HP1/Su(var)205: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Suppressor of variegation 205

Synonyms - heterochromatin protein 1 (hp1), HP1A

Cytological map position - 29A

Function - gene silencing - chromatin binding protein, DNA binding protein

Keywords - chromatin binding protein

Symbol - Su(var)205

FlyBase ID:FBgn0003607

Genetic map position - 2-31.1

Classification - chromo domain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Liu, Y. and Zhang, D. (2015). HP1a/KDM4A is involved in the autoregulatory loop of the oncogene gene c-Jun. Epigenetics[Epub ahead of print]. PubMed ID: 25945750
Summary:
The proto-oncogene c-Jun plays crucial roles in tumorigenesis, and its aberrant expression has been implicated in many cancers. Previous studies have shown that the c-Jun gene is positively autoregulated by its product. Notably, it has also been reported that c-Jun proteins are enriched in its gene body region. However, the role of c-Jun proteins in its gene body region has yet to be uncovered. HP1a is an evolutionarily conserved heterochromatin-associated protein, which plays an essential role in heterochromatin-mediated gene silencing. Interestingly, accumulating evidence shows that HP1a is also localized to euchromatic regions to positively regulate gene transcription. However, the underlying mechanism has not been defined. This study demonstrates that HP1a is involved in the positive autoregulatory loop of the Jra gene, the c-Jun homologue in Drosophila. Jra recruited the HP1a/KDM4A complex to its gene body region upon osmotic stress to reduce H3K36 methylation levels and disrupt H3K36 methylation-dependent histone deacetylation, resulting in high levels of histone acetylation in the Jra gene body region, thus promoting gene transcription. These results not only expand our knowledge towards the mechanism of c-Jun regulation, but also reveal the mechanism by which HP1a exerts its positive regulatory function in gene expression.

Pan, L., et al. (2015). Heterochromatin remodeling by CDK12 contributes to learning in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26508632
Summary:
Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. This study reports that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.

Yuan, K. and O'Farrell, P. H. (2016). TALE-light imaging reveals maternally guided, H3K9me2/3-independent emergence of functional heterochromatin in Drosophila embryos. Genes Dev 30: 579-593. PubMed ID: 26915820
Summary:
Metazoans start embryogenesis with a relatively naive genome. The transcriptionally inert, late-replicating heterochromatic regions, including the constitutive heterochromatin on repetitive sequences near centromeres and telomeres, need to be re-established during development. To explore the events initiating heterochromatin formation and examine their temporal control, sequence specificity, and immediate regulatory consequence, a live imaging approach was established that enabled visualization of steps in heterochromatin emergence on specific satellite sequences during the mid-blastula transition (MBT) in Drosophila. Unexpectedly, only a subset of satellite sequences, including the 359-base-pair (bp) repeat sequence, recruited HP1a at the MBT. The recruitment of HP1a to the 359-bp repeat was dependent on HP1a's chromoshadow domain but not its chromodomain and was guided by maternally provided signals. HP1a recruitment to the 359-bp repeat was required for its programmed shift to later replication, and ectopic recruitment of HP1a was sufficient to delay replication timing of a different repeat. The results reveal that emergence of constitutive heterochromatin follows a stereotyped developmental program in which different repetitive sequences use distinct interactions and independent pathways to arrive at a heterochromatic state. This differential emergence of heterochromatin on various repetitive sequences changes their replication order and remodels the DNA replication schedule during embryonic development.

Giauque, C.C. and Bickel, S.E. (2016). Heterochromatin-associated proteins HP1a and Piwi collaborate to maintain the association of achiasmate homologs in Drosophila oocytes. Genetics [Epub ahead of print]. PubMed ID: 26984058
Summary:
Accurate segregation of homologous chromosomes during meiosis depends on their ability to remain physically connected throughout prophase I. For homologs that achieve a crossover, sister chromatid cohesion distal to the chiasma keeps them attached until anaphase I. However, in Drosophila melanogaster wild-type oocytes, the 4th chromosomes never recombine and X chromosomes fail to cross over in 6-10% of oocytes. Proper segregation of these achiasmate homologs relies on their pericentric heterochromatin-mediated association, but the mechanism(s) underlying this attachment remains poorly understood. Using an inducible RNAi strategy combined with FISH to monitor centromere proximal association of the achiasmate FM7a/X homolog pair, this study analyzed whether specific heterochromatin-associated proteins are required for the association and proper segregation of achiasmate homologs in Drosophila oocytes. Upon knocking down HP1a, H3K9 methytransferases or the HP1a binding partner Piwi during mid-prophase, significant disruption of pericentric heterochromatin-mediated association of FM7a/X homologs was observed. Furthermore, for both HP1a and Piwi knockdown oocytes, transgenic co-expression of the corresponding wild-type protein is able to rescue RNAi-induced defects. Piwi is stably bound to numerous sites along the meiotic chromosomes, including centromere proximal regions. In addition, reduction of HP1a or Piwi during meiotic prophase induces a significant increase in FM7a/X segregation errors. The study presents a speculative model outlining how HP1a and Piwi could collaborate to keep achiasmate chromosomes associated in a homology dependent manner.

Roelens, B., Clemot, M., Leroux-Coyau, M., Klapholz, B. and Dostatni, N. (2016). Maintenance of heterochromatin by the large subunit of the replication-coupled histone chaperone CAF-1 requires its interaction with HP1a through a conserved motif. Genetics [Epub ahead of print]. PubMed ID: 27838630
Summary:
In eukaryotic cells, the organization of genomic DNA into chromatin regulates many biological processes, from the control of gene expression to the regulation of chromosome segregation. The proper maintenance of this structure upon cell division is therefore of prime importance during development for the maintenance of cell identity and genome stability. The Chromatin Assembly Factor 1 (CAF-1) is involved in the assembly of H3-H4 histone dimers on newly synthesized DNA and in the maintenance of a higher order structure, the heterochromatin, through an interaction of its large subunit with the heterochromatin protein HP1a. This study identified a conserved domain in the large subunit of the CAF-1 complex required for its interaction with HP1a in the Drosophila fruit fly. Functional analysis reveals that this domain is dispensable for viability but participates in two processes involving heterochromatin: position-effect variegation (PEV) and long range chromosomal interactions during meiotic prophase. Importantly, the identification in the large subunit of CAF-1 of a domain required for its interaction with HP1 allows the separation of its functions in heterochromatin related processes from its function in the assembly of H3-H4 dimers onto newly synthesized DNA.
Vo, N., Anh Suong, D. N., Yoshino, N., Yoshida, H., Cotterill, S. and Yamaguchi, M. (2016). Novel roles of HP1a and Mcm10 in DNA replication, genome maintenance and photoreceptor cell differentiation. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 27903903
Summary:
Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs. Proximity Ligation Assay (PLA) demonstrated that HP1a is in close proximity to DNA replication proteins including Mcm10, RFC140 and DNA polymerase 255 kDa subunit in S-phase. This was further confirmed by co-immunoprecipitation assay. The PLA signals between Mcm10 and HP1a are specifically observed in the mitotic cycling cells, but not in the endocycling cells. Interestingly, many cells in the posterior regions of eye imaginal discs carrying a double knockdown of Mcm10 and HP1a induced ectopic DNA synthesis and DNA damage without much of ectopic apoptosis. Therefore, the G1-S checkpoint may be affected by knockdown of both proteins. This event was also the case with other HP family proteins such as HP4 and HP6. In addition, both Mcm10 and HP1a are required for differentiation of photoreceptor cells R1, R6 and R7. Further analyses on several developmental genes involved in the photoreceptor cell differentiation suggest that a role of both proteins is mediated by regulation of the lozenge gene.
Strom, A. R., Emelyanov, A. V., Mir, M., Fyodorov, D. V., Darzacq, X. and Karpen, G. H. (2017). Phase separation drives heterochromatin domain formation. Nature [Epub ahead of print]. PubMed ID: 28636597
Summary:
Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). This study presents data that support the hypothesis that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments. Drosophila HP1a protein is shown to undergoes liquid-liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. It is concluded that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. It is proposed that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.
Teo, R. Y. W., Anand, A., Sridhar, V., Okamura, K. and Kai, T. (2018). Heterochromatin protein 1a functions for piRNA biogenesis predominantly from pericentric and telomeric regions in Drosophila. Nat Commun 9(1): 1735. Pubmed ID: 29728561
Summary:
In metazoan germline, Piwi-interacting RNAs (piRNAs) provide defence against transposons. Piwi-piRNA complex mediates transcriptional silencing of transposons in nucleus. Heterochromatin protein 1a (HP1a) has been proposed to function downstream of Piwi-piRNA complex in Drosophila. HP1a germline knockdown (HP1a-GLKD) leads to a reduction in the total and Piwi-bound piRNAs mapping to clusters and transposons insertions, predominantly in the regions close to telomeres and centromeres, resulting in derepression of a limited number of transposons from these regions. In addition, HP1a-GLKD increases the splicing of transcripts arising from clusters in above regions, suggesting HP1a also functions upstream to piRNA processing. Evolutionarily old transposons enriched in the pericentric regions exhibit significant loss in piRNAs targeting these transposons upon HP1a-GLKD. This study suggests that HP1a functions to repress transposons in a chromosomal compartmentalised manner.
Warecki, B. and Sullivan, W. (2018). Micronuclei formation is prevented by Aurora B-mediated exclusion of HP1a from late-segregating chromatin in Drosophila. Micronuclei formation is prevented by Aurora B-mediated exclusion of HP1a from late-segregating chromatin in Drosophila
Summary:
While it is known that micronuclei pose a serious risk to genomic integrity by undergoing chromothripsis, mechanisms preventing micronucleus formation remain poorly understood. This study investigate how late-segregating acentric chromosomes that would otherwise form micronuclei instead reintegrate into daughter nuclei by passing through Aurora B kinase-dependent channels in the nuclear envelope of Drosophila melanogaster neuroblasts. Localized concentrations of Aurora B preferentially phosphorylate H3(S10) on acentrics and their associated DNA tethers. This phosphorylation event prevents HP1a from associating with heterochromatin and results in localized inhibition of nuclear envelope reassembly on endonuclease and X-irradiation-induced acentrics, promoting channel formation. Finally, this study found that HP1a also specifies initiation sites of nuclear envelope reassembly on undamaged chromatin. Taken together, these results demonstrate that Aurora B-mediated regulation of HP1a-chromatin interaction plays a key role maintaining genome integrity by locally preventing nuclear envelope assembly and facilitating incorporation of late-segregating acentrics into daughter nuclei.
Jankovics, F., Bence, M., Sinka, R., Farago, A., Bodai, L., Pettko-Szandtner, A., Ibrahim, K., Takacs, Z., Szarka-Kovacs, A. B. and Erdelyi, M. (2018). Drosophila small ovary gene is required for transposon silencing and heterochromatin organisation and ensures germline stem cell maintenance and differentiation. Development. PubMed ID: 30389853
Summary:
Self-renewal and differentiation of stem cells is one of the fundamental biological phenomena relying on proper chromatin organisation. This study describes a novel chromatin regulator encoded by the Drosophila small ovary (sov) gene. sov was shown to be required in both the germline stem cells (GSCs) and the surrounding somatic niche cells to ensure GSC survival and differentiation. Sov maintains niche integrity and function by repressing transposon mobility, not only in the germline, but also in the soma. Protein interactome analysis of Sov revealed an interaction between Sov and HP1a. In the germ cell nuclei, Sov co-localises with HP1a, suggesting that Sov affects transposon repression as a component of the heterochromatin. In a position effect variegation assay, a dominant genetic interaction was found between sov and HP1a, indicating their functional cooperation in promoting the spread of heterochromatin. An in vivo tethering assay and FRAP analysis revealed that Sov enhances heterochromatin formation by supporting the recruitment of HP1a to the chromatin. A model is proposed in which sov maintains GSC niche integrity by regulating transposon silencing and heterochromatin formation.
BIOLOGICAL OVERVIEW

