reflecting high transcriptional activity. Interestingly, high dlp expression is observed in primary spermatocytes, in meiotic spermatocytes and also in the germinal vesicle, suggesting that it may have important functions during development of germ cells. In Drosophila testis, H3.3 disappears with the bulk of histones, prior to accumulation of protamine and other sperm-specific nuclear basic proteins, leading to sperm DNA compaction at late stages of spermiogenesis. At fertilization, assembly of nucleosomes on paternal DNA immediately follows the rapid loss of protamines from the decondensing male nucleus and is dependent on maternally provided factors like Hira and YEM. HIRA and YEM are crucial since male pronuclei fail to decondense at the pronuclear stage in eggs derived from female mutants for HIRA and YEM. Hence, function of HIRA/YEM at fertilization represents a unique example where deficient chromatin activity cannot be compensated by other redundant factors (Fromental-Ramain, 2017).

In contrast, many examples suggest that H3.3 chaperones/chromatin remodeling complexes may display functional redundancy as mutants in these factors have limited phenotypes. In this context, DLP may be viewed as a typical example. The null allele dlpG and the dlp45 allele encoding a truncated protein lacking the C-terminal DHR necessary for H3.3 binding are viable and fertile, indicating that DLP and other chromatin factors may share common functions. Alternatively, DLP may display accessory functions during development of germ cells. Characterization of the phenotypes of double-mutant animals during germ cell development would help to resolve this important issue (Fromental-Ramain, 2017).

H3.3 was initially seen as a characteristic of active genes with histone turnover occurring as a consequence of transcription. More recent studies revealed that H3.3 is widespread within the genome. In particular, H3.3 is deposited by ATRX/DAXX at telomeres and pericentric repeats. Interestingly, ATRX and DAXX are components of the same chromatin-remodeling complex and physically interact. Recently, Schneiderman (2012) proposed a model on how XNP, the Drosophila homolog of ATRX, and HIRA identify nucleosome-depleted DNA following gene activation, and promote nucleosome assembly through a three steps process. Initially, XNP and HIRA bind exposed DNA at chromatin gaps where nucleosomes have been displaced. Subsequently they co-operate to recruit a predeposition complex including ASF1 and histones. In the final step, XNP and HIRA assist the transfer of histones from delivery factors to DNA and are released when nucleosome assembly is complete. Even if XNP and DLP do not physically interact, this study provides several evidences suggesting that DLP could be a component of the predeposition complex recruited by XNP/HIRA (Fromental-Ramain, 2017).

Both HIRA and XNP have been implicated in RI nucleosome assembly, but mutants of these factors have only limited phenotypes, revealing that they have redundant functions. Thus, single mutants of either xnp or hira weakly affect H3.3 deposition, which is abolished in a double-mutant of xnp and hira. This observation highlights the need for two distinct pathways during RI nucleosome assembly, one mediated by HIRA/YEM and the other by XNP. This study assigns a role to DLP during RI H3.3 deposition since H3.3 incorporation is affected in animals lacking DLP. DLP is thought to co-operate with XNP and it was surprising to observe H3.3 deposition in animals lacking HIRA and DLP. Hence, it is speculated that the pathway mediated by XNP is always functional, although less efficient. It has been recently proposed that XNP recognizes exposed DNA when a nucleosome has been displaced, and serves as a binding platform for the recruitment of H3.3 predeposition complexes to chromatin gaps. Such complexes are believed to contain (H3.3-H4) heterodimers, ASF1 and additional factors. In line with this the data revealed physical interactions between ASF1 and DLP in protein extracts made from baculovirus-infected Sf9 cells co-expressing DLP and ASF1, suggesting that DLP may be one of these additional factors (Fromental-Ramain, 2017).

These data provides additional links between HIRA, DLP, ASF1 and XNP during H3.3 incorporation but how they functionally interact during development remains an open question. Identification of their genomic targets and characterization of their activities during H3.3 deposition would obviously help to resolve this important issue (Fromental-Ramain, 2017).

XNP/dATRX interacts with DREF in the chromatin to regulate gene expression

The ATRX gene encodes a chromatin remodeling protein that has two important domains, a helicase/ATPase domain and a domain composed of two zinc fingers called the ADD domain. The ADD domain binds to histone tails and has been proposed to mediate their binding to chromatin. The putative ATRX homolog in Drosophila (XNP/dATRX) has a conserved helicase/ATPase domain but lacks the ADD domain. In this study, it is proposed that XNP/dATRX interacts with other proteins with chromatin-binding domains to recognize specific regions of chromatin to regulate gene expression. A novel functional interaction is reported between XNP/dATRX and the cell proliferation factor DREF in the expression of pannier (pnr). DREF binds to DNA-replication elements (DRE) at the pnr promoter to modulate pnr expression. XNP/dATRX interacts with DREF, and the contact between the two factors occurs at the DRE sites, resulting in transcriptional repression of pnr. The occupancy of XNP/dATRX at the DRE, depends on DNA binding of DREF at this site. Interestingly, XNP/dATRX regulates some, but not all of the genes modulated by DREF, suggesting a promoter-specific role of XNP/dATRX in gene regulation. This work establishes that XNP/dATRX directly contacts the transcriptional activator DREF in the chromatin to regulate gene expression (Valadez-Graham, 2012; full text of article).

