Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin protein 1 (HP1) gene family

Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes. The functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence reflects the diverse functions supported by this enigmatic genome compartment. Moreover, heterogeneous signatures of selection at chromosomal proteins often mirror the heterogeneity of evolutionary forces that act on heterochromatic DNA. To identify new such surrogates for dissecting heterochromatin function and evolution, a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family was conducted across 40 million years of Drosophila evolution. This study expands this gene family from 5 genes to at least 26 genes, including several uncharacterized genes in Drosophila melanogaster. The 21 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family. Little evidence was found of positive selection at these HP1 genes in both population genetic and molecular evolution analyses. Instead, dynamic evolution was found to occur via prolific gene gains and losses. Despite this dynamic gene turnover, the number of HP1 genes is relatively constant across species. It is proposed that karyotype evolution drives at least some HP1 gene turnover. For example, the loss of the male germline-restricted HP1E in the obscura group coincides with one episode of dramatic karyotypic evolution, including the gain of a neo-Y in this lineage. The expanded compendium of ovary- and testis-restricted HP1 genes revealed by this study, together with correlated gain/loss dynamics and chromosome fission/fusion events, will guide functional analyses of novel roles supported by germline chromatin (Levine, 2012).

Yeast Swi6: a homolog of Drosophila HP1

The switching gene swi6 of Schizosaccharomyces pombe is involved in the repression of the silent mating-type loci mat2 and mat3. A region of 48 aa is homologous to a sequence motif found in HP1 and Polycomb, mouse M31, M32 and M33, and the human HSM1 protein. This motif is called chromo domain (chromatin organization modifier). Swi6 is a structural component of chromatin and may function to compact mat2 and mat3 into a heterochromatin-like conformation that represses the transcription of these silent cassettes (Lorentz, 1994).

Heritable inactivation of specific regions of the genome is a widespread, possibly universal phenomenon for gene regulation in eukaryotes. Self-perpetuating, clonally inherited chromatin structure has been proposed as the explanation for such phenomena as position-effect variegation (PEV) and control of segment determination and differentiation in flies, X-chromosome inactivation and parental imprinting in mammals, gene silencing by paramutation in maize6 and silencing of the mating-type loci in yeasts. The clr4 gene, which is essential for silencing of centromeres and the mating-type loci in Schizosaccharomyces pombe, encodes a protein with high homology to the product of Su(var)3-9, a gene affecting PEV in Drosophila1. Like Su(var)3-9p, Clr4p contains SET and chromo domains, motifs found in proteins that modulate chromatin structure. Site-directed mutations in the conserved residues of the chromo domain confirm that it is required for proper silencing and directional switching of the mating type, as is the SET domain. Surprisingly, RNA differential display experiments demonstrate that clr4+ can mediate transcriptional activation of certain other loci. These results show that clr4 plays a critical role in silencing at mating-type loci and centromeres through the organization of repressive chromatin structure and demonstrate a new, activator function for Clr4p (Ivanova, 1998).

Transcriptional silencing is known to occur at centromeres, telomeres and the mating type region in the nucleus of fission yeast, Schizosaccharomyces pombe. Mating-type silencing factors have previously been shown to also affect transcriptional repression within centromeres and to some extent at telomeres. Mutations in the clr4+, rik1+ and swi6+ genes dramatically reduce silencing at certain centromeric regions and cause elevated chromosome loss rates. Recently, Swi6p was found to co-localize with the three silent chromosomal regions. This study investigates in detail the involvement of clr4+, rik1+ and swi6+ in centromere function. Fluorescence in situ hybridization (FISH) was used to show that, as in swi6 mutant cells, centromeres lag on late anaphase spindles in clr4 and rik1 mutant cells. This phenotype is consistent with a role for these three gene products in fission yeast centromere function. The Swi6 protein is delocalized from all three silent chromosomal regions, and dispersed within the nucleus, in both clr4 and rik1 mutant cells. The phenotypic similarity observed in all three mutants is consistent with the products of both the clr4+ and rik1+ genes being required to recruit Swi6p to the centromere and other silent regions. Mutations in clr4, rik1 and swi6 also result in elevated sensitivity to reagents that destabilise microtubules and show a synergistic interaction with a mutation in the beta-tubulin gene (nda3). These observations suggest that clr4+ and rik1+ must play a role in the assembly of Swi6p into a transcriptionally silent, inaccessible chromatin structure at fission yeast centromeres that is required to facilitate interactions with spindle microtubules and to ensure normal chromosome segregation (Ekwall, 1996).

Inheritance of stable states of gene expression is essential for cellular differentiation. In fission yeast, an epigenetic imprint marking the mating-type (mat2/3) region contributes to inheritance of the silenced state, but the nature of the imprint is not known. At the mating-type region of fission yeast, silencing extends to a large 15 kb chromosomal domain. In addition to the silencing of mat2 and mat3 loci, which are used as donors of genetic information to switch the mat1 locus, an 11 kb interval between donor loci, called the K region, also exhibits transcriptional and recombinational suppression. Several trans-acting factors are required for complete repression at the mat2/3 interval. Interestingly, Swi6 and Clr4 contain the chromodomain, a motif found in proteins associated with higher-order chromatin packaging such as polycomb group (PcG) proteins and heterochromatin protein HP-1 from Drosophila, mouse, and humans. Furthermore, Clr3 and Clr6, which also affect silencing, share homology to histone deacetylases. That a similar mechanism operates at the centromeres and at mat2/3 is suggested by findings that mutations in trans-acting factors essential for silencing at the mat2/3 region also affect silencing of markers inserted within centromeres. Moreover, a part of the K region sequence shows extensive homology to centromeric repeat sequences. Swi6 protein is a dosage-critical component involved in imprinting the mat locus. Transient overexpression of Swi6 alters the epigenetic imprint at the mat2/3 region and heritably converts the expressed state to the silenced state. The establishment and maintenance of the imprint are tightly coupled to the recruitment and the persistence of Swi6 at the mat2/3 region during mitosis as well as meiosis. Remarkably, Swi6 remains bound to the mat2/3 interval throughout the cell cycle and itself seems to be a component of the imprint. This analysis suggests that the unit of inheritance at the mat2/3 locus comprises the DNA plus the associated Swi6 protein complex. In Drosophila, a homolog of Swi6, the HP-1 protein, is found in a complex with the origin recognition complex (ORC). Since, ORC homologs are also found in fission yeast and are present at replication origins throughout the cell cycle, it is possible that Swi6 might also interact with these proteins, providing an independent mechanism for its persistence at the mat locus. Alternatively, it can be imagined that chromatin assembly factor 1 (CAF-1), which physically associates with the DNA replication machinery and HP-1 family members, might be involved (Nakayama, 2000).

The assembly of higher order chromatin structures has been linked to the covalent modifications of histone tails. In vivo evidence is provided that lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation. Moreover, an H3-specific deacetylase Clr3 and a beta-propeller domain protein Rik1 are required for H3 Lys9 methylation by Clr4 and Swi6 localization. These data define a conserved pathway wherein sequential histone modifications establish a 'histone code' essential for the epigenetic inheritance of heterochromatin assembly (Nakayama, 2001).

Centromeric domains often consist of repetitive elements that are assembled in specialized chromatin, characterized by hypoacetylation of histones H3 and H4 and methylation of lysine 9 of histone H3 (K9-MeH3). Perturbation of this underacetylated state by transient treatment with histone deacetylase inhibitors leads to defective centromere function, correlating with delocalization of the heterochromatin protein Swi6/HP1. Likewise, deletion of the K9-MeH3 methyltransferase Clr4/Suvar39 causes defective chromosome segregation. Fission yeast strains retaining one histone H3 and H4 gene have been created; the creation of these strains allows mutation of specific N-terminal tail residues and their role in centromeric silencing and chromosome stability to be investigated. Reduction of H3/H4 gene dosage to one-third does not affect cell viability or heterochromatin formation. Mutation of lysines 9 or 14 or serine 10 within the amino terminus of histone H3 impairs centromere function, leading to defective chromosome segregation and Swi6/HP1 delocalization. Surprisingly, silent centromeric chromatin does not require the conserved lysine 8 and 16 residues of histone H4. To date, mutation of conserved N-terminal residues in endogenous histone genes has only been performed in budding yeast, which lacks the Clr4/Suvar39 histone methyltransferase and Swi6/HP1. This study demonstrated the importance of conserved residues within the histone H3 N terminus for the maintenance of centromeric heterochromatin in fission yeast. In sharp contrast, mutation of two conserved lysines within the histone H4 tail has no impact on the integrity of centromeric heterochromatin. These data highlight the striking divergence between the histone tail requirements for the fission yeast and budding yeast silencing pathways (Mellone, 2003).

Heterochromatin protein 1 (HP1) recruits various effectors to heterochromatin for multiple functions, but its regulation is unclear. In fission yeast, a HP1 homolog Swi6 recruits SHREC, Epe1, and cohesin, which are involved in transcriptional gene silencing (TGS), transcriptional activation, and sister chromatid cohesion, respectively. This study shows that Casein kinase II (CK2) phosphorylates Swi6. Loss of CK2-dependent Swi6 phosphorylation alleviates heterochromatic TGS without affecting heterochromatin structure. This is due to the inhibited recruitment of silencing complex SHREC to heterochromatin, accompanied by an increase in transcriptional activator Epe1. Interestingly, loss of phosphorylation does not affect cohesion. These results indicate that CK2-dependent Swi6 phosphorylation specifically controls TGS in heterochromatin (Shimada, 2009).