Suppressor of variegation 205 (here termed Heterochromatin protein 1, or HP1) is a chromosomally bound protein. It is not considered one of the originally described Polycomb group members, because it does not alter segment identity. Nevertheless, it shares many other properties with proteins of the Polycomb group.

HP1 is a nonhistone chromosomal protein primarily associated with the pericentric heterochromatin and telomeres in Drosophila. The molecular mechanism by which HP1 specifically recognizes and binds to chromatin is unknown. A study tested whether HP1 can bind directly to nucleosomes. It turns out that HP1 can bind both nucleosome core particles and naked DNA. HP1-DNA complex formation is length-dependent and cooperative but relatively sequence-independent. Histone H4 amino-terminal peptides bind to monomeric and dimeric HP1 in vitro. Acetylation of lysine residues has no significant effect on in vitro binding. The C-terminal chromo shadow domain of HP1 specifically binds H4 N-terminal peptide. Neither the chromo domain nor chromo shadow domain alone binds DNA; intact native HP1 is required for such interactions. Together, these observations suggest that HP1 may serve as a cross-linker in chromatin, linking nucleosomal DNA and nonhistone protein complexes to form higher order chromatin structures (Zhao, 2000).

To test whether HP1 can bind to nucleosomes, an electrophoretic mobility shift was performed assay using purified recombinant HP1 protein (rHP1) and labeled reconstituted nucleosome core particles. rHP1 significantly retards the electrophoretic mobility of reconstituted nucleosome core particles in a dose-dependent manner, indicating an HP1-nucleosome association. Significant effects on core particle mobility are obvious at 1 µM HP1 or at a molar ratio of 100:1 of HP1 over core particles. Prominent features of the nucleosome surface that could provide substrates for HP1 binding include the N-terminal histone tails and DNA. To test whether this binding depends on the unstructured histone tails that extend from the nucleosome core, the nucleosome electrophoretic mobility assay was repeated using trypsinized nucleosomes. Limited trypsin digestion of nucleosome cores selectively digests the N-terminal tails of core histones. Under these conditions, a significant fraction of the labeled DNA dissociates from the histone cores and migrates as free DNA. Interestingly, after trypsin digestion, a distinctive electrophoretic behavior is observed. The trypsinized core fraction contains somewhat more free DNA, and this material disappears with increasing HP1, while a distinctive slow mobility complex appears. The trypsinized core particles begin to disappear at higher HP1 concentrations than required for comparable shifts of intact cores, suggesting that trypsin digestion of core particles makes HP1 binding to cores less efficient. In addition, the disappearance of the free DNA band with increasing HP1 concentration suggests that HP1 binds free DNA as well as nucleosome cores. A direct comparison of intact and trypsinized cores shows that the trypsinized cores have significantly higher mobility than intact core particles, consistent with the expected loss of N-terminal tail peptides from the particles (Zhao, 2000).

To confirm HP1-DNA interaction, the ability of rHP1 alone, in the absence of nucleosomes, to shift the Cy5-labeled 146-bp DNA alone was tested. A mobility shift of the DNA fragment occures with increasing amounts of HP1 protein, indicating DNA binding. Furthermore, the abrupt appearance of complexes between 5 and 10 µM HP1 (between 500- and 1000-fold molar excess of HP1) suggests cooperativity in complex formation. To test for sequence or structural specificity for DNA in HP1-DNA binding, circular BlueScript plasmid DNA (pBS) or linear DNA fragments produced by AluI digestion of the pBS plasmid were included in the gel shift assay. Both the circular plasmid and the linear DNA fragments are able to eliminate the band shift of the Cy5-labeled 146-bp 5 S rDNA fragment. Thus, the HP1-DNA interaction is not sequence-specific, and HP1 binding does not require free DNA ends. HP1 complexes form with increasing efficiency as DNA length increases. While HP1-DNA interactions could substantially account for HP1-nucleosome binding, there appears to be some contribution of histone (and/or DNA conformation imposed by nucleosome wrapping) at high salt concentration (Zhao, 2000).

Side by side comparison of the mobility shifts observed with DNA and with nucleosome cores in the presence of HP1 emphasizes the distinct electrophoretic behaviors of these complexes. When the naked DNA and the nucleosome are run side by side in the gel shift assay, the pattern of band shift is different. While a small amount of material in the core fraction co-migrates with the HP1-DNA complex, reflecting the presence of histone-free DNA after core reconstitution, most of the HP1-dependent core complex migrates more slowly. This difference indicates that HP1 binds to nucleosome core particles instead of just stripping off the DNA from the nucleosome (Zhao, 2000).