Protein complex of Drosophila ATRX/XNP and HP1a is required for the formation of pericentric beta-heterochromatin in vivo

ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor proteins. Mutations of the human ATRX gene result in a severe genetic disorder termed X-linked alpha-thalassemia mental retardation (ATR-X) syndrome. This study performed biochemical and genetic analyses of the Drosophila ortholog of ATRX. The loss of function allele of the Drosophila ATRX/XNP gene is semilethal. Drosophila ATRX is expressed throughout development in two isoforms, p185 and p125. ATRX185 and ATRX125 form distinct multisubunit complexes in fly embryo. The ATRX185 complex comprises p185 and heterochromatin protein HP1a. Consistently, ATRX185 but not ATRX125 is highly concentrated in pericentric beta-heterochromatin of the X chromosome in larval cells. HP1a strongly stimulates biochemical activities of ATRX185 in vitro. Conversely, ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. The loss of function allele of the ATRX/XNP gene and mutant allele that does not express p185 are strong suppressors of position effect variegation. These results provide evidence for essential biological functions of Drosophila ATRX in vivo and establish ATRX as a major determinant of pericentric beta-heterochromatin identity (Emelyanov, 2010).

Mammalian and fly ATRX have previously been implicated in the function of heterochromatin, core histone modifications, regulation of DNA methylation, and interactions with heterochromatin protein HP1. However, this study demonstrates that metazoan ATRX can form a stable complex with HP1a in vivo. Although HP1a is known to physically interact with various partners, including histones, histone and DNA modification enzymes, DNA replication and repair proteins, nuclear structure proteins, and transcription factors, this work is the first demonstration that HP1 exists in a stable complex with a nucleosome remodeling factor (Emelyanov, 2010).

HP1a is known to homodimerize through interactions within its chromoshadow domain. Furthermore, at least two HP1a protomers are present in its complex with ATRX185. Considering the predicted molecular mass of the complex (∼200 kDa) and the large molecular excess of HP1a relative to ATRX/XNP in vivo, it is likely that ATRX185 binds a dimer of HP1a, and this heterotrimer constitutes the predominant native form of ATRX185-HP1a complex (Emelyanov, 2010).

HP1a apparently plays an important regulatory role in biochemical activities of XNP/ATRX. Interestingly, the basal ATPase activity of ATRX185 is somewhat inhibited in the absence of HP1a. Thus, HP1a may introduce a conformational change to the ATRX185 polypeptide that derepresses its enzymatic activity. HP1a also strongly stimulates the ability of ATRX185 to remodel nucleosomes in REA assay. In fact, ATRX185 possesses extremely little nucleosome remodeling activity in the absence of HP1a. This strong stimulation cannot be attributed solely to the enhanced ATPase activity of the enzyme. Therefore, HP1a may also promote nucleosome remodeling by other mechanisms. For instance, it may facilitate ATRX tethering to nucleosomes. Alternatively, ATRX may conversely stimulate HP1a interactions with chromatin templates, which will manifest as increased nucleosome remodeling in the REA assay (Emelyanov, 2010).

Importantly, the smaller isoform of ATRX/XNP (ATRX125) does not physically interact with HP1a and forms an alternative complex(es). In size-exclusion chromatography, recombinant ATRX125 is separated from the ATRX185-HP1a complex into a distinct peak with the molecular mass <150 kDa. This elution profile is unlike that of the native form of ATRX125, which fractionates in a peak with a predicted molecular mass of ∼500 kDa. Thus, the native ATRX125 likely forms a multisubunit complex with additional polypeptides that may be involved in regulation of biological functions of ATRX125 in vivo (Emelyanov, 2010).

Loss-of-function mutation of ATRX/XNP gene is semilethal. However, elimination of heterochromatin-specific ATRX185 isoform does not substantially affect fly viability. Therefore, ATRX125 has additional biological functions that do not depend on HP1a and are targeted toward euchromatic loci. For instance, the putative ATRX125 complex may play regulatory roles in transcription of certain euchromatic genes or regulate other chromatin functions (Emelyanov, 2010).

The xnp/atrx mutations are strong recessive suppressors of pericentric PEV in the X chromosome. They also have an effect on variegation of tandemly repeated transgene arrays and telomeric position effect. The latter observation suggests that ATRX/XNP may have a function in heterochromatin silencing that is independent of HP1a, as Su(var)2-5 alleles have little or no dominant effect of silencing of the telomeric 39C-5 insertion. Alternatively, the dose reduction of HP1a in heterozygous alleles of Su(var)2-5 may have a disproportionally weaker influence on its presence in telomeres, which would not be detected by analyzing PEV. On the other hand, the complete elimination of functional ATRX185-HP1a complex in homozygous xnp/atrx alleles may have a stronger effect on HP1a availability for both telomeric and pericentric silencing. Notably, xnp[5] is a weak dominant suppressor of pericentric PEV. This effect is not due to an antimorphic effect of expression of the truncated form of ATRX/XNP, as this truncated product is not localized to the normal pericentric site of Drosophila ATRX (Emelyanov, 2010).

