Domains of Heterochromatin Protein 1 Required for Drosophila melanogaster Heterochromatin Spreading

Centric regions of eukaryotic genomes are packaged into heterochromatin, which possesses the ability to spread along the chromosome and silence gene expression. The process of spreading has been challenging to study at the molecular level due to repetitious sequences within centric regions. A heterochromatin protein 1 (HP1) tethering system was developed that generates “ectopic heterochromatin” at sites within euchromatic regions of the Drosophila melanogaster genome. Using this system, we show that HP1 dimerization and the PxVxL interaction platform formed by dimerization of the HP1 chromo shadow domain are necessary for spreading to a downstream reporter gene located 3.7 kb away. Surprisingly, either the HP1 chromo domain or the chromo shadow domain alone is sufficient for spreading and silencing at a downstream reporter gene located 1.9 kb away. Spreading is dependent on at least two H3K9 methyltransferases, with SU(VAR)3-9 playing a greater role at the 3.7-kb reporter and dSETDB1 predominately acting at the 1.9 kb reporter. These data support a model whereby HP1 takes part in multiple mechanisms of silencing and spreading.HETEROCHROMATIN protein 1 (HP1) was identified in Drosophila as a nonhistone chromosomal protein enriched in centric heterochromatin (James and Elgin 1986; James et al. 1989). On polytene chromosomes, HP1 localizes near centromeres and telomeres, along the fourth chromosome and at ∼200 sites within the euchromatic arms (James et al. 1989; Fanti et al. 2003). Heterochromatin has the ability to “spread,” or propagate in cis, along the chromosome (Weiler and Wakimoto 1995). Spreading is observed when a chromosomal rearrangement places a euchromatic domain next to a heterochromatic domain. Cytologically, spreading is visualized as densely compact chromatin that emanates from the chromocenter, the structure formed by the fusion of centromeres, and extends into the banded regions of polytene chromosomes (Belyaeva and Zhimulev 1991). Euchromatic genes brought into juxtaposition with heterochromatin by chromosomal rearrangements exhibit gene silencing, termed position effect variegation (PEV) (Weiler and Wakimoto 1995). Mutations in Su(var)2-5, the gene encoding HP1, suppress silencing, suggesting HP1 plays a key role in spreading (Eissenberg et al. 1990). The molecular processes of spreading are not well understood.Repetitive sequences within heterochromatin make it difficult to study spreading at the molecular level. In addition, specific repetitive elements are thought to function as initiation sites for heterochromatin formation (Sun et al. 2004; Haynes et al. 2006), making it challenging to separate initiation from spreading. To overcome these problems, we generated a system that nucleates small domains (<20 kb) of repressive chromatin that share many properties with centric heterochromatin. Here we refer to these as ectopic heterochromatin domains. These domains are generated by expressing a fusion protein, consisting of the DNA binding domain of the Escherichia coli lac repressor (LacI) fused to HP1, in stocks possessing lac operator (lacO) repeats upstream of a reporter gene cassette (Danzer and Wallrath 2004). LacI-HP1 associates with the lacO repeats and causes silencing of the adjacent reporter genes. Silencing correlates with alterations in chromatin structure that include the generation of regular nucleosome arrays similar to those observed in centric heterochromatin (Sun et al. 2001; Danzer and Wallrath 2004). Chromatin immunoprecipitation (ChIP) experiments demonstrated that HP1 spreads bidirectionally, 5–10 kb from the lacO repeats, encompassing the reporter genes (Danzer and Wallrath 2004). Thus, HP1 is sufficient to nucleate small heterochromatin-like domains at genomic locations devoid of repetitious sequences, allowing for molecular studies of spreading.HP1 contains an amino terminal chromo domain (CD) and a carboxy chromo shadow domain (CSD), separated by a flexible hinge (Li et al. 2002). The CD forms a hydrophobic pocket implicated in chromosomal association through binding to di- and trimethylated lysine 9 of histone H3 (H3K9me2 and me3, respectively), an epigenetic mark generated by the histone methyltransferases (HMT) SU(VAR)3-9 and dSETDB1 (also known as Egg) (Jacobs et al. 2001; Schotta et al. 2002; Schultz et al. 2002; Ebert et al. 2004; Clough et al. 2007; Seum et al. 2007; Tzeng et al. 2007). Association with methylated H3 is one mechanism of HP1 chromosome association; however, other mechanisms involving interactions with DNA and/or partner proteins likely exist (Fanti et al. 1998; Li et al. 2002; Cryderman et al. 2005). In Drosophila HP1, a single amino acid substitution within the CD (V26M) is present in the Su(var)2-5⁰² allele; flies heterozygous for this allele show suppression of gene silencing by heterochromatin (Eissenberg et al. 1990). Furthermore, flies trans-heterozygous for Su(var)2-5⁰² and a null allele of Su(var)2-5 show dramatic reduction of HP1 near centromeres and do not survive past the third larval stage (Fanti et al. 1998). Consistent with these observations, structural studies show that V26 plays a critical role in forming the hydrophobic pocket of the CD that binds to H3K9me (Jacobs et al. 2001).The HP1 CSD dimerizes and mediates interactions with a variety of nuclear proteins (Cowieson et al. 2000; Yamamoto and Sonoda 2003; Thiru et al. 2004). CSD dimerization sets up an interaction platform for the binding of proteins possessing a penta-peptide motif, PxVxL (where x represents any amino acid) (Thiru et al. 2004; Lechner et al. 2005). Amino acid substitutions within HP1 have been identified that disrupt dimerization, and interaction with PxVxL proteins (Lechner et al. 2000; Thiru et al. 2004). For example, a single amino acid substitution within the CSD (I161E) disrupts dimerization of mouse HP1beta (Brasher et al. 2000). The lack of dimerization also caused the loss of interactions with nuclear factors containing PxVxL motifs and non-PxVxL partners (Yamamoto and Sonoda 2003; Lechner et al. 2005). In contrast, a single amino acid substitution elsewhere in the CSD (W170A) of mouse HP1beta does not prevent dimerization, but disrupts the interaction with PxVxL partner proteins (Brasher et al. 2000). Therefore, the requirement for HP1 dimerization and binding to the PxVxL proteins can be functionally separated. Here, we investigate effects of HP1 domain deletions and amino acid substitutions on HP1 localization, partner protein interactions, and heterochromatin spreading.     

Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.     

TRF4 is involved in polyadenylation of snRNAs in Drosophila melanogaster

The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in an RNA quality control mechanism, where polyadenylated RNAs are degraded by the nuclear exosome. Although Trf4/5 homologue genes are distributed throughout multicellular organisms, their biological roles remain to be elucidated. We isolated here the two homologues of Trf4/5 in Drosophila melanogaster, named DmTRF4-1 and DmTRF4-2, and investigated their biological function. DmTRF4-1 displayed poly(A) polymerase activity in vitro, whereas DmTRF4-2 did not. Gene knockdown of DmTRF4-1 by RNA interference is lethal in flies, as is the case for the trf4 trf5 double mutants. In contrast, disruption of DmTRF4-2 results in viable flies. Cellular localization analysis suggested that DmTRF4-1 localizes in the nucleolus. Abnormal polyadenylation of snRNAs was observed in transgenic flies overexpressing DmTRF4-1 and was slightly increased by the suppression of DmRrp6, the 3′-5′ exonuclease of the nuclear exosome. These results suggest that DmTRF4-1 and DmRrp6 are involved in the polyadenylation-mediated degradation of snRNAs in vivo.     

Arabidopsis membrane steroid binding protein 1 is involved in inhibition of cell elongation

A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220-amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants.     

The Drosophila HOAP protein is required for telomere capping

HOAP (HP1/ORC-associated protein) has recently been isolated from Drosophila melanogaster embryos as part of a cytoplasmic complex that contains heterochromatin protein 1 (HP1) and the origin recognition complex subunit 2 (ORC2). Here, we show that caravaggio, a mutation in the HOAP-encoding gene, causes extensive telomere-telomere fusions in larval brain cells, indicating that HOAP is required for telomere capping. Our analyses indicate that HOAP is specifically enriched at mitotic chromosome telomeres, and strongly suggest that HP1 and HOAP form a telomere-capping complex that does not contain ORC2.     

Hyper-phosphorylated retinoblastoma protein suppresses telomere elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase alpha (pol-alpha) is reported to be required for telomere maintenance, the critical role of pol-alpha in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-alpha in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-alpha or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-alpha might thus contribute to telomere maintenance, and might be regulated by ppRb.     

Enhancer of terminal gene conversion,a new mutation in Drosophila melanogaster that induces telomere elongation by gene conversion

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Terminally deleted chromosomes can be maintained for many generations. Thus, broken chromosome ends behave as real telomeres. It was previously shown that gene conversion may extend the broken ends. Here we found that the frequency of terminal DNA elongation by gene conversion strongly depends on the genotype. A dominant E(tc) (Enhancer of terminal gene conversion) mutation markedly increases the frequency of this event but does not significantly influence the frequency of HeT-A and TART attachment to the broken chromosome end and recombination between directly repeated sequences at the end of the truncated chromosome. The E(tc) mutation was mapped to the 91-93 region on chromosome 3. Drosophila lines that bear the E(tc) mutation for many generations have telomeres, consisting of HeT-A and TART elements, that are longer than those found in wild-type lines. Thus, the E(tc) mutation plays a significant role in the control of telomere elongation in D. melanogaster.     

Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila.