Since HP1-nucleosome complexes appear somewhat more salt-resistant than HP1-DNA complexes and since the binding of HP1 to trypsinized cores appears less efficient than to intact cores, a role for histone tail binding in the HP1-nucleosome interaction was considered. The histone H4 N-terminal tails have been implicated in nucleosome assembly and the regulation of template accessibility in chromatin. They extend out from the nucleosome core and contain four acetylatable lysines (Lys5, Lys8, Lys12, and Lys16). In Drosophila, euchromatic histone H4 can be modified at any of these four lysines, whereas heterochromatic H4 is modified primarily at Lys12. The distinctive distribution of acetylated histone H4 isoforms suggests that HP1 may recognize and bind to heterochromatin through histone H4 tails specifically acetylated at K12. To test whether HP1 interacts with the H4 N-terminal tail and whether acetylation regulates this interaction, five synthetic 20-amino acid peptides based on the conserved H4 tail sequence were challenged to bind to HP1 in vitro. One peptide was nonacetylated, and the other four peptides were acetylated at one of four different lysines (Lys5, Lys8, Lys12, Lys16). These peptides were labeled with the Cy5 at their C-terminal cysteine residues. rHP1 was mixed with Cy5-labeled peptides, subjected to chemical cross-linking, and cross-linked proteins were resolved by SDS-PAGE. All five peptides are cross-linked to rHP1 with similar efficiency, suggesting that, although histone tails bind to rHP1, acetylation does not affect this interaction (Zhao, 2000).

In these experiments, peptide cross-linking was also observed to a protein of slower mobility with twice the apparent molecular weight of HP1. Since the rHP1 preparation is >95% pure, this was inferred to be dimeric HP1. Drosophila HP1 chromo shadow domain peptides can dimerize in solution, supporting this inference. To confirm that full-length HP1 also dimerizes in vitro, cross-linking was performed on purified rHP1 protein and the cross-linked products were analyzed by SDS-PAGE and Western blot with an HP1 antibody. Intact rHP1 indeed forms dimers and trimers in vitro. To identify which region of HP1 binds to the H4 peptides, an rHP1 sample was used in which the protein had been cleaved into two peptides. The identities of these two peptides as the N-terminal chromo domain and the C-terminal chromo shadow domain were established using a chromo domain-specific antibody. In the cross-linking assay, H4 peptides were found to be associated with the C-terminal chromo shadow domain (Zhao, 2000).

To test the domain specificity of DNA binding, three peptides from bacterial HP1 were used: an N-terminal peptide that contains the chromo domain, a C-terminal peptide that contains the chromo shadow domain, and an 84-amino acid peptide in the middle of the protein that links the two domains. None of these three peptides binds to DNA in vitro, indicating that an intact HP1 protein is required for this activity. Intact HP1 still binds DNA in the presence of chromo domain peptides or the central peptide, indicating that peptides do not compete with intact HP1 binding and that there is no contaminating inhibitor in the peptide fractions (Zhao, 2000).

The cross-linking assays provide clear evidence for an interaction between H4 N-terminal tail peptides and HP1. The cross-linking of histone H4 tails to HP1 dimers suggests that H4 tail binding occurs on a surface of the chromo shadow domain, distinct from that mediating chromo shadow domain dimerization. Gel mobility shift assays demonstrate binding by HP1 of both nucleosome core particles and naked DNA. Although the experiments cannot apportion the relative contributions of HP1-histone and HP1-DNA interactions, it appears that both contribute to the HP1-nucleosome association. The histone tails are apparently relatively unstructured in the nucleosome core and are thought to extend far enough beyond the core particle to permit contacts with the DNA wrapped on the surface and/or contacts with adjacent nucleosomes. The effect of trypsin digestion is consistent with a role for histone tails in HP1 binding to nucleosome cores. However, the overall similarity in salt sensitivity of the HP1 core particle and HP1-DNA interactions suggests that HP1-DNA interaction plays an important role in HP1-nucleosome binding (Zhao, 2000).

Binding of nucleosomes requires rHP1 concentrations in the low micromolar range. A rough estimate of HP1 concentration in Drosophila suggests that there is about one HP1 molecule per 15 nucleosomes in a third instar larval nucleus. Thus, normal nuclear HP1 levels should be in the low micromolar range as well. This suggests that the interactions measured are biologically relevant for a relatively abundant chromatin-binding protein whose binding is dynamic during the cell cycle (Zhao, 2000 and references therein).

Recombinant HP1 binds to eukaryotic (5 S rDNA) and prokaryotic DNA (pBS plasmid and lac operon cAMP receptor protein binding site) sequences, suggesting a lack of a strong sequence preference in HP1 binding to DNA and nucleosomes. While it makes sense that HP1 could interact with DNA and nucleosomes, a general and sequence-nonspecific affinity for DNA or nucleosomes cannot explain the relatively restricted chromosomal distribution of HP1 in vivo. Presumably, there are other HP1-interacting factors that confer additional specificity on HP1 binding, serving to target or stabilize HP1 binding in heterochromatin. In addition, hyperphosphorylation of HP1 correlates with heterochromatin assembly, and phosphorylation by casein kinase II is required for efficient heterochromatin targeting. These observations, considered in light of the results reported here, suggest that HP1 phosphorylation could act by regulating HP1-nucleosome binding (Zhao, 2000).

The apparent cooperativity in HP1-DNA complex formation is consistent with a model that heterochromatin complexes spread from an 'initiator' in a cooperative and sequence-independent mechanism. When this spreading occurs across a rearrangement breakpoint, it can result in heterochromatic position effect silencing. Virtually any euchromatic gene can be silenced by heterochromatin, and genes lying hundreds of kilobases from a breakpoint may be silenced, arguing that heterochromatin complexes are relatively efficient and promiscuous in their ability to form higher order chromatin structures. The apparent sequence neutrality and cooperativity of HP1-DNA complexes observed here may underlie the in vivo properties of the HP1-dependent heterochromatin (Zhao, 2000).

Neither the chromo domain, the chromo shadow domain, nor the middle region of HP1 is sufficient for DNA binding, even when they are mixed together. The overall negative surface charge of the chromo domain of the mouse HP1 family protein MOD1 makes it unlikely that the chromo domain alone binds to DNA or RNA. The findings presented here are consistent with this prediction. It seems that in order to bind DNA, the intact HP1 protein is absolutely required. A straightforward model suggests that several different regions of HP1 are required for DNA binding. The binding activity is weak for each individual region and can not be detected in gel shift assay, and the regions have to be in a specific spatial relationship in order to bind to DNA with high affinity. By this model, each chromo domain may contribute to DNA binding, and both of them have to be present in the same molecule in order to 'clip' HP1 to DNA. Another possibility is that HP1 self-association is required for DNA binding: if the self-association and DNA binding domains in HP1 belong to different regions of the protein, both would have to be present to see DNA binding activity. It cannot be ruled out, however, that truncation of full-length HP1 causes misfolding of a single, discrete DNA binding domain (Zhao, 2000).

The archial chromosomal protein Sac7d possesses a folded structure similar to the chromo domain fold. Sac7d protein binds the minor groove of DNA as a monomer through hydrophobic interactions between bases and sugars in the DNA and a triple-stranded-sheet in Sac7d. If HP1 chromo and chromo shadow domains were to bind weakly to DNA in an analogous fashion, the inability of each domain to bind separately could be rationalized as a requirement for cooperativity conferred by tandem chromo domains. Cooperative binding would also be enhanced through dimerization. Dimerization of a mouse chromo shadow domain occurs through the C-terminal-helix of the chromo domain fold. If Drosophila HP1 dimerizes by a similar mechanism, this would leave the triple-stranded-sheet of each dimer partner free to interact with DNA. Such interactions could explain both the delocalized DNA binding sites in HP1 and the cooperativity observe for DNA binding in solution. In vivo, such cooperativity could facilitate the condensation of nucleosomal DNA in heterochromatin (Zhao, 2000 and references therein).

The data presented here demonstrate that Drosophila HP1 binds DNA and nucleosomes directly. A human HP1 family protein, HP1hs, has also been shown to bind to DNA directly. HP1 is implicated as a fundamental component of the protein-DNA complex in heterochromatin. This inference is consistent with cytological evidence that cis-spreading of heterochromatic silencing in chromosome rearrangements is accompanied by the spreading of HP1-associated chromatin into the silenced chromosomal region (Zhao, 2000 and references therein).

A recent estimate of HP1 concentration in Drosophila suggests that there are roughly three molecules of HP1 per nucleosome within polytene heterochromatin. While this estimate is crude, it is consistent with approximately stoichiometric binding of HP1 dimers to nucleosomes throughout heterochromatin. Such binding could interfere with nucleosome sliding and transient nucleosome displacement required for gene activation or with enhancer-promoter communication in euchromatic genes. This model can account for the reported regularization of nucleosome arrays in a heat shock gene silenced by heterochromatin (Zhao, 2000).