In polytene chromosomes, ATRX185 specifically localizes to pericentric beta-heterochromatin of the X chromosome, where it overlaps with HP1a. This localization is unlikely to be explained by interactions with HP1a, because ATRX is largely excluded from other loci, where HP1a is abundantly present (e.g. chromocenter). Therefore, additional sequence determinants in the N terminus of ATRX185 (which are absent in ATRX125) are required for ATRX185 targeting toward the 20B-F cytogenetic region. In the future, it will be interesting to define these sequence motifs in the structure of ATRX185 (Emelyanov, 2010).

In a recent report (Schneiderman, 2009) it was shown that the pericentric focus of D. melanogaster ATRX in polytene chromosomes overlaps with a ∼50-kb satellite block of TAGA repeat. This sequence is not conserved in other Drosophila species, whereas the pericentric localization of ATRX/XNP is. The pericentric ATRX/XNP focus is a major site of replication-independent nucleosome replacement. However, the rapid histone turnover at this site appears to be sequence-dependent and does not require ATRX/XNP. It has also been speculated that the pericentric ATRX/XNP focus contributes to heterochromatic silencing throughout the nucleus, including ectopic loci, such as bwD. It was observed that certain variegated transgenes localized to heterochromatic sites outside of the ATRX/XNP focus are not responsive to ATRX. Thus, it remains likely that silencing by ATRX does require its physical localization to the cognate loci (Emelyanov, 2010).

Pericentric beta-heterochromatin is the most widely studied model of silent heterochromatin in vivo in Drosophila. It harbors heterochromatic genes, rRNA genes, repetitive sequences, and retrotransposon insertions, characteristic of 'heterochromatin.' The breakpoints of classical chromosome aberrations that exhibit PEV (such as w[m4]) and insertion sites of variegating transgenes are all positioned in beta-heterochromatin. The near elimination of HP1a from pericentric X beta-heterochromatin of xnp/atrx mutant flies reveals an important biochemical activity of ATRX in vivo. It is possible that the ATRX/XNP ATPase is required for efficient ATP-dependent deposition of HP1a into this genomic region. Alternatively, it is possible that most if not all HP1a that is normally associated with this region is present in a complex with ATRX/XNP. In either case, this result validates Drosophila ATRX as a major component and the determinant of pericentric beta-heterochromatin structure and function. The role of ATRX185 in its native complex with HP1a in establishment of beta-heterochromatin identity in the fly X chromosomes is yet another example of variable biochemical functions that SWI2/SNF2-like molecular motors can have in modification of chromatin structure in vivo (Emelyanov, 2010).

The XNP remodeler targets dynamic chromatin in Drosophila

Heterochromatic gene silencing results from the establishment of a repressive chromatin structure over reporter genes. Gene silencing is often variegated, implying that chromatin may stochastically switch from repressive to permissive structures as cells divide. To identify remodeling enzymes involved in reorganizing heterochromatin, 11 SNF2-type chromatin remodelers were tested in Drosophila for effects on gene silencing. Overexpression of five remodelers affects gene silencing, and the most potent de-repressor is the alpha-thalassaemia mental retardation syndrome X-linked (ATRX) homolog X-linked nuclear protein (XNP). Although the mammalian ATRX protein localizes to heterochromatin, Drosophila XNP is not a general component of heterochromatin. Instead, XNP localizes to active genes and to a major focus near the heterochromatin of the X chromosome. The XNP focus corresponds to an unusual decondensed satellite DNA block, and both active genes and the XNP focus are sites of ongoing nucleosome replacement. It is suggested that the XNP remodeler modulates nucleosome dynamics at its target sites to limit chromatin accessibility. Although XNP at active genes may contribute to gene silencing, it was found that a single focus is present across Drosophila species and that perturbation of this site cripples heterochromatic gene silencing. Thus, the XNP focus appears to be a functional genetic element that can contribute to gene silencing throughout the nucleus (Schneiderman, 2009).

The chromatin remodelling factor dATRX is involved in heterochromatin formation

Despite extensive study of heterochromatin, relatively little is known about the mechanisms by which such a structure forms. This study shows that the Drosophila homologue of the human alpha-thalassemia and mental retardation X-linked protein (dATRX, also known as XNP), is important in the formation or maintenance of heterochromatin through modification of position effect variegation. There are two isoforms of the dATRX protein, the longer of which interacts directly with heterochromatin protein 1 (dHP-1) through a CxVxL motif both in vitro and in vivo. These two proteins co-localise at heterochromatin in a manner dependent on this motif. Consistent with this observation, the long isoform of the dATRX protein localises primarily to the heterochromatin at the chromocentre on salivary gland polytene chromosomes, whereas the short isoform binds to many sites along the chromosome arms. It is suggested that the establishment of a regular nucleosomal organisation may be common to heterochromatin and transcriptionally repressed chromatin in other locations, and may require the action of ATP dependent chromatin remodelling factors (Bassett, 2008).

This study has identified novel mutations in the putative chromatin remodelling factor dATRX, and has shown that these suppress PEV, using two independent variegating alleles on chromosome I and IV. This implicates the dATRX gene in the process of heterochromatin formation or maintenance in vivo (Bassett, 2008).