The Su(var)205 gene of Drosophila melanogaster encodes heterochromatin protein 1 (HP1), a protein located preferentially within beta-heterochromatin. Mutation of this gene has been associated with dominant suppression of position-effect variegation. We have cloned and sequenced the gene encoding HP1 from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with Drosophila melanogaster HP1 shows two regions of strong homology, one near the N-terminus (57/61 amino acids identical) and the other near the C-terminus (62/68 amino acids identical) of the protein. Little homology is seen in the 5' and 3' untranslated portions of the gene, as well as in the intronic sequences, although intron/exon boundaries are generally conserved. A comparison of the deduced amino acid sequences of HP1-like proteins from other species shows that the cores of the N-terminal and C-terminal domains have been conserved from insects to mammals. The high degree of conservation suggests that these N- and C-terminal domains could interact with other macromolecules in the formation of the condensed structure of heterochromatin.     

The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

SGS1 is required for telomere elongation in the absence of telomerase

In S. cerevisiae, mutations in genes that encode telomerase components, such as the genes EST1, EST2, EST3, and TLC1, result in the loss of telomerase activity in vivo. Two telomerase-independent mechanisms can overcome the resulting senescence. Type I survival is characterized by amplification of the subtelomeric Y' elements with a short telomere repeat tract at the terminus. Type II survivors arise through the abrupt addition of long tracts of telomere repeats. Both mechanisms are dependent on RAD52 and on either RAD50 or RAD51. We show here that the telomere elongation pathway in yeast (type II) is dependent on SGS1, the yeast homolog of the gene products of Werner's (WRN) and Bloom's (BLM) syndromes. Survival in the absence of SGS1 and EST2 is dependent upon RAD52 and RAD51 but not RAD50. We propose that the RecQ family helicases are required for processing a DNA structure specific to eroding telomeres.     

Rat homolog of PinX1 is a nucleolar protein involved in the regulation of telomere length

Human PinX1 involves in regulation of telomere length. Here, we describe the function of a rat homolog of PinX1. Rat PinX1 (rPinX1) was cloned from WB-F344, a rat hepatic stem-like epithelial cell. It encodes a protein of 331 amino acids with 70% homology to human PinX1 and 91% homology to mouse. Northern analysis revealed that rPinX1 is expressed in both somatic and germ tissues, most abundantly in heart, liver and testis. Co-localization with a nucleolar protein, fibrillarin, showed that rPinX1 resides in the nucleolus. Analysis of truncated mutants revealed that an internal K,E/D region seems to be important for nucleolar localization. A stable cell line expressing rPinX1 was established in NIH3T3, a mouse-transformed embryonic fibroblast cell line, and stable cells were subcultured for more than 150 population doublings. The growth of stable rPinX1 cells slowed down at late passages, and a fraction of these cells exhibited increased size and stained positively for senescence-associated β-galactosidase. Overexpression of rPinX1 in NIH3T3 cells resulted in gradual telomere shortening over successive passages. However, the telomeric 3′ overhang was not altered by PinX1 expression. This study demonstrates that a rat homolog of human PinX1 is a nucleolar protein, and that overexpression of rPinX1 induces cellular senescence and telomere shortening, but has no effect on 3′ overhang length. The function of PinX1 in regulating telomere length is conserved in rodents, and this study may provide insight into the mechanism by which a nucleolar protein can regulate telomere length.     

Sex Differences in Drosophila melanogaster Heterochromatin Are Regulated by Non-Sex Specific Factors

The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. We adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulates autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also binds to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also makes autosomal heterochromatin in XX flies dependent on roX and enhances the effect of Top2 mutations, suggesting a combinatorial action. We postulate that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.     

Evidence for a juvenile hormone receptor involved in protein synthesis in Drosophila melanogaster

The larval fat body of newly eclosed adults of Drosophila melanogaster was found to contain a single major binding protein specific for juvenile hormone (JH). Binding to this protein was saturable, of high affinity, and specific for JH III. The protein has a subunit molecular weight (Mr) of 85,000, as determined by photoaffinity labeling. The same or similar JH-binding protein was found in larval fat body and cuticle of third instar larvae and in male accessory glands and heads of newly eclosed adults. It was not found in several other tissues in adults. Male accessory gland cytosol from wild-type flies was found to contain a single binder with a dissociation constant (KD) of 6.7 nM for JH III; a binder in similar preparations from the methoprene-tolerant (Met) mutant had a KD value 6-fold higher. JH III stimulated protein synthesis in glands cultured in vitro, but this effect was reduced in Met flies as compared to wild-type flies, establishing a correlation between JH binding and biological activity of the hormone. In addition, glandular protein accumulation during the first 2 days of adult development was less in Met flies than in wild-type flies. These results strongly suggest that the binding protein we have identified mediates this JH effect in male accessory glands and thus is acting as a JH receptor.