Several nonhistone proteins have been implicated in HP1 interactions. Among these HP1-binding proteins, some may act as structural proteins or regulators to target the binding of HP1 to heterochromatic regions instead of generally throughout the chromosomes [e.g. SU(VAR)3-7, Suppressor of variegation 3-9, lamin B receptor]. Others may be molecular machines or regulators that could be recruited by HP1 or other heterochromatin proteins to heterochromatic environments to perform certain physiological activities [e.g. ORC complex, chromatin assembly factor 1 (CAF-1), TIF1, and TIF1]. Dimerization of HP1 and the cooperative binding of HP1 to DNA and nucleosomes suggests a role for HP1 in condensing chromatin to form a higher order structure. An analogous 'drawstring' model has been proposed to mediate enhancer-promoter interactions in euchromatin. HP1 dimerization or polymerization may facilitate the condensation of heterochromatin into a structure inhospitable to euchromatic gene expression, accounting for heterochromatic position effect silencing. The requirement by heterochromatic genes for HP1 to maintain normal euchromatic gene expression may reflect the evolution of heterochromatic genes to require HP1-mediated condensation to maintain their functional organization. The data suggest that HP1 serves as a bifunctional cross-linker between nucleosome and nonhistone protein complexes to organize higher order chromatin, mediating the silencing of euchromatic genes and the expression of heterochromatic genes (Zhao, 2000).

Polytene chromosomes are found in the salivary glands of Drosophila third instar larvae. These special chromosomes exhibit two kinds of chromosomal staining. The heterochromatic areas, having few active genes, are condensed, while the euchromatic areas, rich in active genes, are spread out and especially thick. They display over a thousand dark staining bands alternating with lighter staining regions. The DNA of the euchromatic areas has been duplicated 1000 times over, while the DNA of heterochromatic areas is underreplicated.

Heterochromatin is found in either an alpha or a beta form. Alpha-heterochromatin is a small, densely staining mass in the middle of the chromocenter. Known also as mitotic heterochromatin it does not replicated in polytene chromosomes and consists overwhelmingly of highly repetitive DNA sequences. In general the 50 million base pairs of the mitotic heterochromatin is transcriptionally inert but by genetic criteria does contain a few dozen ordinary genes. In contrast to the alpha form, beta-heterochromatin is not visible in mitotic chromosomes and only appears as a diffuse mesh-like chromatin mass in polytene chromosomes. The beta-heterochromatin of the X-chromosome has a demonstrated gene density little different from that of an average euchromatin location (Miklos, 1991 and references).

HP1 localizes preferentially to the beta-heterochromatic areas of polytene chromosomes, in the so called chromocenter. Whether or not the alpha-heterochromatin is stained cannot be ascertained, as it represents such a small percentage of the chromocenter mass. Since most teleomeric regions stain for HP1, there are suggestions that telomeric regions might be functionally heterochromatin. The distribution pattern of HP1 does not follow the distribution of any known satellite DNA but rather follows very closely that of the clone u family of middle repetitious sequences (James, 1989 and references).

The heterochromatin protein 1 prevents telomere fusions in Drosophila

Telomeres are specialized DNA-protein complexes at the ends of linear chromosomes that ensure the stability of eukaryotic genomes. HP1 associates with Drosophila telomeres, and functions to prevent telomeric fusion. HP1 is a constant feature of the telomeres of interphase polytene and mitotic chromosomes. This localization does not require the presence of telomeric retrotransposons, since HP1 is also detected at the ends of terminally deleted chromosomes that lack these elements. Importantly, larvae expressing reduced or mutant versions of HP1 exhibit aberrant chromosome associations and multiple telomeric fusions in neuroblast cells, imaginal disks, and male meiotic cells. This work provides evidence that HP1 plays a functional role in mediating normal telomere behavior in Drosophila (Fanti, 1998b). The assertion that HP1 does not remain associated with chromosomes during the mitotic process (Kellum, 1995a) are difficult to reconcile with the Pak (1997) and Fanti (1998b) studies, both of which show HP1 staining in the heterochromatin of metaphase chromosomes (Fanti, 1998b).

Telomeres play a protective role by preventing loss of terminal sequences during DNA replication and by preventing chromosome fusions. Telomeres, by their interaction with both the nuclear envelope and nuclear matrix, are also involved in determining the dynamic spatial order of chromosomes during mitotic and meiotic cycles. Moreover, telomeres can exert position effects (TPE) on gene expression, a property shared with heterochromatin. The existence of telomeres was postulated for the first time by Herman J. Muller to account for the failure to recover terminally deleted chromosomes after X-irradiation in Drosophila melanogaster. He observed that the recovered chromosomes are either capped by telomeric fragments from other chromosomes or are involved in other rearrangement breakpoints. However, terminal deletions have since been recovered in Drosophila. Experiments performed by Barbara McClintock provided early evidence for the critical role that telomeres play in chromosome behavior in maize. McClintock showed that chromosomes that lack a telomere fuse, generate a dicentric bridge during mitosis, and initiate a chromosome breakage-fusion-bridge cycle (Fanti, 1998b and references).

A large number of studies have shown that the telomeres of eukaryotes are usually composed of conserved short tandemly repeated GC-rich sequences. This sequence conservation reflects a common mechanism for telomere synthesis. This mechanism involves the activity of telomerase, a ribonucleoprotein DNA polymerase that compensates for the loss of terminal sequences at every replication round by adding short tandem GC-rich sequences onto the chromosome end. Several lines of evidence indicate that these telomeric tandem repeats are essential for chromosome stability. For example, yeast chromosomes lacking telomeric DNA are lost. Moreover, terminal deletions have been recovered in several species and in all the cases their ends have been healed by the addition of telomeric sequences (Fanti, 1998b and references).

The telomeres of Drosophila represent a case dramatically different from other well studied organisms, at least as regards the replication functions. The Drosophila telomeres contain arrays of mobile retrotransposon-like elements called HeT-A and TART. It has also been shown that telomeres contain other sequences called TAS (telomere associated sequences). However, all these elements are dispensable for chromosome stability. Terminal deletions in Drosophila have been recovered. Their analysis has shown that in many cases their ends completely lack all these elements, and consequently these chromosomes continue to lose terminal DNA sequences. Nevertheless, these broken chromosomes behave as capped chromosomes in that they are stably transmitted through many generations. It has been shown that occasionally the HeT-A and TART elements transpose to the receding ends of the broken chromosomes. These observations suggest that in Drosophila these elements could be involved in the essential function of telomere elongation and that the telomere capping and replicating functions could be separate. Thus it appears that, while the replication function of the telomere could depend on DNA, the capping function is probably attributable to one or more proteins. Recently, it has been shown that UbcD1, encoding a class I ubiquitin-conjugating (E2) enzyme, causes frequent telomere-telomere attachments during both mitosis and male meiosis in Drosophila (Cenci, 1997). These results suggest that proper telomere behavior requires a ubiquitin-mediated proteolysis and reinforces the idea that the chromosome capping requires a protein component. However, candidate telomeric protein remains to be identified in Drosophila (Fanti, 1998b and references).

Although past works have concentrated on the role of HP1 in heterochromatin formation and gene expression, the localization of HP1 to other chromosomal locations, namely specific euchromatic regions and to telomeric regions, has been noted. To test a possible involvement of this protein in telomeric function, a detailed cytological analysis of HP1 distribution in telomeres of both mitotic and polytene chromosomes was carried in six different wild-type strains of Drosophila and in strains carrying stable terminal deletions. An examination was also carried out to see whether mutations in HP1 affect the ability of the protein to recognize telomeres and influenced telomere behavior. The results of this analysis have shown that HP1 is a stable component of all telomeres in Drosophila, including the ends of stable terminal deletions. Moreover, its absence in mutant cells causes multiple telomere-telomere fusions and results in a striking spectrum of abnormal chromosome configurations (Fanti, 1998b).

How does HP1 recognize the telomere? The molecular determinant that is responsible for the localization of HP1 to the heterochromatin or to the telomere is not known. The studies performed so far seem to exclude the DNA binding properties of HP1. Instead, a conserved amino acid sequence motif, called chromo domain, has been identified and is postulated to mediate interactions of HP1 with other chromosomal proteins. Cytological analysis of the effect of a Su(var)2-502 point mutation in the chromo domain reveals that the chromo domain may be dispensable for telomeric localization and function. Although the possibility may not be completely excluded that HP1 protein might bind terminal DNA in nonsequence-dependent manner, the simplest explanation is that an as yet unidentified telomere binding protein recruits HP1. At present, there are not candidate proteins that could suggest how HP1 might be recruited at the telomeres. It has been recently shown that both the Drosophila origin recognition complex (ORC) (Pak, 1997) and the Su(var)3-7 (Céard, 1997) proteins preferentially associate with heterochromatin and colocalize with HP1 by forming a physical complex. However, the immunostaining of polytene chromosomes with a DmORC and Su(var)3-7 antisera have indicated a lack of association to the telomeres (Cléard, 1997 and Pak, 1997) (Fanti, 1998b and references).