Invertebrate HP1 homologs

In this study, all somatic copies of the Tetrahymena HHP1 gene, which encodes Hhp1p, an HP1-like protein in macronuclei, have been disturbed. Unlike the Drosophila HP1 gene, HHP1 is not essential in Tetrahymena spp., and during vegetative growth no clear phenotype is observed in cells lacking Hhp1p. However, during a shift to nongrowth conditions, the survival rate of HHP1 mutant cells is reduced compared to that of wild-type cells. Upon starvation, Hhp1p becomes hyperphosphorylated concomitant with a reduction in macronuclear volume and an increase in the size of electron-dense chromatin bodies; neither of these morphological changes occurs in the absence of Hhp1p. Activation of two starvation-induced genes (ngoA and CyP) is significantly reduced in HHP1 mutant cells while, in contrast, the expression of several growth-related or constitutively expressed genes is comparable to that in wild-type cells. These results suggest that Hhp1p functions in the establishment and/or maintenance of a specialized condensed chromatin environment that facilitates the expression of certain genes linked to a starvation-induced response. Inasmuch as chromatin effects are also likely to be gene specific, it remains of interest to know to what extent the loss of Hhp1p affects the genome-wide expression (up or down) of other starvation-induced genes in this organism (Huang, 1999).

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

Roles of Piwi proteins in transcriptional regulation mediated by HP1s in cultured silkworm cells

Piwi proteins are part of a superfamily of Argonaute proteins, which are one of the core components of the RNA silencing pathway in many eukaryotes. Piwi proteins are thought to repress the transposon expression both transcriptionally and post-transcriptionally. Drosophila melanogaster Piwi was recently reported to associate with chromatin and to interact directly with the Heterochromatin Protein 1 (HP1a). This study shows that silkworm Piwi proteins interact with HP1s in the nucleus. The silkworm, Bombyx mori, has two Piwi proteins, Ago3 and Siwi, and two typical HP1 proteins, HP1a and HP1b. HP1a was found to play an important role in the interaction between Ago3/Siwi and HP1b in the ovary-derived BmN4 cell line. This study also found that Ago3/Siwi regulates the transcription in an HP1-dependent manner. These results suggest that silkworm Piwi proteins function as a chromatin regulator in collaboration with HP1a and HP1b (Tatsuke, 2014).

Endogenous Nuclear RNAi Mediates Behavioral Adaptation to Odor

Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. This study shows that the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, increased binding of the HP1 homolog HPL-2 was observed at the odr-1 locus in AWC and reduced odr-1 mRNA was seen in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation (Juang, 2013).

Chicken HP1 homologs

Three clones for chicken chromobox proteins were obtained from liver and ovary cDNA libraries. pCHCB1 and pCHCB2 encode polypeptides showing 96% and 95% identity with mouse M31 and M32, respectively, which are homologs of Drosophila heterochromatin protein 1 (HP1); pCHCB3 encodes a polypeptide whose sequences of chromobox and C-terminal region show high-level similarities with those of mouse M33, Drosophila polycomb (Pc) protein, and Xenopus Pc homolog. When these cDNAs are expressed in female chicken embryonic fibroblasts (CEFs) as GFP-fused or HA-tagged proteins, all three proteins are found to be localized in nuclei. Among them, CHCB1 associates with brightly stained spots with 4', 6-diamidino-2-phenylindole (DAPI), suggesting CHCB1 accumulation on heterochromatins. One of these spots was identified as W-heterochromatin, known to consist of XhoI and EcoRI family repetitive sequences, constituting the majority of sequences in the chicken W chromosome. When CHCB1 lacking the N-terminal basic/acidic region or a part of the chromobox region is overexpressed in CEFs, W-heterochromatin becames partially or extensively decondensed in the majority of nuclei. Overexpression of CHCB3 lacking a part of the chromobox does not cause decondensation of W-heterochromatin. Specific antisera raised against a part of CHCB1 or CHCB2, produced in Escherichia coli, detects protein species having apparent molecular masses of 25 kDa or 22 plus 23 kDa, respectively, in the subnuclear fraction containing the majority of chromatin from female chicken MSB-1 cells (Yamaguchi, 1998).

Facultative heterochromatin is a cytological manifestation of epigenetic mechanisms that regulate gene expression. Constitutive heterochromatin is marked by distinctive histone H3 methylation and the presence of HP1 proteins, but the chromatin modifications of facultative heterochromatin are less clear. Histone modifications and HP1 has been examined in the facultative heterochromatin of nucleated erythrocytes; mouse and chicken erythrocytes have different mechanisms of heterochromatin formation. Mouse embryonic erythrocytes have abundant HP1, increased tri-methylation of H3 at K9 and loss of H3 tri-methylation at K27. In contrast, HP1 proteins are lost during the differentiation of chicken erythrocytes, and that H3 tri-methylation at both K9 and K27 is reduced. This coincides with the appearance of the variant linker histone H5. HP1s are also absent from erythrocytes of Xenopus and zebrafish. These data show that in the same cell lineage there are different mechanisms for forming facultative heterochromatin in vertebrates. This is the first report of cell types that lack HP1s and have gross changes in the levels of histone modifications (Gilbert, 2003).

Proteins related to HP1

Su(var)3-9 (also known as HP1-like gene - see Drosophila Su(var)3-9) codes for a protein that is closely related to HP1, containing a chromo domain and a region of homology to Enhancer of zeste and Trithorax, two antagonistic regulators of the Antennapedia and bithorax gene complexes, as well as to the human protein ALL-1/Hrx ( implicated in acute leukemias). This region of homology is found in all four proteins at the C-terminus. The homology of Su(var)3-9 to both negative (Polycomb and Enhancer of zeste) and positive (Trithorax) regulators of the Antennapedia and bithorax complexes also suggests similarities in the molecular processes connected with stable transmission of a determined state and the clonal propagation of heterochromatinization (Tschiersch, 1994).

A mammalian chromatin-associated protein, CHD1 (chromo-ATPase/helicase-DNA-binding domain), might have an important role in the modification of chromatin structure. The Drosophila melanogaster CHD1 homolog (dCHD1) encodes an 1883-aa open reading frame that is 50% identical and 68% similar to the mouse CHD1 sequence, including conservation of the three signature domains for which the protein was named. dCHD1 is related to both Drosophila Brahma and Imitation SWI as well as to Polycomb and HP1, based on the presence of both a helicase domain (found in Brahma and ISWI) and a chromo domain (found in Polycomb and HP1). When the chromo and ATPase/helicase domain sequences in various CHD1 homologs are compared with the corresponding sequences in other proteins, certain distinctive features of the CHD1 chromo and ATPase/helicase domains are revealed. This suggests that CHD constitutes a distinct subgroup that diverged early in evolution from HP1 and PC subgroups as well as from ISWI type proteins. The dCHD1 gene maps to position 23C-24A on chromosome 2L. Western blot analyses with antibodies raised against a dCHD1 fusion protein specifically recognize an approximately 210-kDa protein in nuclear extracts from Drosophila embryos and cultured cells. Most interestingly, these antibodies reveal that dCHD1 localizes to sites of extended chromatin (interbands) and regions associated with high transcriptional activity (puffs) on polytene chromosomes from salivary glands of third instar larvae. These observations strongly support the idea that CHD1 functions to alter chromatin structure in a way that facilitates gene expression (Stokes, 1996).

The murine gene CHD1 (MmCHD1) was previously isolated in a search for proteins that would bind a DNA promoter element. The presence of chromo (chromatin organization modifier) domains (such as those found in Drosophila Brahma and Imitation SWI) and an SNF2-related helicase/ATPase domain (present also in Drosophila HP1 and Polycomb) led to speculation that this gene might regulate chromatin structure or gene transcription. Three novel human genes are related to MmCHD1. Examination of sequence databases produce several more related genes, most of which are not known to be similar to MmCHD1, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals. A Drosophila homolog, DmCHD1, contains all the domains found in the human sequence; another homolog, DmCHD3, lacks the DNA-binding domain sequence. MmCHD1 preferentially binds via minor groove interactions to DNA that contains (A+T)-rich tracts including those in a matrix attachment region. The major region of sequence variation in CHD proteins is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1. Targeted deletion of ScCHD1, the sole Saccharomyces cerevesiae CHD gene, was performed with deletion strains being less sensitive than wild type to the cytotoxic effect of 6-azauracil. This finding suggests that enhanced transcriptional arrest at RNA polymerase II pause sites (due to 6-azauracil-induced nucleotide pool depletion) is reduced in the deletion strain and that ScCHD1 inhibits transcription. This observation, along with the known roles of other proteins with chromo or SNF2-related helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template (Woodage, 1997).

The chromo and SET domains are conserved sequence motifs present in chromosomal proteins that function in epigenetic control of gene expression, presumably by modulating higher order chromatin. Based on sequence information from the SET domain, human (SUV39H1) and mouse (Suv39h1) homologs of the dominant Drosophila modifier of position-effect-variegation (PEV) Suppressor of variegation 3-9 (Su(var)3-9) have been isolated. Su(var)3-9 ( also known as HP1-like gene) codes for a protein that is closely related to HP1. Mammalian homologs contain, in addition to the SET domain, the characteristic chromo domain, a combination that is also preserved in the Schizosaccharyomyces pombe silencing factor clr4. Chromatin-dependent gene regulation is demonstrated by the potential of human SUV39H1 to increase repression of the pericentromeric white marker gene in transgenic flies. Immunodetection of endogenous Suv39h1/SUV39H1 proteins in a variety of mammalian cell lines reveals enriched distribution at heterochromatic foci during interphase and centromere-specific localization during metaphase. In addition, Suv39h1/SUV39H1 proteins associate with M31 (a homolog of Drosophila heterochromatin protein 1), currently the only other characterized mammalian SU(VAR) homologue. These data indicate the existence of a mammalian SU(VAR) complex and define Suv39h1/SUV39H1 as novel components of mammalian higher order chromatin (Aagaard, 1999).