It was further shown that the dATRX protein is expressed in two isoforms, the longer of which shows a strong interaction with the dHP1a protein in both GST pulldown and co-immunoprecipitation assays. This interaction is necessary for localisation of the long isoform to heterochromatin in 3T3 cells, and colocalisation with dHP-1a. This interaction is mediated by a CxVxL motif specific to the long isoform, mutation of which abolishes interaction and colocalisation with dHP-1a. Additionally, the long isoform specifically localises to the chromocentre in Drosophila polytene chromosomes, providing further evidence for a role of the long isoform in heterochromatin formation. The observed suppression of PEV by the dATRX3 allele that removes this isoform specifically suggests that the interaction is relevant in vivo. It is also consistent with an observed interaction between human ATRX and HP-1α, and genetic interactions between the two homologues in C. elegans (Cardoso, 2005). These studies combined with the results of the PEV assay strongly imply a role of ATRX in heterochromatin formation in a variety of organisms, and may provide a mechanism of recruitment to such regions (Bassett, 2008).

The exclusion of the short isoform from heterochromatin in 3T3 cells suggests that this has a distinct function at non-heterochromatic sites throughout the genome, and is consistent with its lack of interaction with dHP-1a, and the staining observed in the chromosome arms on polytene chromosomes. Indeed, this staining largely associates with the interbands, representative of less dense chromatin. One role of the short isoform may be during central nervous system formation during embryogenesis (Sun, 2006), in controlling glial and neuronal patterning (Bassett, 2008).

Analysis of the dATRX3 mutation that removes the long isoform shows no visible phenotype, aside from reduced viability of the flies. Studies of the chromatin structure in these mutants have failed to show any difference in the nucleosome spacing as judged by micrococcal nuclease digestion. This could simply be a consequence of the limitations of this assay or alternatively could suggest a different role in higher order structure formation, or redundancy with other chromatin remodelling factors such as dMi-2 (Bassett, 2008).

In order to form a condensed, heterochromatic structure, nucleosome positions must be optimised such that the relative orientations of two nucleosomes are consistent along the fibre. This would allow a regular, ordered structure to form, essential for the formation of a compact fibre and subsequent further folding into a higher order structure such as heterochromatin. Drosophila ACF has been shown to act to alter nucleosome repeat lengths both in vitro and in vivo, suggesting a role in 'shuffling' of nucleosome positions to generate a more uniform array. One role of the dATRX remodelling factor may be to achieve this. A second mechanism may be by inducing twist, which would aid or antagonise compaction of the chromatin fibre depending on its direction (Bassett, 2008).

It is suggested that chromatin remodellers are the end effectors of histone modifications. Consistent with this view, many remodelling complexes contain components that recognize specific modification states of histone tails. For instance, the SANT domain of dISWI may bind to unmodified tails, while one of the PHD domains from human Mi-2 binds preferentially to trimethylated lysine 36 of histone H3, which marks the end of active transcription units. ATRX may be recruited by its interaction with HP-1 or MeCP2 to heterochromatin, or the PHD domain in the human protein may play a role in methylated histone binding. In this manner, the epigenetic code present on histones may be translated by chromatin remodelling factors into alterations in folding of the chromatin fibre. Consistent with this idea, the chromatin remodelling activity of Mit1 has been shown to be important for heterochromatin formation in S. pombe. It is proposed that dATRX plays such a role at heterochromatin (Bassett, 2008).

dXNP/DATRX increases apoptosis via the JNK and dFOXO pathway in Drosophila neurons

Mutation of the XNP/ATRX gene, which encodes an SNF2 family ATPase/helicase protein, leads to ATR-X syndrome and several other X-linked mental retardation syndromes. Although XNP/ATRX is a chromatin remodeler, the molecular mechanism by which mental retardation occurs in patients with ATR-X has yet to be determined. To better understand the role of XNP/ATRX in neuronal development, Drosophila XNP (dXNP/DATRX) was ectopically expressed in Drosophila neurons. Neuronal expression of dXNP/DATRX resulted in various developmental defects and induced strong apoptosis. These defects and apoptosis were suppressed by Drosophila inhibitor of apoptosis protein 1. Expression of dXNP/DATRX also increased JNK activity and the levels of reaper and hid transcripts, which are pro-apoptotic factors that activate caspase. Furthermore, dXNP/DATRX-induced rough eye phenotype and apoptosis were suppressed by dFOXO deficiency. These results suggest that dXNP/DATRX is involved in caspase-dependent apoptosis in Drosophila neurons via regulation of the JNK and dFOXO pathway (Hong, 2009).

dXNP, a Drosophila homolog of XNP/ATRX, induces apoptosis via Jun-N-terminal kinase activation

XNP/ATRX, a causative gene of X-linked alpha-thalassemia/mental retardation syndrome, encodes an SNF2 family ATPase/helicase protein. To better understand the role of XNP/ATRX in development, a Drosophila XNP/ATRX homolog, dXNP, was isolated and characterized that contains highly conserved SNF2 and helicase domains. Ectopically expressed dXNP induced strong apoptosis in the developing eye and wing, but did not affect cell cycle progression or the expression of wingless and engrailed, essential regulators of development. The dXNP-induced apoptosis was strongly suppressed by DJNKK/hemipterous mutation, and dXNP increased JNK activity. Taken together, these results suggest that dXNP regulates apoptosis via JNK activation (Lee, 2007).