Recent data offer some hints suggesting that HP1 may also mediate the association of both the heterochromatin and telomeres with the inner nuclear membrane. It has been recently shown that human chromo domain proteins homologous to Drosophila HP1 interact with the lamin B receptor (LBR), an integral protein of the inner nuclear membrane (Ye, 1996 ). The same protein also interacts with the Drosophila HP1 in a yeast two-hybrid assay (Shaffer et al. cited in Elgin, 1996). Intriguingly, it has been recently found that the UbcD1 gene, encoding a ubiquitin-conjugating enzyme, is required for a normal telomere behavior (Cenci, 1997). Mutations at this locus cause multiple telomeric associations. However, since the telomeric fusions are resolved during mitotic anaphase, this suggests that in these mutants the telomeres remain connected by proteinaceous bridges rather than by DNA fusions. Thus, the most plausible hypothesis is that the telomeres are normally associated during interphase by UbcD1 target proteins and that in UbcD1 mutants that fail to degrade these proteins, the telomeric associations are maintained after interphase (Cenci, 1997). Two main features of the Su(var)2-5 mutants seem to exclude HP1 as a UbcD1 direct target. (1) HP1 is a component of telomeres whose absence causes telomeric fusions. (2) The existence of HP1-induced telomeric fusions, not resolved in anaphase, suggests a different mechanism of action that may involve DNA end fusion or very strong proteinaceous bridges. Among many other possibilities, it is not unreasonable to suppose that UbcD1 can be involved in degradation of HP1-interacting proteins, like the lamin B receptor, that mediate the ordered interaction of telomeres with other structures like the inner nuclear membrane (Fanti, 1998b).

The Drosophila histone variant H2A.V works in concert with HP1 to promote kinetochore-driven microtubule formation

Unlike other organisms that have evolved distinct H2A variants for different functions, Drosophila melanogaster has just one variant which is capable of filling many roles. This protein, H2A.V, combines the features of the conserved variants H2A.Z and H2A.X in transcriptional control/heterochromatin assembly and DNA damage response, respectively. This study shows that mutations in the gene encoding H2A.V affect chromatin compaction and perturb chromosome segregation in Drosophila mitotic cells. A microtubule (MT) regrowth assay after cold exposure revealed that loss of H2A.V impaired the formation of kinetochore-driven (k) fibers, which could account for defects in chromosome segregation. All defects were rescued by a transgene encoding H2A.V that lacked the H2A.x function in the DNA damage response, suggesting that the H2A.Z (but not H2A.X) functionality of H2A.V was required for chromosome segregation. Loss of H2A.V weakened HP1 localization, specifically at the pericentric heterochromatin of metaphase chromosomes. Interestingly, loss of HP1 yielded not only telomeric fusions but also mitotic defects similar to those seen in H2A.V null mutants, suggesting a role for HP1 in chromosome segregation. H2A.V precipitated HP1 from larval brain extracts indicating that both proteins were part of the same complex. Moreover, the overexpression of HP1 rescued chromosome missegregation and defects in the kinetochore-driven k-fiber regrowth of H2A.V mutants indicating that both phenotypes were influenced by unbalanced levels of HP1. Collectively, these results suggest that H2A.V and HP1 work in concert to ensure kinetochore-driven MT growth (Verní, 2015).

This study provides compelling evidence that H2A.V, the Drosophila histone H2A variant, plays an important and unanticipated role during Drosophila mitosis. The cytological characterization of H2A.V810 mutant larval brain chromosomes revealed that loss of H2A.V has an impact on chromosome organization and cell proliferation, which is consistent with previous results on a role of this histone variant in chromatin remodeling and heterochromatin organization. This study also demonstrates that a significant proportion of H2A.V mutant cells fails to complete mitosis and contains chromosomes that remain scattered across the spindle (pseudo anaphase or PA) due to failed metaphase plate alignment. Similar effects have been previously described in Drosophila S2 cells depleted by RNAi of either kinetochore proteins, augmin components or splicing factors. However unlike those S2 interfered cells, which exhibit PAs with long spindles, H2A.V810 mutant cells have PA (premature- or pseudo-anaphase) spindles that appear similar to wild type anaphases. The reason why H2A.V mutant cells are not elongated is unclear, but it may depend on the different cellular systems employed in the different studies. Intriguingly, the presence of PAs in H2A.V810 mutants indicates for the first time that Drosophila H2A.V is also necessary for chromosome segregation growth (Verní, 2015).

Interestingly, the results from both Dgt6 immunolocalization and spindle microtubule re-growth assay following cold-induced MT depolymerization in mitotic neuroblasts reveal that H2A.V might be involved in the organization of kinetochore-driven, k-fibers microtubule bundles that attach sister kinetochores to spindle poles. However, it is believed that defects in the organization of k-fibers are not a consequence of the reduction of Dgt6. Recent studies demonstrated that Wac, a newly discovered component of Augmin complex, is required for spindle formation in S2 cells but is dispensable for somatic mitosis. In fact, a wac deletion mutant was viable and displayed only weak defects in brain cell divisions, suggesting that the components of Augmin complex (including Dgt6) might have non essential roles in spindle assembly growth (Verní, 2015).

It has been previously reported that defective k-fiber formation and elongation disrupt chromosome segregation and spindle formation in Drosophila cells. The results, which are in line with this finding, indicate that a specific chromatin organization is also necessary to ensure a proper spindle assembly. It is speculated that the observed PAs are a result of improper organization of k-fibers, and that PAs fail to complete mitosis, thus reducing in part the frequency of anaphases in H2A.V810 mutants. It is also plausible that persistent chromosome misalignment leads to a mitotic arrest of these cells, which in turn could explain the presence of H2A.V810 mutant cells with overcondensed chromosomes. However, while in a previous study, the presence of PAs was always associated to a strong increase of mitotic index (MI), the current mutants the MI did not change. One explanation is that the reduction of anaphase frequency in H2A.V810 mutants (20%) is not as dramatic as that reported for Dgt6-depleted S2 cells (50%) and therefore it unlikely affects mitotic progression. An alternative explanation is that loss of H2A.V might affect the regulation of G2-M and/or M-A cell cycle checkpoints thus preventing a metaphase arrest. Further investigations are required to verify this hypothesis. It is worth noting that, although a role for H2A.Z in chromosome segregation has been previously documented in human and yeast cells, the current data provide the first evidence of an potential involvement of H2A.Z in the organization of k-fibers growth (Verní, 2015).

This study also provides unanticipated molecular evidence that H2A.V interacts directly or indirectly with HP1, confirming that both proteins are part of same complex. It is intriguing that the H2A.V-HP1 interaction depends on the HP1 CD domain, which also binds H3K9me2/3 and mediates heterochromatin formation. This supports the existence of a cascade of events that requires the recruitment of H2A.V and different histone modifications for the establishment of heterochromatin. Yet, the reason why depletion of H2A.V causes a direct loss of HP1 and particularly during mitosis is unclear. Nonetheless, as HP1 overexpression in H2A.V mutant cells prevents PA formation, it is speculated that a H2A.V-dependent stabilization/localization of HP1 at centromeric region is essential to ensure proper chromosomal behavior growth (Verní, 2015).

Previous studies have shown that H2A.Z alters the nucleosomal surface, thus enabling preferential binding of HP1a to condensed higher chromatin structures 44RIDcit0044. It is conceivable then that the H2A.V-HP1 interaction is favored by the condensation of pericentric chromatin fiber in metaphase. Alternatively, these interactions may be encouraged by metaphase-specific posttranslational modifications of H2A.V, HP1 or other interacting proteins. Indeed, it has been proposed the mechanism underpinning HP1 recruitment on mitotic chromosomes might be different from that in interphase. Still, little is known about the factors required for specific localization of HP1 at mitotic centromeres save for a few discoveries. Human HP1α binding to INCENP, for instance, has been demonstrated as necessary for HP1α targeting to mitotic centromeres. It is believed that H2A.V may play a similar role in mediating HP1 binding, but how this takes place remains to be seen growth (Verní, 2015).