Proteins such as HP1, found in fruit flies and mammals, and Swi6, its fission yeast homolog, carry a chromodomain (CD) and a chromo shadow domain (CSD). These proteins are required to form functional transcriptionally silent centromeric chromatin, and their mutation leads to chromosome segregation defects. CSDs have only been found in tandem in proteins containing the related CD. Most HP1-interacting proteins have been found to associate through the CSD and many of these ligands contain a conserved pentapeptide motif. This study examined the 1.9 Å crystal structure of the Swi6 CSD. This reveals a novel dimeric structure that is distinct from the previously reported monomeric nuclear magnetic resonance (NMR) structure of the CD from the mouse modifier 1 protein (MoMOD1, also known as HP1beta or M31). A prominent pit with a non-polar base is generated at the dimer interface, and is commensurate with binding an extended pentapeptide motif. Sequence alignments based on this structure highlight differences between CDs and CSDs that are superimposed on a common structural core. The analyses also revealed a previously unrecognized circumferential hydrophobic sash around the surface of the CD structure. It is concluded that dimerization through the CSD of HP1-like proteins results in the simultaneous formation of a putative protein-protein interaction pit, providing a potential means of targeting CSD-containing proteins to particular chromatin sites (Cowieson, 2000).

Mammalian HP1 homologs

HP1 contains a highly conserved sequence, the chromobox, which can be used to isolate HP1-like genes from both mouse (M31 and M32) and man (HSM1). A monoclonal antibody raised against the M31 protein recognises a 26-kDa protein in murine and human nuclear extracts and localizes to large masses of condensed chromatin within murine interphase nuclei, some of which are associated with the nucleoli. At metaphase, the antibody binds to the centromeres of both human and murine chromosomes. The evolutionary conservation of this chromosomal localization indicates that the M31 protein is likely to be important in the packaging of mammalian chromosomal DNA into constitutive heterochromatin (Wreggett, 1994).

Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal component tightly associated with the pericentromeric heterochromatic region of fruit fly, mouse, and human throughout the cell cycle. Drosophila HP1 has been shown to be involved in position effect variegation and to be required for the correct chromosome segregation in vivo, while the biological activity of human homolog (HP1Hsa) has not yet been characterized. Human CENP-B and CENP-C, two major centromere heterochromatin autoantigens often recognized by autosera in scleroderma patients, possess DNA-binding activity in vitro. Human HP1, which is also an autoantigen targeted by some types of anticentromere autosera, is a DNA-binding protein. Human HP1 was expressed as a GST-fusion in Escherichia coli and purified with glutathione-Sepharose. The DNA-binding activity of the recombinant HP1 was demonstrated by gel mobility shift assay and South-Western-type blotting. The minimum DNA-binding region was further limited to the internal 64-amino acid stretch that is less-conserved between human and fruit fly but retains a helix-enriched motif with weak similarity to CENP-C. This suggests that HP1 is involved in the pericentromeric heterochromatin formation by directly associating with genomic DNA (Sugimoto, 1996).

Mouse chromatin modifier protein 1 (MoMOD1) is an HP1-like heterochromatin-associated protein. The structure of the chromatin binding domain was determined using nuclear magnetic resonance. The protein consists of an N-terminal three-stranded anti-parallel beta-sheet that folds agains a C-terminal alpha-helix. The structure reveals an unexpected homology to two archaebacterial DNA binding proteins that are also involved in chromatin structure. Structural comparisions suggest that chromo domains, of which more than 40 are now known, act as protein interaction motifs and that the MOMOD1 protein acts as an adaptor mediating interaction between different proteins (Ball, 1997)

HP1 and insulators

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. Using human cells lines, a study was carried out on the ability of insulators to block the action of chromatin-associated repressors, such as HP1, MeCP2 and PcG proteins. The Drosophila special chromatin structure insulator (scs, found to flank the hsp70 heat shock locus in Drosophila) completely blocks transcriptional repression mediated by all of the repressors tested. The Drosophila gypsy insulator is able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by methyl-CpG-binding protein (MeCP2) and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displays a limited ability to block repression, and a matrix or scaffold attachment region element is entirely unable to block repression mediated by any repressor tested. These results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high degree of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators (van der Vlag, 2000).

These results show that several insulators are able to block repression mediated by chromatin-associated repressors. Of these, the scs insulator is most efficient in blocking the repressors tested. This result demonstrates a striking evolutionary conservation, since in this study the scs insulator was used outside its natural Drosophila environment. This implies that there are human proteins that bind to the scs element in such a manner that the insulator becomes functional. Also, the function of the gypsy insulator is functionally conserved, since gypsy is able to block most of the repressors used. However, unlike scs, gypsy is not able to block repression mediated by HPC2 or MeCP2. There are several explanations for this observation: (1) it is an intrinsic characteristic of the gypsy insulator; (2) the DNA-protein interaction within the gypsy nucleoprotein complex is not sufficiently conserved to allow the insulator to function properly within the context of the human cell line; (3) the gypsy insulator does not obtain a proper chromatin structure that allows the insulator to become fully functional. At present, the data do not favor the last two options for the following reasons: (1) if the evolutionary conservation is insufficient, it is hard to explain why gypsy is very efficient in blocking repression mediated by RING1, Su(z)2, and mHP1. With insufficient conservation, one might expect that the gypsy insulator would block no vertebrate repressor at all. (2) To exclude an improper chromatin environment, stably transfected cell lines were made with the reporter construct that contains gypsy. Also, in this case, gypsy is unable to block repression mediated by HPC2 and MeCP2. (3) No significant differences in the nucleosomal chromatin structure of the reporter construct containing gypsy were detected. (4) If an improper chromatin structure plays a role, this would not explain why gypsy, under similar conditions, is very efficient in blocking repression mediated by RING1, Su(z)2, and mHP1. When taking these arguments together, the possibility is favored that these results indicate that it is an intrinsic characteristic of the gypsy insulator to block repression by RING1, Su(z)2, and mHP1 but not by HPC2 and MeCP2. Apparently, gypsy is able to block chromatin-associated repressors with a high level of selectivity. This establishes an important point: whereas both HPC2 and MeCP2 are able to very efficiently repress gene activity, they are different from the other repressors in the sense that their action cannot be blocked by the gypsy insulator. The assay used thus uncovers both differences in the ability of insulators to block repression and differences between chromatin-associated repressors. The differences between repressors do not become apparent when only their abilities to repress gene activity are being monitored (van der Vlag, 2000).

These results show an orientation dependence of the 5'-located DNase I-hypersensitive site (5'-HS) in the chicken beta-globin locus. Whereas a single copy of the entire element does not have much effect on the repression mediated by any of the repressors tested, a distinct effect has been found when a tandem of six core elements within the 5'-HS element is tested. Previously, it has been shown that the enhancer blocking ability of the 5'-HS element resides precisely in this core element. The finding that the tandem of 5'-HS core elements is very efficient in blocking repression, but only when cloned in the 5' to 3' orientation, comes as a surprise. The fact that this is true for all repressors tested gives weight to the idea that this orientation dependence is an intrinsic property of the 5'-HS element. No indication has been found that a Drosophila MAR/SAR element is able to block chromatin-associated repressors. This was observed in a stably transfected cell line and in a cell line 72 h after transfection. In either case, a bona fide nucleosomal chromatin structure was observed. It should also be pointed out that the ability of MAR/SARs to shield reporter genes against enhancers and repressing chromatin is controversial. It is concluded that the portion of the Drosophila histone MAR/SAR element tested does not possess an ability to block repression (van der Vlag, 2000).

The result that is most easy to interpret is the efficient blocking of repression by the scs insulator, since scs blocks each repressor tested. How do these findings relate to previous studies? The scs and gypsy insulators have been tested for their ability to protect a reporter gene against position effects in transformed flies. The white maxigene construct is able to confer high expression levels of white and is prone to repression due to position effects. The white maxigene has been artifically flanked with the scs and scs' elements. The elements have been shown to confer a consistently high level of white expression in the majority of transformants, independent of the integration position within euchromatic regions of the genome. These results strongly suggest that scs and scs' are able to efficiently block repression. Thus, the ability of the scs element to block repression, are in agreement with earlier studies (van der Vlag, 2000).

The gypsy insulator has not been tested in the context of the white maxigene. Instead, the gypsy insulator has been used to flank the white minigene. These constructs are considered to be easily affected by position effect variegation, a phenomenon that involves repression in a heterochromatin environment. The extent of position effect variegation increases when these constructs are tested in fly lines that lack functional Su(Hw) protein. Su(Hw) is the protein that binds to gypsy and is necessary for gypsy to function properly. These results have been interpreted as indicating that in these fly lines the gypsy insulator does not function properly and that, consequently, repression is blocked less efficiently (van der Vlag, 2000).

The 5'-HS element has been tested in Drosophila embryos within the context of the white minigene. This assay tests the ability of the 5'-HS element to protect against activation emerging from the surrounding chromatin, not the ability to protect against repression. However, recently the 5'-HS element has been found to convey position-independent expression levels to a reporter gene that was stably integrated in a chicken cell line. This favors a model in which the 5'-HS element protects a reporter gene against both activating and repressing influences emerging from the surrounding chromatin (van der Vlag, 2000).