Glial and neuronal functions of the Drosophila homolog of the human SWI/SNF gene ATR-X (DATR-X) and the jing zinc-finger gene specify the lateral positioning of longitudinal glia and axons

Neuronal-glial communication is essential for constructing the orthogonal axon scaffold in the developing Drosophila central nervous system (CNS). Longitudinal glia (LG) guide extending commissural and longitudinal axons while pioneer and commissural neurons maintain glial survival and positioning. However, the transcriptional regulatory mechanisms controlling these processes are not known. The midline function of the jing C2H2-type zinc finger transcription factor has been shown to be only partially required for axon scaffold formation in the Drosophila CNS. A screen was performed for gain-of-function enhancers of jing gain-of-function in the eye; the Drosophila homolog DATR-X (also termed XNP) of the disease gene of human alpha-thalassemia/mental retardation X-linked (ATR-X) was identified, as well as other genes with potential roles in gene expression, translation, synaptic transmission and cell cycle. jing and DATR-X reporter genes are expressed in both CNS neurons and glia including the longitudinal glia. Co-expression of jing and DATR-X in embryonic neurons synergistically affects longitudinal connective formation. During embryogenesis, jing and DATR-X have autonomous and non-autonomous roles in the lateral positioning of LG, neurons and longitudinal axons as shown by cell-specific knock-down of gene expression. jing and DATR-X are also required autonomously for glial survival. jing and DATR-X mutations show synergistic effects during longitudinal axon formation, suggesting they are functionally related. These observations support a model in which downstream gene expression, controlled by a potential DATR-X-Jing complex, facilitates cellular positioning and axon guidance, ultimately allowing for proper connectivity in the developing Drosophila CNS (Sun, 2006).

Of the candidates from the screen, DATR-X was chosen for study due to a possible involvement in Jing CNS function and disease relevance. Mutations in the human ATR-X gene are associated with several X-linked mental retardation phenotypes that lead to cognitive delay, facial dysmorphism, microcephaly, skeletal and genital abnormalities and neonatal hypotonia. 87% of mental retardation (MR) genes have a fruit fly homolog and 76% have a candidate functional ortholog revealing a remarkable conservation between humans and Drosophila melanogaster. Some orthologs of human MR genes have cellular phenotypes involving neurons, glia and neural precursor cells and arise from defects in proliferation, migration and process extension or arborization. For example, targeted mutation of ATR-X to the early forebrain in mice leads to cortical progenitor cell death and reduced forebrain size. In addition, mutations in genes controlling the identity of forebrain neuronal precursors can result in holoprosencephaly where the brain hemispheres do not separate. An increased understanding of the molecular and cellular bases for hereditary MR is critical for the generation of drug treatments (Sun, 2006).

ATR-X belongs to the SWI/SNF group of chromatin remodeling proteins that use the energy provided by ATP hydrolysis to disrupt histone-DNA associations and move nucleosomes to different positions. This chromatin modulation allows for the access of activators or repressors to their DNA binding sites in their target genes. The helicase C and SNF2N domains of ATR-X have been shown to have DNA translocase and nucleosome-remodeling activities. Accordingly, mutations in ATR-X have been mapped to the helicase C and SNF2N domains which show approximately 60% homology with those in DATR-X and have been conserved from C. elegans to humans. This conservation supports a conserved role for Drosophila ATR-X in chromatin remodeling (Sun, 2006).

Vertebrate ATR-X has a C2C2 zinc finger motif in the amino terminus that is similar to a plant homeodomain (PHD) finger previously identified in proteins involved in chromatin-mediated transcriptional regulation. Interestingly, D. melanogaster and C. elegans ATR-X proteins do not contain the zinc finger domain, suggesting that these structures may have been acquired through evolution due to a necessity in vertebrate chromatin remodeling mechanisms (Sun, 2006).

Given the absence of the zinc finger domains in DATR-X, it is postulated that invertebrate DATR-X proteins may be complexed with proteins containing a nuclear targeting and DNA-binding motif in order to regulate gene expression at the proper regulatory sites. This may be a role for Jing since it has a very similar embryonic expression pattern as well as mutant and over-expression phenotypes as those of DATRX. Therefore, it seems that the ATPase domain of DATR-X has been conserved through evolution and that the other regions of the protein may have evolved to suit the specific needs of the cell. In summary, different mechanisms of ATR-X function and different binding partners across species may account for the divergence of sequence with respect to the amino terminal and Q-rich repeats while the main chromatin remodeling aspects of ATR-X remain similar (Sun, 2006).