This functional characterization of H2A.V has also unveiled the role of Drosophila HP1a in the assembly of the mitotic spindle. The results indicate that the loss of HP1 yields defects in the kinetochore-driven k-fiber organization, which can in turn compromise chromosome segregation thus generating PAs. Past studies have shown that HP1a contributes to chromosome segregation and centromere stability in a variety of organisms including mammals, but the mechanism is still not completely understood. HP1 is known to interact with components of the centromere and the kinetochore complex, providing targets to begin understanding how downregulation or mislocalization of HP1 result in mitotic defects. It has also been reported that in contrast to Swi6 in S. pombe, the correct localization of HP1 is not required for the recruitment of cohesins to centromeric regions in mammals. Yet, HP1a seems to help in protecting cohesins from degradation by recruiting the Shugoshin protein. This study has highlighted an additional function of HP1 during chromosome segregation, one which depends on interaction with H2A.V and is required to regulate k-fiber organization. These results thus provide further evidence of a functional versatility of HP1 that is likely conserved also in mammals growth (Verní, 2015).

Chromatin state changes during neural development revealed by in vivo cell-type specific profiling

A key question in developmental biology is how cellular differentiation is controlled during development. While transitions between trithorax-group (TrxG) and polycomb-group (PcG) chromatin states are vital for the differentiation of ES cells to multipotent stem cells, little is known regarding the role of chromatin states during development of the brain. This study shows that large-scale chromatin remodelling occurs during Drosophila neural development. The majority of genes activated during neuronal differentiation are silent in neural stem cells (NSCs) and occupy black chromatin and a TrxG-repressive state. In neurons, almost all key NSC genes are switched off via HP1-mediated repression. PcG-mediated repression does not play a significant role in regulating these genes, but instead regulates lineage-specific transcription factors that control spatial and temporal patterning in the brain. Combined, these data suggest that forms of chromatin other than canonical PcG/TrxG transitions take over key roles during neural development (Marshall, 2017).

Using Targeted DamID, this study has compiled cell type-specific DNA-binding maps of key chromatin proteins in NSCs and neurons, and used these to investigate the chromatin transitions that occur during neural development in the Drosophila brain. The data reveal unexpected roles for chromatin states in this context (Marshall, 2017).

The Black chromatin state—a silent state lacking PcG or HP1a constituents—was identified in cultured embryonic Kc167 Drosophila cells but its role during development was previously unknown. The current data clearly demonstrate the importance of this form of chromatin and the novel TrxG-repressive state during development in vivo, with both states covering the majority of genes that are silent in NSCs but active in neurons. It is not yet known whether Black chromatin is actively repressive and requires chromatin remodelling to become active, or merely passively silent, requiring only the binding of appropriate transcription factors to activate transcription. Enrichment of the linker histone H1 in this state would suggest that chromatin is less accessible. Black chromatin in Drosophila and other organisms has been reported also to contain varying levels of the H3K27me2 mark, which may contribute to repression, and which suggested a link to PcG-associated chromatin. Interestingly, however, very few transitions were observed between Black and PcG chromatin: between NSCs and immature neurons, <0.5 Mb of DNA transitioned between Black and PcG states in either direction and the PcG-associated states separated strongly from Black chromatin in these models. If Black chromatin does contain H3K27me2 in the brain, there would appear to be little conversion between H3K27me2 and H3K27me3 during neural development (Marshall, 2017).

In addition to many genes that transitioned from Black chromatin in NSCs to an active TrxG state in neurons, a subset of genes was observed that transitioned between Black and non-TrxG active chromatin. These genes were strongly enriched for those encoding metabolic function (in accordance with previous reports of housekeeping and metabolism genes being associated with the non-TrxG active state) and were activated without the presence of Brm. These transitions may be similar to those recently described in nematodes and Drosophila imaginal discs for the activation of developmentally regulated genes without TrxG-associated chromatin marks (Marshall, 2017).

The TrxG-repressive state describe in this study is intriguing. This state is enriched for Brm binding in transcriptionally silent chromatin, and covers a large number of neuronal genes in NSCs. PcG-independent repression via the REST complex in ES cells has been reported to be associated with neuronal genes. These genes were found to be present in a poised state that included the TrxG-associated H3K4me3 mark, a situation very similar to the TrxG-repressive state that was observed in this study (Marshall, 2017).

In neural development, little role was observed for PcG repression in controlling broad cell fate, but instead a role was identifed in specifying different neuronal lineages. Specific PcG repression/TrxG activation has previously been described for lineage-specific transcription factors within the mushroom body of the brain. A mixed PcG chromatin state has also been previously associated with spatially compartmentalised transcription factors in ChIPseq of whole wing discs, although that study was not cell type-specific. This last study speculated that such a mixed state might be indicative of bivalent chromatin in Drosophila. However, both the former study and the current found concomitant occupancy of RNA Pol II on gene bodies in the PcG-mixed state, an observation at odds with silent bivalent chromatin. Combined with a significantly enriched association with lineage-specific transcription factors from existing RNA-seq data and immunofluorescence studies, the current data suggest that this chromatin state is a genuine mixture between PcG repression in some lineages and TrxG activation in other lineages. No evidence was seen in these data sets of true bivalent chromatin as reported in ES cells (strong binding of both PcG and TrxG components with no transcription) (Marshall, 2017).

In conclusion, these data present a picture of neural development that differs from that observed during the differentiation of ES cells in culture. Although lineage-specific transcription factors that generate neuronal diversity are regulated through PcG repression, the majority of genes activated in neurons follow different chromatin transitions. These genes are present in the silent Black chromatin state and a novel TrxG-repressive chromatin state in NSCs and transition to a TrxG permissive chromatin state in immature neurons. Furthermore, this study demonstrated that almost all NSC identity and cell cycle genes are repressed in HP1-associated chromatin in neurons, and that this repression occurs concomitantly with a wide-scale accumulation of HP1-associated binding across the genome. Although canonical PcG/TrxG transitions are vital during early development, these data suggest that other forms of chromatin take over important regulatory roles during neural development (Marshall, 2017).

dadd1 and dxnp prevent genome instability by maintaining HP1a localization at Drosophila telomeres

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 (Chavez, 2017).

Maintenance of chromosomal stability is an essential feature required for correct cell proliferation and overall cell survival. Specialized heterochromatic regions, such as telomeric and pericentromeric regions, are required for correct chromosome segregation. The DNA sequences of these regions are rich in repetitive sequences, such as satellite DNA, and may also contain different kinds of LTR and non-LTR retrotransposons. These sequences are very promiscuous and in principle could align with similar sequences in other chromosomes, generating chromosomal aberrations. In somatic cells, the transcription of these sequences must be silenced; in particular, retrotransposon sequences must be silenced to avoid retrotransposition mechanisms that could lead to the integration of these sequences into other genomic regions. Several protein complexes participate in the establishment and maintenance of heterochromatin, and these complexes include proteins such as HP1a (CBX5 in mammals), which have specialized domains that recognize different histone post-translational modifications. In particular, HP1a is able to recognize the H3K9me3 mark on nucleosomes. The recognition of this mark and the oligomerization of HP1a are steps required for the establishment of a heterochromatic state. In the last 10 years, different laboratories have reported HP1a protein interactors obtained through several experimental approaches. The ATRX protein has emerged as one of these interacting factors. In mammalian cells, ATRX recruits CBX5 to telomeric regions, and both proteins cooperate to maintain pericentric heterochromatin. In Drosophila, the homolog of the ATRX gene is divided into the ADD domain of the protein, encoded by the dadd1 gene, which recognizes the trimethylated form of lysine 9 of the histone 3, and the ATPase-SNF2 domain, encoded by the dxnp gene (also called datrx). The dadd1 gene encodes three isoforms generated by differential splicing (López-Falcón, 2014). The three isoforms contain the ADD domain, but two of these isoforms contain three extra MADF domains that are not present in the human ATRX gene (López-Falcón, 2014). It was previously reported that the products of these genes interact physically and genetically (López-Falcón, 2014). Additionally, the dxnp gene encodes two protein isoforms, the dXNPL isoform which has a heterochromatic localization and the dXNPs isoform which is observed at heterochromatic and euchromatic regions in polytene chromosomes. The fact that the main domains of ATRX are divided in Drosophila provides a useful tool to study the possible specific roles of these domains. The present study analyzed in further detail the contribution of each of these domains to the maintenance of heterochromatin. Organisms carrying mutant alleles of both dadd1 and dxnp have up to threefold more chromosomal aberrations, including telomeric fusions, than wild-type individuals. The telomeric fusion phenotype was analyzed in more detail. First, it was demonstrated that the dadd1 and dxnp proteins play an important role in the maintenance of the silenced state of the telomeric HTT array in somatic cells, preventing aberrant transcription and integration of the retrotransposons, and second, they were found to be necessary to maintain HP1a localization at the telomeric regions. Finally, through rescue experiments, the dadd1 isoforms were found to play a differential role in the maintenance of telomeric heterochromatin. These experiments helped determine that the dAdd1a isoform is responsible for targeting HP1a to the telomeric regions (Chavez, 2017).