What is the evidence from other studies that link PcG proteins to the function of insulators? The most convincing genetic evidence indicates that the function of gypsy depends on PcG-mediated repression. Mutations in PcG genes suppress the insulator properties of gypsy, as monitored by its ability to prevent enhancer-promoter interactions. It has further been found that when either gypsy or scs is placed between a polycomb response element and a promoter, the repression initiated from the polycomb response element is blocked. The data of the current study are in agreement with these earlier studies. Whether this also implies that insulators such as scs and gypsy function as stop signals to terminate spreading of the PcG complex remains speculative. Taken together, however, all data point toward an important role of PcG proteins in the function of the gypsy and scs insulators (van der Vlag, 2000).

These data indicate that insulators are conserved nucleoprotein structures that are able to efficiently block repression mediated by a variety of chromatin-associated repressors in evolutionarily nonrelated species. Whereas this statement is true in general, the data also show an unexpected level of selectivity toward specific repressors (HPC2 and MeCP2) as well as an orientation dependence of boundary function (the 5'-HS core elements). This suggests that there are distinct classes of insulators that may be well defined by their ability to block the action of specific chromatin-associated repressors. The repression assay that was developed may be a powerful tool in characterizing both putative insulators as well as novel repressors (van der Vlag, 2000).

HP1 compartmentalization

The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein, which is part of the oncogenic PML-RARalpha hybrid produced by the t(15;17) chromosomal translocation, characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. SP100 is shown to bind members of the heterochromatin protein 1 (HP1) family of non-histone chromosomal proteins. Further, a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics (Seeler, 1998).

The existence of the variant SP100-HMG protein suggests the possibility of a direct association between an NB protein with chromatin by means of DNA binding through the HMG box motifs. These motifs are found in the canonical HMG-1 and -2 non-histone chromosomal proteins, and mediate nonsequence-specific DNA binding with a marked preference for bent, single-stranded or cruciform, i.e., non-B DNA structures. In addition, several transcription factors, such as UBF, LEF-1 (Drosophila homolog: Pangolin), and SRY, also contain HMG box motifs. Interestingly, these proteins exhibit both sequence-specific and nonspecific interactions with DNA, mediated by the HMG box motifs. Thus it would be interesting to know whether SP100-HMG also might be capable of sequence-specific DNA binding and hence might possibly be a bona fide transcription factor. A more indirect association of SP100 proteins with chromatin also might be provided by interactions with HP1. Alternatively, or in addition, HP1 may be recruited to the NBs, i.e., away from chromatin. Indeed, these data suggest that enhanced SP100 expression correlates with increased accumulation of HP1 in the NBs. However, the quantity of HP1 in the NBs, as well as the amount necessary to influence chromatin structure by HP1 depletion, remains to be established (Seeler, 1998 and references).

Structural studies of HP1 homologs

The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. The structure of the chromo domain from the mouse HP1beta protein, MOD1, has been determined. In contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1beta protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. The large subunit of chromatin assembly factor 1 (CAF1p150) binds to mouse HP1 proteins. The interaction is required for the association of CAF1 with heterochromatin in non-S-phase cells, and CAF1 also promotes the incorporation of MOD1 into nascent chromatin during DNA replication in vitro. A peptide motif that is conserved in both the TIFs and the large subunit of CAF1p150 that interacts with the shadow chromo domain has been identified. Mapping studies, with fragments of the CAF1 and TIF1beta proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation (Brasher, 2000).

In principle, the HP1 family proteins might interact with each other to form higher order complexes in two different ways. The HP1 dimers might interact with each other to form higher order complexes, e.g. tetramers, or alternatively, different HP1 monomers might interact to form heterodimers. No further specific multimerization of MOD1 could be detected. However, one cannot rule out the possibility of further multimerization of the protein upon post-translational modification, such as phosphorylation, which is known to occur in eukaryotic cells (Brasher, 2000).

The unmodified HP1 proteins might nevertheless form heterodimers directly with one another. Most residues involved in the dimer interface are conserved between the shadow domains of different HP1 proteins, that is, A125, L132, N153, P157, I161, Y164, L168 and W170 are conserved in all except swi6. In addition, interactions have previously been reported between mouse HP1alpha and either itself or MOD1 in a yeast two-hybrid screen. Moreover, human HP1alpha binds to both itself and to HP1gamma in pull-down assays using in vitro translated proteins. Based on the biochemical, sequence and structural data, there is therefore a possibility of heterodimer formation between different HP1 monomers. Nevertheless, so far no biological functions have been ascribed to such interactions and it has been shown that HP1alpha, HP1beta and HP1gamma generally behave and localize differently in mammalian cells (Brasher, 2000 and references therein).

The region of MOD1C involved in the interaction with proteins containing the MIR motif has been mapped by NMR. The residues involved comprise the C-terminal end of the second helix, the C-terminal tail and the adjacent residues from the first and second beta-strands. W170, which is located at the center of this region, appears to play a critical role in the interaction. Its mutation to either A or E abolishes MOD1C binding to both the TIF and CAF MIRs, but does not affect the dimeric nature of the domain. The gel-filtration and sedimentation analyses demonstrate that one MIR peptide binds to one shadow chromo domain dimer. Given this stoichiometry, one would expect that interaction with the peptide would induce asymmetry in the dimer, and indeed strong evidence is seen for this in the NMR spectra. Two possible modes of peptide binding to MOD1C are imagined. In the first, one of the monomers binds a peptide molecule, and this binding prevents the other monomer from binding a second peptide, e.g. by allosteric changes. Alternatively, both monomers might be involved in binding a single peptide molecule. The latter possibility is favored because it has been found that the monomeric MOD1C mutants are not able to bind to the TIF and CAF MIR fragments. The inability of these monomeric mutants to bind the CAF MIR peptide is interpreted as being due to disruption of the binding surface, where the peptide binds to both subunits at the dimer interface (Brasher, 2000).

This mode of protein-protein interaction in which a single monomeric peptide is recognized by a dimeric protein interaction motif is unprecedented in intracellular proteins. The only similar example occurs in the major histocompatibility complex (MHC II), where a single peptide binds to a site formed by two different polypeptide chains. The mapped region does not have any deep groove or cavity that would allow the proposing of a detailed mode of binding. One possibility is that the MIR peptide binds in an extended conformation to the N-terminal beta-strand in the shadow chromo domain (thereby extending the beta-sheet) and at the same time makes contact with residues in the C-terminal tail of the other subunit. Another possibility is that the MIR peptide binds in between the C-terminal tails, moving them apart and thereby creating the necessary cavity for binding. This would be consistent with the NMR data that suggest that the C-terminal tail is not as well structured as the rest of the domain and such a mode of binding would explain why a large part of the molecule becomes asymmetric upon peptide binding. Clearly, the detailed mode of binding will need to await solution of the 3D structure of the complex (Brasher, 2000).

Given their sequence similarity, it was thought possible that the TIF1beta and CAF1p150 MIR peptides would interact with the same binding site on MOD1C: this view is supported by experiments presented here. While the conserved MIR motif is capable of binding to MOD1C, additional adjacent residues are also involved. Given the lack of sequence similarity between TIF1beta and CAF1p150 outside the conserved MIR motif, it is speculated that the flanking regions of the two proteins might bind differently to the shadow chromo domain. Structural studies of the two complexes will reveal which MOD1 residues are involved and this might enable the design of MOD1 mutants that would bind specifically to either CAF1p150 or TIF1beta, allowing further insight into the biological roles of each complex. Some of the proteins that interact with MOD1C do not possess a recognizable MIR motif and may bind in a different way, or even to a different binding site. A possible location for an alternative site might be at the other end of the dimer axis where there is a relatively hydrophobic surface patch, made up of residues V154, P157 and Q158 (Brasher, 2000).

HP1 proteins are phosphorylated in vivo and this phosphorylation may be an important mechanism for regulating the proteins' multimerization and/or interactions. Three casein kinase II phosphorylation sites have be detected on Drosophila HP1, S15, S199 and S202, which are needed for heterochromatin binding. In HP1beta and HP1gamma, but not HP1alpha, T169 and S172 lie in a similar sequence context to that of the C-terminal phosphorylation sites in Drosophila HP1. T169 is located close to the MIR binding site on the shadow chromo domain, suggesting that its phosphorylation might prevent binding by the hydrophobic MIR peptides. Studies of the structure and interactions of MOD1C thus suggest a mechanism by which phosphorylation might alter the function of HP1 proteins during the cell cycle and/or development, where it is known that their phosphorylation patterns change (Brasher, 2000).

Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. The functional domains of swi6p have been identified and it has been demonstrated that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids that lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and it is suggested that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity (Wang, 2001).

The complete coding sequences for two Xenopus laevis isoforms of heterochromatin protein 1, corresponding to HP1alpha and HP1gamma, have been isolated. The sequence of xHP1alpha shows considerable divergence from its mammalian homologs, whereas xHP1gamma is highly conserved. Functionally, xHP1alpha behaves identically to human HP1alpha. Unexpected differences were observed between the two HP1 variants in binding native soluble chromatin, that seem to correlate with their distinct nuclear distributions in vivo. A surprising finding is that the characteristic interaction of HP1 chromodomains with histone H3 at methylated lysine 9 is not detected in preformed chromatin due to its inaccessibility. Instead, a strong chromatin-binding activity was localized to the short hinge region between the chromodomain and the chromoshadow domain of xHP1alpha but not xHP1gamma. This novel chromatin-binding activity has a non-specific DNA-binding component in addition to a linker histone-dependent preference for an altered chromatin structure with a likely heterochromatin organization (Meehan, 2003).

Repressive functions of HP1

The Drosophila HMG1-like protein DSP1 was identified in yeast by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion. DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B; SP100B in turn binds to human homologs of HP1. "nuclear bodies" (NBs), found in nuclei of mammalian cells, are known to contain SP100A and PML. SP100A is an autoantigen recognized by antibodies from patients suffering from primary biliary cirrhosis, and PML has been implicated to play a role in acute promyelocytic leukemia. Both SP100A and PML are up-regulated by interferons: overexpression of PML results in slow growth. HP1 is a Drosophila protein involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression (Lehming, 1998).