Jing encodes a nuclear protein with putative DNA-binding and transcriptional regulatory domains. The C2H2 zinc fingers of Jing are most similar (50% identical) to those of the mouse adipocyte enhancer binding protein 2 (AEBP2) and also show 25% homology to those of the Kruppel family of transcription factors including those encoding gli and ZIC2. AEBP2 function is implicated in chromatin remodeling events and has strong expression in the brain. Genetic screening identifies a related group of jing-interacting genes. A background sensitive to jing function was used to conduct a genetic screen in the eye. For the GOF screen, it was hypothesized that mis-expression of jing in the eye in combination with other genes involved in jing transcriptional regulation would lead to alterations in gene expression and consequently disrupt ommatidial formation. The genetic relationship between DATR-X and jing in embryonic neurons and glia shows that the screen was successful in identifying genes whose function in adult neuronal cells is relevant to jing function in the embryonic CNS (Sun, 2006).

EP(3)3084 contains a transposon in proximity to a novel gene known by its Flybase transcript identifier as CG15507. Despite strong effects of EP(3)3084 expression in the eye these were specifically strongly enhanced after co-expression with jing, DAtx2 and JIGR1. Furthermore, each gene specifically interacted with the other three, but not with randomly chosen EP lines, suggesting a functional relationship among the four genes. The EP elements in these lines are located in the 5' untranslated region of the downstream genes suggesting these elements may result in over-expression. Given the regulatory role of MADF domains, it is possible that JIGR1 regulates gene expression with Jing and DATR-X. Alternatively, JIGR1 may regulate the expression of a Jing/DATR-X target gene. Likewise, DAtx2 may by involved in regulating the translation of a protein that is an essential component of a Jing/DATR-X/JIGR1 complex or a downstream target of these genes. A role for the orthologs of translational regulators in mental retardation has been shown for the Drosophila ortholog of fragile X mental retardation 1 (Dfmr1). Dfmr1 regulates the MAP1B homolog of Futsch to control synaptic structure and function in the embryonic Drosophila CNS. Therefore, genetic screening and phenotypic analysis in Drosophila has the power to decipher pathways and the cellular bases of MR genes (Sun, 2006).

In wild-type Drosophila embryos, longitudinal glia assume characteristic positions and do not cross the midline or into adjacent VNC segments. This is due to multiple mechanisms at different stages of development including response to repulsive and attractive molecules, cell-cell contact, trophic support and axon contact. A disruption in any of these processes will perturb formation of the glial and axonal scaffolds. The expression of jing and DATR-X reporter genes in longitudinal glia correlates with the longitudinal glial phenotypes associated with mutations in these genes. During stage 12, Robo present on the LG responds to repulsive midline Slit molecules to maintain lateral positioning. The medial misplacement of Robo- and Repo-positive LG during stage 12 after jing and DATR-X glial-specific knockdown suggests that there may have been a breakdown in Robo-dependent repulsive mechanisms. However, the fact that Robo protein was present after jing and DATR-X glial and neuronal knockdown suggests that robo expression may not be regulated by Jing and DATR-X. Alternatively, Jing and DATR-X may regulate the expression of a factor that controls how Robo 'reads' the Slit signal. In support, misrouting of axons across the midline in the presence of Robo occurs in calmodulin and Son of sevenless mutants where these proteins are required to process the Sli signal. It is also possible that jing and DATR-X regulate the expression of factors controlling glial and neuronal positioning in a Robo-independent fashion (Sun, 2006).

jing and DATR-X mutations clearly affect more than Robo-mediated LG positioning. (1) Glial survival is not affected in robo mutant embryos whereas glia die despite continuous axonal contact in jing and DATR-X glial-specific mutants. Therefore, the loss of CNS glia may reflect a breakdown in an intrinsic survival pathway mediated by jing and DATR-X. The expression of jing and DATR-X reporter genes in glia is consistent with such a role. Furthermore, both jing and DATR-X/ATR-X have been implicated in cell survival processes in the CNS midline and tracheal cells and in cortical progenitors, respectively. (2) In robo mutants only the central pCC/MP2 fascicle but not the outer two longitudinal fascicles are affected. However, in jing and DATR-X glial and neuronal mutants the outer fascicles are fused, often broken and can be seen crossing the midline (Sun, 2006).

These defects are similar to those after ablation of neurons or glia and after genetic loss of glia as in gcm mutants. These observations suggest that multiple biological processes require the proper function of these genes and are consistent with an important upstream role for jing and DATR-X in glial and neuronal differentiation. Evidence is accumulating that chromatin accessibility plays a key role in the transcriptional regulation of cell-type-specific gene expression in the CNS. The conservation in ATPase domains along with the similar phenotype of DATR-X and jing mutations and in their expression patterns raises the possibility that Jing is involved in the targeting of a chromatin remodeling complex containing DATR-X to transcriptional target genes whose products are required for the response of longitudinal growth cones and glia to guidance cues. In summary, a group of genes have been identified that pertain to jing function and specifically genetically interact in adult neuronal cells. The results show that specific neural and glial developmental defects underlie the problems in axon guidance associated with mutations in DATR-X and jing. More studies using targeted mutations of MR genes will alleviate the view that brain phenotypes result from generic effects due to a heightened sensitivity of the brain (Sun, 2006).

Distinct factors control histone variant H3.3 localization at specific genomic regions in mammalian embryonic stem cells and neuronal precursor cells

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, this study reports genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. These data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells (Goldberg, 2010).