Recent studies have described more than 100 HP1a interactors through different methodologies; furthermore, the importance of some of these interactors in the maintenance of HP1a immunolocalization-specific patterns has been proposed and recently assayed in cultured cells. HP1a was described in the late 1980s as one of the major components of heterochromatin, and since that time, many HP1a variants have been described. The main characteristic of these proteins is the presence of a chromodomain that is capable of recognizing primarily the di/tri-methylated state of H3K9. The research done on HP1a points towards a role for the protein as part of several protein complexes, each of which likely maintains different heterochromatic domains (Chavez, 2017).

The ATRX protein has been identified as a protein involved in the maintenance of heterochromatic regions, mainly pericentric and telomeric regions rich in repetitive sequences and transposable retro-elements. Vertebrate ATRX has two important domains, the SNF2 (helicase/ATPase) domain and the ADD (H3K9me3/H3K4 unmodified recognition) domain. In Drosophila, these domains are separated and encoded by two different genes: the dadd1 gene encodes three protein isoforms derived from alternative splicing events, which conserve an ADD domain, and the dxnp gene, which encodes two protein isoforms that conserve an SNF2 domain. The dadd1 and dxnp proteins interact physically and they co-localize with HP1a in several heterochromatic regions, including the chromocenter (López-Falcón, 2014). The independent study of these proteins in Drosophila can help in understanding the different roles played by these domains (Chavez, 2017).

The results obtained in the present study demonstrate the cooperation between the ADD and the SNF2 domains to maintain chromosomal stability and in the targeting of the HP1a protein to telomeric domains (Chavez, 2017).

The dadd1 and dxnp proteins are HP1a interactors but clearly some of the protein isoforms may play different roles than HP1a in the regulation of the different telomeric domains. The chromosomal aberrations observed in the dxnp and dadd1 mutants are not just restricted to telomeric fusions and are more generalized than the ones reported for HP1a. Mutations in Su(var)205 often give rise to telomeric associations (in salivary glands) and telomeric fusions (in mitotic chromosomes), but mutations in this protein also affect HTT array retrotransposon expression, leading to chromosomal instability; thus, HP1a regulates both the CAP and the HTT array. The phenotypes observed for the dxnp and dadd1 alleles, in addition to the telomeric fusions, also include decondensations, in which the chromosome arms are longer, and centromeric fusions. These results are consistent with reports in the literature, as these proteins have a wide genomic distribution and have been shown to mediate the suppression of position effect variegation using pericentromeric reporters. An interesting issue raised by the current results is the fact that there seems to be a differential requirement for the specific isoforms of these proteins in selected heterochromatic compartments and in the prevalence of certain phenotypes analyzed. For instance, the dxnp2 allele, which affects both dxnp isoforms, in combination with a null dadd1 background, presented more telomeric fusions than any other of the genotypes analyzed; the same combination, but with the dxnp3 allele, which affects only the long dxnp isoform (dXNPL), did not show as many telomeric fusions. These data suggest that the short (dXNPs) isoform has an important role in preventing these types of chromosomal aberrations. Additionally, the frequency of telomeric fusions observed in dadd1 and dxnp mutants is lower than the previously reported frequency when different allelic combinations of Su(var)205 gene were assayed. One possible explanation for the differences observed in the frequency of telomeric fusions is provided by chromatin immunoprecipitation experiments which demonstrate that flies lacking the dadd1 gene still have some levels of HP1a at the TAS-L region, providing evidence that at the telomeric heterochromatic domain the different regions (in this case the HTT array and the TAS regions) may have differential responses to the loss of dadd1 proteins (Chavez, 2017).

When the transcripts and copy numbers of the TART and Het-A retrotransposons were analyzed, dadd1 proteins appeared to have a major role in regulating the transposition and transcription of the TART and Het-A retrotransposons. These phenotypes are only evident in combinations of the null dadd1 allele and the dxnp2 allele. However, higher transcript levels do not always reflect on higher number of integrated copies as can be seen for the TART retrotransposon in the null dadd1 background. The lack of correlation between integrated copies and transcript abundance of this retrotransposon show that there must be another mechanism by which these proteins are maintaining the HTT array (Chavez, 2017).

Homologous recombination is the second mechanism by which Drosophila telomeres are maintained; interestingly, vertebrate ATRX has been shown to inhibit homologous recombination by sequestering the MRN complex (Clynes, 2015). Somatic mutations in vertebrate ATRX lead to an Alternative Lengthening of Telomeres mechanism (ALT) in certain types of human cancers (Napier, 2015; Ramamoorthy, 2015). Hence, it is possible that the Drosophila dxnp proteins could also prevent homologous recombination, and this could explain in part why an additional mutation in the null dadd1 background in this case in dxnp is required to complete the retrotransposition mechanism, whereas the dadd1 proteins, seem to have a role in regulating the transcription of these retro-elements. Also a differential regulation of transcription for the TART and Het-A elements has been previously described. It has been demonstrated that the TART retrotransposon is much more sensible to mutations on the repressors DREF, Ken and TRF2 than the Het-A element leading to higher levels of expression and copy number integration. The results have placed the dadd1 proteins (along with DREF, Ken and TRF2) also as negative regulators of TART expression. Interestingly, since mutations in these proteins (DREF, Ken and Trf2) also lead to retrotransposon integration, it can be assumed then, given previous data on the interaction of DREF and dxnp that albeit loss of dadd1 proteins, the levels of dxnp could possibly be maintained through its interaction with DREF and this prevents the integration of the retrotransposon copies into the genome. It would be interesting in the future to address the interdependence of these proteins to bind Het-A and TART regulatory regions in the different mutant backgrounds to get a better understanding of the differential regulation of these retrotransposons (Chavez, 2017).

Overall, these data lead to a proposal that the SNF2 (helicase/ATPase) domain is required to prevent the integration of the retrotransposons, while the ADD domain, possibly through the targeting of HP1a (and other activities), maintains a repressed transcriptional state of the retrotransposon elements in somatic cells. According to the current results, the ADD-containing proteins are essential to maintain HP1a at all of the chromosomes telomeres, while dxnp appears to affect only a subset of the telomeres analyzed. As mentioned previously, the ADD domain is able to recognize the H3K9me3 histone mark when it is in combination with the H3K4 without methylation, an interesting feature is that the ADD domain can keep binding to this mark even if the H3Ser10 is phosphorylated, whereas the chromodomain of HP1a cannot. There are reports in the literature that demonstrate that the Jil-1 kinase is present at the HTT telomeric domain, acting as a transcriptional activator for the expression of the Het-A retrotransposon. The curret results place the dadd1 and dxnp proteins as negative regulators of HTT retrotransposon expression. When the rescue experiments were performed with the dAdd1a or dAdd1b protein isoforms in a null dadd1 background it was interesting to observe that both proteins are localized at the telomeric regions. The dAdd1a signal is homogeneous at all the observed regions. However, only dAdd1a co-localized with HP1a at all the observed sites. An interesting observation is that in the chromosomes derived from the dAdd1a exogenous expression, the wild type enrichment of HP1a at the chromocenter is disrupted in some of the preparations observed. This result indicates the importance of maintaining the correct wild type levels of dadd1 proteins. When the localization of dAdd1b protein isoform was analyzed, it could be seen that this protein failed to target HP1a at the telomeres and at the chromosome arms, even though it does localizes to the telomeres and other regions at the chromosome arms. Another interesting feature is that dAdd1b seems to be enriched at the chromocenter and this does not perturb HP1a localization and enrichment at this region (Chavez, 2017).

Thus, perhaps the dAdd1a protein is required to maintain correct levels of HP1a at the telomeres in regions where there is H3Ser10 phosphorylation, thereby maintaining the balance between an active transcriptional state (mediated by Jil-1) and a silenced state required to maintain correct levels of the retrotransposons and to avoid incorrect retrotransposition mechanisms. Whereas dAdd1b could also carry silencing activities independent of HP1a (Chavez, 2017).

Based on these data, a model is proposed in which the dxnp and dadd1 proteins cooperate to maintain chromosomal stability by the transcriptional silencing of the retrotransposons of the HTT array and by promoting the correct targeting of HP1a to all of the telomeres (Chavez, 2017).