DSP1, fused to a GAL4 DNA-binding domain and bound to DNA at GAL4 sites, functions as a transcriptional repressor in HeLa cells. The reporter used in this experiment bore five GAL4 sites upstream of DNA sequences, taken from the TK promoter, that bind the activators C/EBP and SP1. A GAL4 fusion bearing the complete DSP1 represses the reporter some tenfold in mammalian cells. A fusion protein containing the carboxyl half of DSP1, which includes both HMG boxes, represses even more efficiently than does the full length fusion, whereas the amino half of DSP1 increases activation, a result of unknown significance. Intact human HMG2 fused to GAL4 also works as an efficient repressor in mammalian cells. In all cases, repression is abolished by deleting the GAL4-binding sites. In no case is repression found in yeast, suggesting that the repression seen in higher eukaryotes requires proteins absent from yeast (Lehming, 1998).

To identify such proteins from higher eukaryotes, a yeast two-hybrid screen was performed by using GAL4-DSP1(178-393) as bait and challenging with a human cDNA library derived from B cells. The cDNAs were fused to DNA encoding rII, an activating region derived from GAL4. One interacting candidate was found, a previously sequenced protein called SP100B, a splice variant of the NB protein SP100A. SP100A is 479 amino acids long; SP100B contains 688 amino acids. Both proteins are identical in their first 476 amino acids. The cDNA clone represents amino acids 5-528 of SP100B, referred to here as SP100B. SP100B, but not SP100A, interacts with the carboxyl half of DSP1, as well as with human HMG2. The B-specific domain of SP100B (amino acids 477-528) is sufficient for the interaction with both DSP1(178-393) and hHMG2 in vitro. The fusion protein GAL4-SP100B, like GAL4-DSP1 and GAL4-hHMG2, works as a repressor in mammalian cells but not in yeast. The repression depends on the presence of the GAL4 DNA-binding sites in the reporter (Lehming, 1998).

A second two-hybrid screen was performed, similar to the first, except that in this case the bait was GAL4-SPl00B. Two strongly interacting candidates were recovered, each of which contained both a chromodomain (CD) and a chromo shadow domain (CSD). Both proteins, hHPlalpha and hHP1gamma, are homologs of the Drosophila protein HPl (also called Su[var]205). In a yeast two-hybrid assay system, hHPlalpha and hHP1gamma interact with SP100B. The interaction requires the CSD of hHP1 but not the CD. For SP100B, the interaction domain is located between amino acids 286 and 333, a region identical in both SP100A and SP100B. SP100B also interacts with itself: this self-association determinant is probably located in the N terminus of the protein. SP100B interacts with both hHP1 proteins in vitro. Both GAL4-hHP1 fusion proteins work as repressors when bound to GAL4 sites in mammalian cells. In all cases, repression is abolished by deleting the GAL4-binding sites. Like the interaction with SP100B, repression requires the chromo shadow and not the CD. GAL4-hHPl represses approximately tenfold more efficiently than does GAL4-DSP1(178-393) and fivefold more efficiently than does GAL4-SP100B. Thus the CSD of hHP1alpha, comprising 53 amino acids, bears two functions: it confers the repression function when fused to GAL4, and it interacts with SP100B (Lehming, 1998).

Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, a strategy was developed for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. Its obligate corepressor, KAP1, which coordinates histone deacetylation via the recruitment of the NuRD complex, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable (Ayyanathan, 2003).

HP1Hsalpha promotes nucleosome associations that drive chromatin condensation

HP1Hsalpha-containing heterochromatin is located near centric regions of chromosomes and regulates DNA-mediated processes such as DNA repair and transcription. The higher-order structure of heterochromatin contributes to this regulation, yet the structure of heterochromatin is not well understood. This study took a multidisciplinary approach to determine how HP1Hsalpha-nucleosome interactions contribute to the structure of heterochromatin. HP1Hsalpha preferentially binds histone H3K9Me3-containing nucleosomal arrays in favor of non-methylated nucleosomal arrays; non-specific DNA interactions and pre-existing chromatin compaction were shown to promote binding. The chromo and chromo shadow domains of HP1Hsalpha play an essential role in HP1Hsalpha-nucleosome interactions, while the hinge region appears to have a less significant role. Electron microscopy of HP1Hsalpha-associated nucleosomal arrays showed that HP1Hsalpha causes nucleosome associations within an array, facilitating chromatin condensation. Differential sedimentation of HP1Hsalpha associated nucleosomal arrays showed that HP1Hsalpha promotes interactions between arrays. These strand-to-strand interactions are supported by in vivo studies where tethering the Drosophila homologue HP1a to specific sites promotes interactions with distant chromosomal sites. These findings demonstrate that HP1Hsalpha-nucleosome interactions cause chromatin condensation, a process that regulates many chromosome events (Azzaz, 2014).

HP1 and position effect variegation

Locus control regions (LCRs) are gene regulatory elements in mammals that can overcome the highly repressive effects normally associated with heterochromatic transgene locations (for example the centromere) in mice. Deletion of essential LCR sequences renders the cognate gene susceptible to this form of repression, so a proportion of the cells from transgenic mice that would normally express the transgene are silenced -- a phenomenon known as position effect variegation (PEV). PEV can also occur when the transgene is non-centromeric and the extent of variegation can be developmentally regulated. Furthermore, by overexpressing a mammalian homolog (M31) of Drosophila melanogaster heterochromatin protein 1 (HP1) in transgenic mouse lines that exhibit PEV, it is possible to modify the proportion of cells that silence the transgene in a dose-dependent manner. Thus, M31 overexpression has two contrasting effects that are dependent on chromosomal context: (1) it enhances PEV in those lines with centromeric or pericentromeric transgene locations; and (2) it suppresses PEV when the transgene is non-centromeric. These results indicate that components or modifiers of heterochromatin may have a chromosomal-context-dependent role in gene silencing and activation decisions in mammals (Festenstein, 1999).

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. The relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing is correlated with a decrease in promoter accessibility and is enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin- mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases (Saveliev, 2002).

HP1 and the cell cycle

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

Mammalian heterochromatin proteins 1 (HP1alpha, HP1beta, and HP1gamma) are nonhistone proteins that interact in vitro with a set of proteins that play a role in chromatin silencing, transcription, and chromatin remodeling. Using antibodies specific for each HP1 isoform, it is shown that they segregate in distinct nuclear domains of human HeLa cells. By contrast, in mouse 3T3 interphase cells, HP1alpha and HP1beta are strictly colocalized. In mitotic HeLa cells, all of HP1alpha and a fraction of HP1beta and HP1gamma remain associated with chromosomes. Immunostaining of spread HeLa chromosomes show that HP1alpha is mainly localized on centromeres as shown previously for HP1beta, while HP1gamma is distributed on discrete sites on the arms of chromosomes. Biochemical analysis shows that HP1alpha and HP1gamma are phosphorylated throughout the cell cycle, although more extensively in mitosis than in interphase, while HP1beta apparently remains unphosphorylated. Therefore, despite their extensive sequence conservation, mammalian HP1 isoforms differ widely in their nuclear localization, mitotic distribution and cell cycle-related phosphorylation. Thus, subtle differences in primary sequence and in posttranslational modifications may promote their targeting at different chromatin sites, generating pleiotropic effects (Minc, 1999).

Histones are subject to numerous post-translational modifications. Some of these 'epigenetic' marks recruit proteins that modulate chromatin structure. For example, heterochromatin protein 1 (HP1) binds to histone H3 when its lysine 9 residue has been tri-methylated by the methyltransferase Suv39h. During mitosis, H3 is also phosphorylated by the kinase Aurora B. Although H3 phosphorylation is a hallmark of mitosis, its function remains mysterious. It has been proposed that histone phosphorylation controls the binding of proteins to chromatin, but any such mechanisms are unknown. This study shows that antibodies against mitotic chromosomal antigens that are associated with human autoimmune diseases specifically recognize H3 molecules that are modified by both tri-methylation of lysine 9 and phosphorylation of serine 10 (H3K9me3S10ph). The generation of H3K9me3S10ph depends on Suv39h and Aurora B, and occurs at pericentric heterochromatin during mitosis in different eukaryotes. Most HP1 typically dissociates from chromosomes during mitosis, but if phosphorylation of H3 serine 10 is inhibited, HP1 remains chromosome-bound throughout mitosis. H3 phosphorylation by Aurora B is therefore part of a 'methyl/phos switch' mechanism that displaces HP1 and perhaps other proteins from mitotic heterochromatin (Hirota, 2005).