ATRX interacts with H3.3 in maintaining telomere structural integrity in pluripotent embryonic stem cells

ATRX (alpha thalassemia/mental retardation syndrome X-linked) belongs to the SWI2/SNF2 family of chromatin remodeling proteins. Besides the ATPase/helicase domain at its C terminus, it contains a PHD-like zinc finger at the N terminus. Mutations in the ATRX gene are associated with X-linked mental retardation (XLMR) often accompanied by alpha thalassemia (ATRX syndrome). Although ATRX has been postulated to be a transcriptional regulator, its precise roles remain undefined. This study demonstrates ATRX localization at the telomeres in interphase mouse embryonic stem (ES) cells in synchrony with the incorporation of H3.3 during telomere replication at S phase. Moreover, it was found that chromobox homolog 5 (CBX5) (also known as heterochromatin protein 1 alpha, or HP1 alpha) is also present at the telomeres in ES cells. It was shown by coimmunoprecipitation that this localization is dependent on the association of ATRX with histone H3.3, and that mutating the K4 residue of H3.3 significantly diminishes ATRX and H3.3 interaction. RNAi-knockdown of ATRX induces a telomere-dysfunction phenotype and significantly reduces CBX5 enrichment at the telomeres. These findings suggest a novel function of ATRX, working in conjunction with H3.3 and CBX5, as a key regulator of ES-cell telomere chromatin (Wong, 2010).

NP-1/ATR-X acts with RB, HP1 and the NuRD complex during larval development in C. elegans

Mutations in the XNP/ATR-X gene cause several X-linked mental retardation syndromes in humans Mei-41. The XNP/ATR-X gene encodes a DNA-helicase belonging to the SNF2 family. It has been proposed that XNP/ATR-X might be involved in chromatin remodelling. The lack of a mouse model for the ATR-X syndrome has, however, hampered functional studies of XNP/ATR-X. C. elegans possesses one homolog of the XNP/ATR-X gene, named xnp-1. By analysing a deletion mutant, it has been shown that xnp-1 is required for the development of the embryo and the somatic gonad. Moreover, abrogation of xnp-1 function in combination with inactivation of genes of the NuRD complex, as well as lin-35/Rb and hpl-2/HP1 leads to a stereotyped block of larval development with a cessation of growth but not of cell division. A specific function for xnp-1 together with lin-35 or hpl-2 has been demonstrated in the control of transgene expression, a process known to be dependent on chromatin remodelling. This study thus demonstrates that in vivo XNP-1 acts in association with RB, HP1 and the NuRD complex during development (Cardoso, 2005).

lin-35/Rb and xnp-1/ATR-X function redundantly to control somatic gonad development in C. elegans

In screens for genetic modifiers of lin-35/Rb, the C. elegans retinoblastoma protein homolog, a mutation in xnp-1 was identified. Mutations in xnp-1, including a presumed null allele, are viable and, in general, appear indistinguishable from the wild type. In contrast, xnp-1 lin-35 double mutants are typically sterile and exhibit severe defects in gonadal development. Analyses of the abnormal gonads indicate a defect in the lineages that generate cells of the sheath and spermatheca. xnp-1 encodes the C. elegans homolog of ATR-X, a human disease gene associated with severe forms of mental retardation and urogenital developmental defects. xnp-1/ATR-X is a member of the Swi2/Snf2 family of ATP-dependent DEAD/DEAH box helicases, which function in nucleosome remodeling and transcriptional regulation. Expression of an xnp-1::GFP promoter fusion is detected throughout C. elegans development in several cell types including neurons and cells of the somatic gonad. These findings demonstrate a new biological role for Rb family members in somatic gonad development and implicate lin-35 in the execution of multiple cell fates in C. elegans. In addition, these results suggest a possible conserved function for xnp-1/ATR-X in gonadal development across species (Bender, 2004).

xnp-1 encodes the C. elegans homolog of the human ATR-X gene, a member of the Swi/Snf superfamily of ATP-dependent chromatin remodeling helicases. Mutations in human ATR-X lead to severe mental retardation as well as many secondary anomalies including urogenital defects in approximately 80% of ATR-X patients. The mutation identified in xnp-1(fd2) mutants leads to a substitution (R → K) of a highly conserved arginine at amino acid position 1130 (corresponding to human ATR-X position 2197) in the C terminus of the peptide. Interestingly, an analysis of molecular lesions from ATR-X patients indicates that mutations affecting the C-terminal region of the ATR-X protein are often associated with the most severe forms of urogenital defects. In contrast to humans, however, expression of the gonadal defect in C. elegans is dependent upon the coordinate inactivation of class B SynMuv genes such as lin-35. Thus, in C. elegans, lin-35 and xnp-1 function redundantly in the control of gonadal development (Bender, 2004).

Studies on Swi/Snf family members have indicated their importance in many diverse biological processes, most of which can be linked mechanistically to the control of nucleosome remodeling and gene expression. The precise level of control exerted by Swi/Snf members has been reported to range from gene-specific to global and appears to depend on several factors including the particular Swi/Snf complex involved, associations with various binding partners, genetic background, and cell cycle phase. Moreover, the effects exerted by Swi/Snf complexes on individual target genes can be either repressive or activating; the outcome most likely depends on the influence of other bound regulators such as histone modifying enzymes (Bender, 2004).