Other dxnp and dAdd1-interacting proteins have been identified also in Drosophila telomeres, including DREF, CG1910 and dSETDB1. It is interesting that dSETDB1 is also present at Drosophila telomeres, as SETDB1 has been established to participate in the retrotransposon silencing through a DNA methylation-dependent mechanism in vertebrates. Other dadd1 interactors include Bonus (BON) , which has been conserved through evolution; the family of proteins in vertebrates most related to this protein are the TIF1 families, which include TIF1 alpha, beta, and gamma. One of these families includes TIF1/TRIM28, which is also known as KAP1. In vertebrates, KAP1, Setdb1, and DAXX form a complex with ATRX that helps to maintain heterochromatin at the telomeres and IAP repeats. It would be important in the future to address whether these proteins participate in the maintenance of the silenced state of the HTT array along with the dxnp and dadd1 proteins (Chavez, 2017).

In conclusion, this study has demonstrated that the dadd1 and dxnp proteins cooperate to maintain genomic stability through at least two different mechanisms, preventing retrotransposon transcription and integration of the retrotransposons in the HTT array, as well as targeting HP1a to telomeric regions in somatic cells. This study has also revealed the specific roles of the different dxnp and dadd1 protein isoforms in the maintenance of the telomeric heterochromatic domain (Chavez, 2017).

Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. This study identifies chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. The results reveal an extensive list of known and novel HP1a-interacting proteins, of which three were selected for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, this study found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. It is proposed that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms (Alekseyenko, 2014).

This study identified HP1a interactors using a chromatin-based biochemical approach (BioTAP-XL). Connections were found between HP1a and factors responsible for chromatin organization, gene transcription, replication, and DNA repair in agreement with previously reported results for Drosophila and human HP1. Surprisingly, among the top interactors, multiple, previously unstudied proteins were discovered. Most of the new proteins were present in all developmental stages of the fly and fell into two categories. The first category was interactors for which the connection to HP1a was not known for Drosophila but was reported for the homologous proteins in different organisms; for example, Nipped-B (hNIPBL) and INCENP (hINCENP). The second group includes novel HP1a interactors such as CG8290, CG30403, CG14438, and MES-4 (Alekseyenko, 2014).

Two top candidates, CG8290 and CG3680, were validated and were named dADD1 and HIPP1, respectively. Analysis of dADD1-BioTAP ChIP-seq results together with immunofluorescence localization studies strongly suggests that the main target of this protein is pericentric heterochromatin, correlating with the primary localization of HP1a. LC-MS/MS analysis of proteins associated with dADD1 showed that besides HP1a and HP2, the top candidate interactor is BON. BON is the only Drosophila homolog of the TIF1 family, and human TIF1 members interact with and phosphorylate HP1. No direct interactions were observed between BON and HP1a in flies despite the observation that bonus could act as both an enhancer and suppressor of PEV. Therefore, it is proposed that dADD1 could act as a bridge between HP1a and BON (Alekseyenko, 2014).

The most interesting finding regarding dADD1 is its relationship to the human ATRX protein through its ADD domain. ATR-X (α-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin remodeler, are responsible for the syndrome. Approximately half of the missense mutations in affected individuals are clustered in a cysteine-rich domain termed ADD, and the other half cluster in the SNF2-type ATP-dependent chromatin-remodeling domain. The ADD domain was shown to bind the H3K9me3 chromatin mark and recruit ATRX to pericentric heterochromatin. In flies, the reported ortholog of ATRX is XNP. XNP has the SNF2-type ATP-dependent chromatin-remodeling domain but is missing the ADD domain. It was shown previously as well as in this study that XNP interacts physically and functionally with HP1a. At the same time, it was reported that 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, corresponding to an unusual, decondensed block of satellite DNA. It is speculated that in flies, the SNF2 and ADD domains of human ATRX are divided between two proteins, XNP and dADD1. Interestingly, both proteins strongly interact with HP1a but are rather weak interactors with each other based on results from the dADD1-BioTAP pull-down (Alekseyenko, 2014).

HIPP1/CG3680 was the second top candidate in the validation analysis. It was previously scored as a potential partner of HP1a in a high-throughput yeast two-hybrid assay, but this interaction was not pursued. This study discovered association between HP1a and HIPP1 using multiple biochemical approaches. The ChIP-seq together with the LC-MS/MS results of the HIPP1-BioTAP pull-down suggest that the protein associates with HP1a within heterochromatin and also plays an HP1a-indpendent role within insulator complexes in euchromatin. The association with insulators is further strengthened by the recent identification of CG8436 and CG9740 as novel CP190-interacting proteins in S2 cells. Overall, this analyses of HIPP1 suggest separate roles in the euchromatic and heterochromatic portions of the genome, but the possibility cannot be excluded that the presence of HIPP1 simultaneously in both locations has some common purpose (Alekseyenko, 2014).

This study provides an initial, unbiased view of HP1a-associated complexes on chromatin across the life cycle of Drosophila melanogaster. It was possible to expand the BioTAP-XL approach to biochemically challenging life stages and identify protein-RNA associations in addition to protein-DNA and protein-protein interactions from a single chromatin preparation. A strength of BioTAP-XL is that it requires no prior knowledge of the biochemical properties of a given complex beyond the ability to assess whether the BioTAP-tagged bait protein retains its wild-type function (Alekseyenko, 2014).

Follow-up analysis by BioTAP-XL tagging of two previously uncharacterized but strong HP1a interactors revealed that they coexist with HP1a in heterochromatin but also exhibit distinct binding locations in the euchromatic portion of the genome, likely shared with distinct partners identified in their protein interaction mass spectrometry lists. It is believed that this constitutes a very promising beginning for the construction of an HP1a chromatin interaction network. For example, future investigations of potential subcomplexes could be pursued by asking whether post-translational modifications might govern subsets of HP1a-protein, HP1a-DNA, or HP1a-RNA interactions. Indeed, HP1a is known to have multiple sites of phosphorylation in vivo, and this study found that it was possible to identify phosphorylated HP1a peptides from complex peptide mixtures in multiple life stages that are consistent with those identified in a global phosphoproteome analysis of Drosophila embryos. Site-specific mutagenesis of these sites might interfere with subsets of interactions of the HP1a-BioTAP bait protein and could be assessed in parallel for phenotypes using classical genetics, thereby revealing how subsets of key functional interactions may be regulated by post-translational modifications. Furthermore, it was also possible to detect phosphorylation of HIPP1, dADD1, and other HP1 interactors in S2 cells, embryos, larvae, and adults. Future mining and BioTAP-XL analysis of the full HP1 protein interaction list will provide ample material to further define potential HP1 subcomplexes and their nucleic acid-binding properties. In summary, despite years of prominence in the growing field of epigenetics, many critical aspects of HP1a targeting and function still remain mysterious. It is proposed that continuing to decipher the function of HP1a and its multiple partners will require a concerted, 'chromatin-centric' approach (Alekseyenko, 2014).


GENE STRUCTURE

bases in 5' UTR - 287

Exons - five

bases in 3' UTR - 278


PROTEIN STRUCTURE

Amino Acids - 206

Structural Domains

Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein in Drosophila with dosage-dependent effects on heterochromatin-mediated gene silencing. An evolutionarily conserved amino acid sequence in the N-terminal half of HP1 (the 'chromo domain') shares > 60% sequence identity with a motif found in the Polycomb protein, a silencer of homeotic genes (Paro, 1991 and Platero, 1995).

Based on the characterization of HP1 to date, there are three properties intrinsic to HP1: nuclear localization, heterochromatin binding, and gene silencing. The identification of domains responsible for the nuclear localization and heterochromatin binding properties of HP1 have been examined by expressing a series of beta-galactosidase/HP1 fusion proteins in Drosophila embryos and polytene tissue. Two functional domains in HP1 have been identified: a nuclear localization domain of amino acids 152-206 and a heterochromatin binding domain of amino acids 95-206. Both of these functional domains overlap an evolutionarily conserved COOH-terminal region (Powers, 1993).

HP1 was cloned from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with D. 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 of the protein (62/68 amino acids identical). This 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 (Clark, 1992).

The chromo (chromatin modification organizer) domain was originally identified as a protein sequence motif common to Drosophila chromatin proteins (Polycomb (PC) and HP1). There are, in fact, two chromo domain-like motifs in HP1. A recently discovered C-terminal chromo domain variant occurs only in proteins that also have an N-terminally located chromo domain. Because it is related to the chromo domain, the variant has been termed the " chromo shadow domain." There are other examples of proteins which have two chromo domains. The Schizosaccharomyces pombe SW16 protein, involved in repression of the silent mating-type loci, is a member of the chromo shadow group. The similar modular architecture of SW16, HP1 and HP1-like proteins supports the model that the specificity of action of chromatin proteins is generated by combinations of protein modules (Aasland, 1995).


HP1/Su(var)205: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 May 99 

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