HP1 interactions with transcription factors

The KRAB repression domain was originally identified in humans as a conserved amino acid sequence motif at the amino termini of proteins that contain multiple TFIIIA/Krüppel class Cys2-His2 (C2H2) zinc fingers in their COOH termini. The KRAB domain has since been identified in frog, rodent, and human zinc finger proteins (ZFPs). It has been estimated that between 300 and 700 human genes encode C2H2 zinc finger proteins, one-third of which are predicted to contain KRAB domains; accordingly, these genes have been designated the KRAB-ZFP family. The KRAB domain, consisting of approximately 75 amino acids, can function as a potent transferable DNA binding-dependent repression module. Moreover, more than 10 independently encoded KRAB domains have been demonstrated to be potent repressors, and substitutions of conserved residues within this domain abolish repression activity. These observations suggested that transcription repression is a common property of this domain. A corepressor for the KRAB domain, KAP-1, has been cloned that is required for KRAB-mediated repression in vivo. KAP-1 is a 97-kDa nuclear phosphoprotein whose primary amino acid sequence displays a number of interesting structural motifs. The RING finger, B boxes (beta1 and beta2), and a coiled-coil region at the amino terminus collectively constitute the KRAB interaction, or RBCC, domain. Carboxy terminal to this constellation of motifs appears a relatively novel stretch of amino acids, a plant homeodomain (PHD) finger, and a bromodomain, which together likely represent at least two or more independent repression domains. To characterize the repression mechanism utilized by KAP-1, an examination has been carried out of the ability of KAP-1 to interact with murine (M31 and M32, both HP1 homologs) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction shows that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. Colocalization of KAP-1 with M31 and M32 is observed in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. The predicted region of KAP-1 required for binding to HP1 proteins lies between the coiled-coil domain and the PHD. Consistent with this, analysis of this central domain of KAP-1 reveals a region of amino acid sequence homology termed the HP1BD, shared with the mTIF1alpha coactivator protein, which binds the mHP1alpha and M31 heterochromatin proteins. This finding suggests that the small-signature amino acid sequence motif may be responsible for protein-protein interactions involving HP1 family members and may be useful in identifying other components that are direct targets bound by HP1 family members. While the HP1BD is sufficient for binding of HP1 family members, significant repression activity is also exhibited by a GAL4 fusion that contains only the PHD and bromodomains. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. it is speculated that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments (Ryan, 1999).

Mammalian TIF1alpha and TIF1beta (KAP-1/KRIP-1) are related transcriptional intermediary factors that possess intrinsic silencing activity. TIF1alpha is believed to be a euchromatic target for liganded nuclear receptors, while TIF1beta may serve as a co-repressor for the large family of KRAB domain-containing zinc finger proteins. An association of TIF1beta with both heterochromatin and euchromatin in interphase nuclei is reported. Co-immunoprecipitation of nuclear extracts shows that endogenous TIF1beta, but not TIF1alpha, is associated with members of the heterochromatin protein 1 (HP1) family. However, in vitro, both TIF1alpha and TIF1beta interact with and phosphorylate the HP1 proteins. This interaction involves a conserved amino acid motif, which is critical for the silencing activity of TIF1beta but not TIF1alpha. Trichostatin A, an inhibitor of histone deacetylases, can interfere with both TIF1 and HP1 silencing. The silencing activity of TIF1alpha appears to result chiefly from histone deacetylation, whereas that of TIF1beta may be mediated via both HP1 binding and histone deacetylation (Nielsen, 1999).

Recruitment of HP1 by co-repressors

Posttranslational modification of histones has emerged as a key regulatory signal in eukaryotic gene expression. Recent genetic and biochemical studies link H3-lysine 9 (H3-K9) methylation to HP1-mediated heterochromatin formation and gene silencing. However, the mechanisms that target and coordinate these activities to specific genes is poorly understood. The KAP-1 (Drosophila homolog: Bonus) corepressor for the KRAB-ZFP superfamily of transcriptional silencers binds to SETDB1, a novel SET domain protein with histone H3-K9-specific methyltransferase activity. Although acetylation and phosphorylation of the H3 N-terminal tail profoundly affect the efficiency of H3-K9 methylation by SETDB1, it has been found that methylation of H3-K4 does not affect SETDB1-mediated methylation of H3-K9. In vitro methylation of the N-terminal tail of histone H3 by SETDB1 is sufficient to enhance the binding of HP1 proteins, which requires both an intact chromodomain and chromoshadow domain. Indirect immunofluoresence staining of interphase nuclei localizes SETDB1 predominantly in euchromatic regions that overlap with HP1 staining in nonpericentromeric regions of chromatin. Moreover, KAP-1, SETDB1, H3-MeK9, and HP1 are enriched at promoter sequences of a euchromatic gene silenced by the KRAB-KAP-1 repression system. Thus, KAP-1 is a molecular scaffold that is targeted by KRAB-ZFPs to specific loci and coordinates both histone methylation and the deposition of HP1 proteins to silence gene expression (Schultz, 2002).

The transcriptional intermediary factor 1ß (TIF1ß) is a corepressor for KRAB-domain-containing zinc finger proteins and is believed to play essential roles in cell physiology by regulating chromatin organization at specific loci through association with chromatin remodeling and histone-modifying activities and recruitment of heterochromatin protein 1 (HP1) proteins. In this study, a modified embryonal carcinoma F9 cell line (TIF1ßHP1box/-) was engineered expressing a mutated TIF1ß protein (TIF1ßHP1box) unable to interact with HP1 proteins. Phenotypic analysis of TIF1ßHP1box/- and TIF1ß+/- cells shows that TIF1ß-HP1 interaction is not required for differentiation of F9 cells into primitive endoderm-like (PrE) cells on retinoic acid (RA) treatment but is essential for further differentiation into parietal endoderm-like (PE) cells on addition of cAMP and for differentiation into visceral endoderm-like cells on treatment of vesicles with RA. Complementation experiments reveal that TIF1ß-HP1 interaction is essential only during a short window of time within early differentiating PrE cells to establish a selective transmittable competence to terminally differentiate on further cAMP inducing signal. Moreover, the expression of three endoderm-specific genes, GATA6, HNF4, and Dab2, is down-regulated in TIF1ßHP1box/- cells compared with wild-type cells during PrE differentiation. Collectively, these data demonstrate that the interaction between TIF1ß and HP1 proteins is essential for progression through differentiation by regulating the expression of endoderm differentiation master players (Cammas, 2004).

Interaction of HP1 with Brahma homologs

Mammalian heterochromatin protein 1 (HP1) alpha, HP1ß and HP1gamma are closely related non-histone chromosomal proteins that function in gene silencing, presumably by organizing higher order chromatin structures. It has been shown by co-immunoprecipitation that HP1alpha, but neither HP1ß nor HP1gamma, forms a complex with the BRG1 chromatin-remodeling factor in HeLa cells. In vitro, BRG1 interacts directly and preferentially with HP1alpha. The region conferring this preferential binding has been mapped to residues 106-180 of the HP1alpha C-terminal chromoshadow domain. Using site-directed mutagenesis, three amino acid residues I113, A114 and C133 have been identified in HP1alpha (K, P and S in HP1ß and HP1gamma) that are essential for the selective interaction of HP1alpha with BRG1. Interestingly, these residues were also shown to be critical for the silencing activity of HP1alpha. Taken together, these results demonstrate that mammalian HP1 proteins are biochemically distinct and suggest an entirely novel function for BRG1 in modulating HP1alpha-containing heterochromatic structures (A. L. Nielsen, 2002).

There are several mechanisms by which BRG1 could contribute to HP1alpha-dependent silencing. Reconstitution studies have shown that, on its own, BRG1 can remodel nucleosomes in an ATP-dependent manner, indicating that the presence of this ATPase subunit within a complex is sufficient for chromatin remodeling. HP1alpha can bind to nucleosomes in vitro and associates with chromatin in vivo through a direct interaction with the histone fold domain of histone H3. Thus, the chromatin-remodeling activity of BRG1 may facilitate the binding of HP1alpha to its nucleosomal sites. Recent studies have also described a specific binding of HP1alpha to the methylated tail domain of H3, which is critical for its targeting to centromeric heterochromatin. Similarly, the underacetylated state of the histone tails is an important determinant for the maintenance of the HP1alpha protein at heterochromatic sites and for full silencing. Thus, the BRG1 activity may enhance access of the histone tails to HP1alpha-associated deacetylases and methyltransferases, which may, in turn, promote the formation of a local, heterochromatin environment that results in effective gene silencing. Another way in which BRG1 might participate in the assembly of HP1alpha-dependent heterochromatic structures is to alter nucleosome spacing. In general, sequences with repressive chromatin domains are packaged into highly regular nucleosome arrays, the regularity of which correlates with gene silencing. Thus, it is tempting to speculate that BRG1 may contribute to HP1alpha-mediated silencing by manipulating nucleosomal spacing (A. L. Nielsen, 2002).

Interaction of HP1 with Histone H3

Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. In P19 embryonal carcinoma (EC) nuclei, HP1alpha, beta and gamma form homo- and heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1 interacts with the linker histone H1. HP1 binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1 and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly (A. Nielsen, 2001).

In cultured mammalian cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1, creating a binding site for the chromo domain of HP1. SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter. In fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins (S. Nielsen, 2001).

The Rb protein functions as a repressor, at least partly, through the recruitment of histone deacetylase activity. Whether histone methylation might also be involved in Rb-mediated repression is considered in this study, since the SUV39H1 methylase has repressive potential. To establish whether Rb can associate with histone-methylase activity, a glutathione S-transferase (GST)-Rb fusion was incubated with nuclear extract, and any bound methylase activity was assayed on bulk histones as a substrate. GST-Rb can purify histone-methylase activity, whereas GST fusions to transcriptional activators such as P/CAF, E2F1, p53 and ATF2 do not. The Rb-associated methylase activity is specific for histone H3 and does not recognize the GAR substrate for arginine methylases (S. Nielsen, 2001).

An antibody directed against Rb can precipitate histone-methylase activity that is specific for histone H3. This methylase binds the pocket domain of Rb because tumor-derived mutations in the pocket (F706C), or truncations of the pocket (928 and 737), abolish binding to the methylase. The Rb-associated methylase has specificity for Lys 9 of histone H3 (S. Nielsen, 2001).

The SUV39H1 protein possesses lysine methylase activity, which resides within its conserved SET domain. As this enzyme has specificity for Lys 9 of histone H3 an investigation was carried out to see whether SUV39H1 could be the methylase associated with Rb. A GST-Rb fusion can bind to transfected, hemagglutinin (HA)-tagged SUV39H1. Endogenous Rb also associates with endogenous SUV39H1, as shown by a co-immunoprecipitation analysis (S. Nielsen, 2001).