An obvious model to account for the functional redundancy of LIN-35 and XNP-1 is that both proteins share in common one or more transcriptional targets. Thus, in single-mutant backgrounds, sufficient regulation of the target can be brought about through the intact pathway acting alone. However, in double mutants, two means of regulation are missing and the shared target (or targets) may become grossly deregulated. Based on precedent from studies on the transcriptional effects of Rb family members, as well as other lin-35 synthetic mutants, the actions of both LIN-35 and XNP-1 on the shared target(s) are found to be repressive in nature (Bender, 2004).

As to how many common targets might be affected in the double mutants is an open question. Many studies analyzing the transcriptional targets of individual Swi/Snf complexes have been carried out, and they suggest that Swi/Snf proteins may regulate the expression of sizeable numbers (on the order of several hundred to several thousand) of physically disparate target genes. Likewise, many recent reports seeking to determine the transcriptional targets of Rb family members suggest that Rb family members may collectively regulate the expression of up to several hundred genes. Although such studies may be significantly compromised by issues such as cell- and tissue-type specific differences, genetic redundancy, and indirect effects, they provide at least some basis for estimating the number of genes that may be co-regulated by LIN-35 and XNP-1 in C. elegans. Namely, assuming a nonbiased set of 250 independent targets for both LIN-35 and XNP-1, as well as a genome consisting of 17,000 genes, it would be predicted that on average, 3.7 genes would be regulated by both factors. Although such calculations are highly speculative, they do suggest that the observed phenotype of xnp-1 lin-35 mutants could be due to the missexpression of a relatively small number of genes, perhaps even a single common target. Identification of such targets, either by genetics or other means, will await further studies (Bender, 2004).


REFERENCES

Search PubMed for articles about Drosophila XNP

Bassett, A. R., Cooper, S. E., Ragab, A. and Travers, A. A. (2008). The chromatin remodelling factor dATRX is involved in heterochromatin formation. PLoS One 3: e2099. PubMed ID: 18461125

Bender, A. M., Wells, O. and Fay, D. S. (2004). lin-35/Rb and xnp-1/ATR-X function redundantly to control somatic gonad development in C. elegans. Dev. Biol. 273: 335-349. 15328017

Bonnefoy, E., Orsi, G. A., Couble, P. and Loppin, B. (2007). The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization. PLoS Genet. 3(10): 1991-2006. PubMed ID: 17967064

Cardoso, C., et al. (2005). XNP-1/ATR-X acts with RB, HP1 and the NuRD complex during larval development in C. elegans. Dev. Biol. 278(1): 49-59. PubMed ID: 15649460

Drané, P., Ouararhni, K., Depaux, A., Shuaib, M. and Hamiche, A. (2010). The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3. Genes Dev 24: 1253-1265. PubMed ID: 20504901

Emelyanov, A. V., Konev, A. Y., Vershilova, E. and Fyodorov, D. V. (2010). Protein complex of Drosophila ATRX/XNP and HP1a is required for the formation of pericentric beta-heterochromatin in vivo. J. Biol. Chem. 285(20): 15027-37. PubMed ID: 20154359

Fromental-Ramain, C., Ramain, P. and Hamiche, A. (2017). The Drosophila DAXX like protein (DLP) cooperates with ASF1 for H3.3 deposition and heterochromatin formation. Mol Cell Biol [Epub ahead of print]. PubMed ID: 28320872

Goldberg, A. D., et al. (2010). Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140: 678-691. PubMed ID: 20211137

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Lee, N. G., Hong, Y. K., Yu, S. Y., Han, S. Y., Geum, D. and Cho, K. S. (2007). dXNP, a Drosophila homolog of XNP/ATRX, induces apoptosis via Jun-N-terminal kinase activation. FEBS Lett 581: 2625-2632. PubMed ID: 17531976

Loppin, B., Bonnefoy, E., Anselme, C., Laurencon, A., Karr, T. L. and Couble, P. (2005). The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus. Nature 437(7063): 1386-90. PubMed ID: 16251970

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Schneiderman, J. I., Orsi, G. A., Hughes, K. T., Loppin, B. and Ahmad, K. (2012). Nucleosome-depleted chromatin gaps recruit assembly factors for the H3.3 histone variant. Proc Natl Acad Sci U S A 109(48): 19721-19726. PubMed ID: 23150573

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Sun, X., Morozova, T. and Sonnenfeld, M. (2006). Glial and neuronal functions of the Drosophila homolog of the human SWI/SNF gene ATR-X (DATR-X) and the jing zinc-finger gene specify the lateral positioning of longitudinal glia and axons. Genetics 173: 1397-1415. PubMed ID: 16648585

Valadez-Graham, V., et al. (2012). XNP/dATRX interacts with DREF in the chromatin to regulate gene expression. Nucleic Acids Res. 40(4): 1460-74. PubMed Citation: 22021382

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Biological Overview

date revised: 12 August 2013

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