Whether SUV39H1 can act as co-repressor with Rb was investigated. SUV39H1 represses the activity of a promoter bearing GAL4 sites in a concentration-dependent manner in vivo, but only when Gal4-Rb is present at the promoter. The co-repressor functions of SUV39H1 can also be seen on the cyclin E promoter, a natural target for Rb-mediated repression. This promoter can be stimulated by E2F and is not affected by SUV39H1 alone. Under limiting conditions, where Rb represses E2F activity slightly, the SUV39H1 enzyme can further repress E2F activity in cooperation with Rb. When the methylase domain of SUV39H1 is removed, the resulting SUV39H1SET is unable to mediate repression. These results suggest that SUV39H1 uses its methylase activity to repress the cyclin E promoter when it is targeted there by Rb (S. Nielsen, 2001).

SUV39H1 is known form a complex with the HP1 protein. Recently, HP1 function has been placed downstream of SUV39H1 histone methylation, since HP1 recognizes specifically, and binds to, histone H3 methylated at Lys 9. This mechanistic link prompted an investigation of the role of HP1 in Rb/SUV39H1-mediated repression. Rb and HP1 can interact in a two-hybrid screen in yeast, and it has been shown that there is an LXCXE motif (X is any amino acid) in HP1. It was therefore asked if HP1 binds to Rb in mammalian cells. A GST-HP1 fusion can bind Rb that is present in nuclear extracts; Rb and HP1 can associate in vivo, as determined by co-immunoprecipitation analysis. An LXCXE motif peptide can compete for the binding of histone H3 methylase activity to Rb, but does not affect the binding of H3 methylase activity to HP1, which is consistent with the finding that the methylase activity is associated with the Rb pocket (S. Nielsen, 2001).

Whether HP1 can recognize methylated lysines while associated with Rb was tested. To address this, a histone H3 peptide methylated at Lys 9 was used as an affinity resin. Recombinant Rb does not bind to this methylated peptide, but it can do so efficiently in the presence of recombinant HP1. This result confirms that HP1 can bind directly to Rb and that it can recognize Rb and methylated lysine simultaneously. A similar experiment was attempted using nuclear extracts as the source of protein. The H3 peptide methylated at Lys 9 binds to HP1, SUV39H1 and Rb, as detected by Western blotting (S. Nielsen, 2001).

These results suggest that an Rb-regulated promoter such as cyclin E should be associated with HP1. To test this chromatin immunoprecipitation analysis of the cyclin E promoter was performed. A nucleosome encompassing the cyclin E initiation site (cyclin Epr) that is known to be deacetylated is associated with HP1 in fibroblast cells of mouse embryos. Since the cyclin Epr nucleosome binds HP1, whether this nucleosome contains histone H3 that is methylated at Lys 9 was examined. To test this an antibody was produced that recognizes histone H3 when methylated at Lys 9. In Rb+/+ cells the cyclin Epr nucleosome contains methylated histone H3 and is associated with HP1. However, in Rb-/- cells histone H3 methylation and HP1 binding is significantly reduced. Thus, in the presence of Rb, methylase activity and HP1 are targeted to the cyclin E promoter (S. Nielsen, 2001).

Collectively, these results implicate each of the components of the SUV39H1-HP1 complex in the repression functions of the Rb protein. In this model Rb brings to the promoter the SUV39H1 enzyme (and possibly other members of this family) to methylate Lys 9 of histone H3 and provides a binding site for HP1. Methylation by SUV39H1 cannot take place on an already acetylated lysine. Thus the deacetylase activity associated with Rb may be a necessary preceding step to SUV39H1-mediated methylation. The precise function of HP1 in repression is unclear. HP1 may protect the methyl group on Lys 9 from attack from potential demethylases; it may bring in other repressive functions, or it may enhance the stability of the Rb-associated repressor complex (S. Nielsen, 2001).

HP1 is found associated with a number of transcriptional repressors, suggesting that it may have a role in repressing many other promoters. Thus, the results presented here extend the role of SUV39H1 and HP1 beyond heterochromatic gene silencing to a more general, genome-wide function in repressing gene transcription (S. Nielsen, 2001).

Distinct modifications of histone amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. In addition to histone modifications that facilitate gene activity, it is of similar importance to restrict inappropriate gene expression if cellular and developmental programs are to proceed unperturbed. Mammalian methyltransferases that selectively methylate histone H3 on lysine 9 (Suv39h HMTases) generate a binding site for HP1 proteins -- a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure. High-affinity in vitro recognition of a methylated histone H3 peptide by HP1 requires a functional chromo domain; thus, the HP1 chromo domain is a specific interaction motif for the methyl epitope on lysine9 of histone H3. In vivo, heterochromatin association of HP1 proteins is lost in Suv39h double-null primary mouse fibroblasts but is restored after the re-introduction of a catalytically active SWUV39H1 HMTase. These data define a molecular mechanism through which the SUV39H-HP1 methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin (Lachner, 2001).

The human ISWI-containing factor RSF (remodeling and spacing factor) mediates nucleosome deposition and, in the presence of ATP, generates regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation is impaired in directing transcription. Histone-tail modifications regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 increases transcription. Methylation of the histone H3 tail by Suv39H1 represses transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system is refractory to the effect imposed by acetylation and methylation (Loyola, 2001).

In mammalian cells, as in Schizosaccharomyces pombe and Drosophila, HP1 proteins bind histone H3 tails methylated on lysine 9 (K9). However, whereas K9-methylated H3 histones are distributed throughout the nucleus, HP1 proteins are enriched in pericentromeric heterochromatin. This observation suggests that the methyl-binding property of HP1 may not be sufficient for its heterochromatin targeting. The association of HP1alpha with pericentromeric heterochromatin is shown to depend not only on its methyl-binding chromo domain but also on an RNA-binding activity present in the hinge region of the protein that connects the conserved chromo and chromoshadow domains. These data suggest the existence of complex heterochromatin binding sites composed of methylated histone H3 tails and RNA, with each being recognized by a separate domain of HP1alpha (Muchardt, 2002).

Cellular senescence is an extremely stable form of cell cycle arrest that limits the proliferation of damaged cells and may act as a natural barrier to cancer progression. A distinct heterochromatic structure is descibed that accumulates in senescent human fibroblasts, that is designated senescence-associated heterochromatic foci (SAHF). SAHF formation coincides with the recruitment of heterochromatin proteins and the retinoblastoma (Rb) tumor suppressor to E2F-responsive promoters and is associated with the stable repression of E2F target genes. Notably, both SAHF formation and the silencing of E2F target genes depend on the integrity of the Rb pathway and do not occur in reversibly arrested cells. These results provide a molecular explanation for the stability of the senescent state, as well as new insights into the action of Rb as a tumor suppressor (Narita, 2003).

SAHFs are observed in interphase nuclei and contain the heterochromatin-associated proteins H3 methylated on lysine 9 (K9M-H3) and HP1, exclude histones found in euchromatin (e.g., K9Ac-H3 and K4M-H3), and are not sites of active transcription. SAHFs are distinct from pericentric heterochromatin, and their appearance is accompanied by an increase in HP1 incorporation into senescent chromatin and an enhanced resistance of senescent DNA to nuclease digestion (Narita, 2003).

SAHF formation requires an intact Rb pathway, since expression of E1A, or inactivation of either p16INK4a or Rb, can prevent their appearance. During the initial phases of senescence, Rb might control the nucleation of heterochromatin at specific sites throughout the genome, which then spreads by the action of histone methyltransferases and recruitment of HP1 proteins. HP1 proteins have the capacity to dimerize and may interact to form higher order chromatin structures once a critical mass has been reached. A similar pattern of nucleation and spreading occurs during silencing of the mating type locus in S. pombe, position effect variegation in Drosophila, and X inactivation in mammalian cells, although HP1 proteins do not accumulate on the inactive X. Importantly, SAHF formation correlates precisely with cell cycle exit and the silencing of E2F target genes (Narita, 2003).

Much of what is known concerning the regulation of E2F activity comes from studies examining cell cycle transitions into and out of a quiescent state. These transitions are controlled in a reversible manner, in part, by the competing action of HATs and HDACs on the histones of E2F target promoters. This study compares the physical state and regulation of E2F target genes in quiescent and senescent cells. In both cell states, the amount of K9-aceylated histone H3 that associates with E2F target promoters declines, consistent with the downregulation of transcription that accompanies cell cycle exit. However, in senescent IMR90 cells, histone H3 acetylation is ultimately replaced by methylation at lysine 9, an apparently irreversible modification that prevents acetylation by HATs and is barely observed on E2F-responsive promoters in quiescent cells. Methylated lysine 9 forms a docking site for HP1 proteins and, accordingly, HP1gamma preferentially associates with E2F target promoters in senescent cells. These modifications are predicted to form a 'lock' on the transcription of E2F responsive promoters, making them less accessible to the transcription machinery. Accordingly, several E2F-responsive genes in senescent cells are stably repressed and insensitive to enforced E2F expression relative to quiescent cells. Although it remains to be determined whether every E2F target gene behaves as those studied here, their transition to a heterochromatin-like organization may contribute to the insensitivity of senescent cells to mitogenic signals and the apparent irreversibility of the senescence process (Narita, 2003).

One function of heterochromatin is the epigenetic silencing by sequestration of genes into transcriptionally repressed nuclear neighborhoods. Heterochromatin protein 1 (HP1) is a major component of heterochromatin and thus is a candidate for establishing and maintaining the transcriptionally repressive heterochromatin structure. Maintenance of stable heterochromatin domains in living cells is demonstrated to involve the transient binding and dynamic exchange of HP1 from chromatin. HP1 exchange kinetics correlate with the condensation level of chromatin and are dependent on the histone methyltransferase Suv39h. The chromodomain and the chromoshadow domain of HP1 are both required for binding to native chromatin in vivo, but they contribute differentially to binding in euchromatin and heterochromatin. These data argue against HP1 repression of transcription by formation of static, higher order oligomeric networks but support a dynamic competition model, and they demonstrate that heterochromatin is accessible to regulatory factors (Cheutin, 2003).

A histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity; Phosphorylation of H3S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin

Histone deacetylase (HDAC) inhibitors perturb the cell cycle and have great potential as anti-cancer agents, but their mechanism of action is not well established. HDACs classically function as repressors of gene expression, tethered to sequence-specific transcription factors. This study reports that HDAC3 is a critical, transcription-independent regulator of mitosis. HDAC3 forms a complex with A-Kinase-Anchoring Proteins AKAP95 and HA95, which are targeted to mitotic chromosomes. Deacetylation of H3 in mitosis requires AKAP95/HA95 and HDAC3 and provides a hypoacetylated H3 tail that is the preferred substrate for Aurora B kinase. Phosphorylation of H3S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin. This transcription-independent pathway, involving interdependent changes in histone modification and protein association, is required for normal progression through mitosis and is an unexpected target of HDAC inhibitors, a class of drugs currently in clinical trials for treating cancer (Li, 2006).

The classic role of HDAC3 has been that of a transcriptional repressor of gene expression, as part of a complex tethered to sequence-specific transcription factors. This study reports the unexpected finding that HDAC3 has a critical, transcription-independent function in mitosis. In interphase cells, AKAP95/HA95 binds to the nuclear matrix and is less associated with HDAC3. HP1 proteins are recruited to methylated H3K9 in heterochromatin. When cells enter into mitosis, AKAP95/HA95 may target the HDAC3 complex to deacetylate H3, in a reaction that is blocked by HDAC inhibitors, and thereby provides a hypoacetylated H3 tail as substrate for Aurora B to phosphorylate on S10. Phosphorylation of S10 by Aurora B then dissociates HP1 proteins from methylated H3K9 residues on mitotic heterochromatin, which has been referred to as the 'meth-phos switch'. These interdependent changes in histone modification and protein association are required for normal progression through mitosis, perhaps by facilitating chromosome condensation, or by serving as the indicator for the mitotic checkpoint to control proper cell division (Li, 2006).

While the transcriptional effect of HDAC inhibitors on specific genes, such as p21 and other cell cycle-regulated genes, has been reported to contribute to their anti-tumor actions, especially in G1-phase arrest, their direct effects on histone acetylation levels may be equally important for the anti-tumor activity because of the important functions of histones in different cellular processes, including mitosis. It is increasingly clear that HDAC inhibition induces G2/M arrest in many human cell lines and causes mitotic defects in different cancer cell lines. This effect of HDAC inhibition is independent of ongoing gene transcription, suggesting direct effects of histone hyperacetylation on mitosis. These results indicate that the hyperacetylation of histones induced by HDAC inhibitors directly interfere with mitotic progression (Li, 2006).

Global histone acetylation is reduced during mitosis. The current studies reveal that HDAC3 and its partner proteins AKAP95 and HA95 are required for global histone deacetylation during mitosis. Of note, the most dramatic change in acetylation that occurs during mitosis is hypoacetylation of Lys 5 of H4, which matches the substrate specificity of HDAC3. Moreover, the results clearly show that HDAC3 is required for normal mitotic progression. This is consistent with a recent study in which knockdown of HDAC3, but not HDAC1 or HDAC2, increased cells in G2/M phase in human colon cancer cells. Furthermore, knockdown of HDAC3 or AKAP95/HA95 also mimics the effects of nonselective HDAC inhibition on phosphorylation of H3S10 and retention of HP1β proteins on mitotic chromosomes. Inhibition of HDAC3 is therefore likely to be the mechanism by which HDAC inhibitors induce the G2/M block in the cell cycle. The transcription independence of this effect, while unexpected, is completely consistent with a direct mitotic function of HDAC3 in the context of the novel pathway that that is reported here (Li, 2006).

Specific patterns of histone modification at gene promoters regulate transcription via a 'histone code'. Notably, the transient phosphorylation of H3S10 has been reported in the promoter region of many mammalian immediate-early genes, which are rapidly induced in response to extracellular stimuli including UV radiation, growth factors, and cytokines. On these promoters, the phosphorylation of H3S10 precedes the H3K14 acetylation, resulting in multiple modifications of H3 that facilitate gene activation. On the contrary, this study found that the phosphorylation of H3S10 by Aurora B during mitosis requires the previous deacetylation of histones by HDAC3. Thus, in contrast to the phosphorylation of H3S10 by other kinases that prefer preacetylated histone tails, the mitotic phosphorylation of H3S10 by Aurora B kinase is linked to the deacetylation of H3, specifically by HDAC3. This characteristic of Aurora B may be specific to metazoans because IPL1, the yeast homolog of Aurora kinase, phosphorylated both monoacetylated and unacetylated H3. In addition to H3S10, Aurora B also phosphorylates H3S28 and other proteins including his- tone H3 variant centromere protein A (CENP-A). In human cell systems, Aurora B also seems to prefer hypoacetylated H3 and CENP-A H3 as substrate for phosphorylation of H3S28 and CENP-A Ser7, respectively. The global hypoacetylation of H3 tail lysines in mitotic cells and their proximity to the major sites of phosphorylation by Aurora B kinase suggest that deacetylation of histone substrates may be a general preference for Aurora B function. The relative importance of specific hypoacetylated lysines for phosphorylation of specific serine residues remains to be elucidated (Li, 2006).

The specificity of Aurora B toward hypoacetylated histone substrate suggests a mechanistic link between HDAC3-dependent histone deacetylation and a transcription-independent mechanism of mitotic arrest. H3S10 phosphorylation during mitosis is characteristic of many organisms, and is dependent on Aurora B kinase, which plays a central role throughout different stage of mitosis, including chromosome condensation, alignment, and segregation, spindle assembly, and cytokinesis. The recent finding that Aurora-dependent phosphorylation of H3S10 dissociates HP1 from mitotic heterochromatin provides molecular insight into the function of Aurora B. The current findings implicate AKAP95/HA95 and HDAC3 as upstream regulators of this "meth-phos switch", and provide a molecular mechanism to explain the anti-cancer effects of HDAC inhibitors. Aurora B kinase itself is overexpressed in a large number of cancers. The finding that Aurora B is present in HDAC3 complexes and that its kinase activity is dramatically greater when the H3 tail is hypoacetylated suggests that the interdependence of Aurora B and HDAC3 may be a novel and specific target for cancer therapies that would overcome the toxicity of nonspecific HDAC inhibitors (Li, 2006).

HP1 and JAK pathway tumorogenesis

The JAK/STAT pathway has pleiotropic roles in animal development, and its aberrant activation is implicated in multiple human cancers. JAK/STAT signaling effects have been attributed largely to direct transcriptional regulation by STAT of specific target genes that promote tumor cell proliferation or survival. In a Drosophila hematopoietic tumor model, however, that JAK overactivation globally disrupts heterochromatic gene silencing, an epigenetic tumor suppressive mechanism. This disruption allows derepression of genes that are not direct targets of STAT, as evidenced by suppression of heterochromatin-mediated position effect variegation. Moreover, mutations in the genes encoding heterochromatin components heterochromatin protein 1 (HP1) and Su(var)3-9 enhance tumorigenesis induced by an oncogenic JAK kinase without affecting JAK/STAT signaling. Consistently, JAK loss of function enhances heterochromatic gene silencing, whereas overexpressing HP1 suppresses oncogenic JAK-induced tumors. These results demonstrate that the JAK/STAT pathway regulates cellular epigenetic status and that globally disrupting heterochromatin-mediated tumor suppression is essential for tumorigenesis induced by JAK overactivation (Shi, 2006).

JAK2 phosphorylates histone H3Y41 and excludes HP1alpha from chromatin

Activation of Janus kinase 2 (JAK2) by chromosomal translocations or point mutations is a frequent event in haematological malignancies. JAK2 is a non-receptor tyrosine kinase that regulates several cellular processes by inducing cytoplasmic signalling cascades. This study shows that human JAK2 is present in the nucleus of haematopoietic cells and directly phosphorylates Tyr 41 (Y41) on histone H3. Heterochromatin protein 1alpha (HP1alpha), but not HP1beta, specifically binds to this region of H3 through its chromo-shadow domain. Phosphorylation of H3Y41 by JAK2 prevents this binding. Inhibition of JAK2 activity in human leukaemic cells decreases both the expression of the haematopoietic oncogene lmo2 and the phosphorylation of H3Y41 at its promoter, while simultaneously increasing the binding of HP1alpha at the same site. These results identify a previously unrecognized nuclear role for JAK2 in the phosphorylation of H3Y41 and reveal a direct mechanistic link between two genes, jak2 and lmo2, involved in normal haematopoiesis and leukaemia (Dawson, 2009).

Lamin interaction with HP1 homologs and the nuclear envelope

At the nuclear envelope in higher eukaryotic cells, the nuclear lamina (See Drosophila Lamin) and the heterochromatin are adjacent to the inner nuclear membrane, and their attachment is presumably mediated by integral membrane proteins. In a yeast two-hybrid screen, the nucleoplasmic domain of lamin B receptor (LBR), an integral protein of the inner nuclear membrane, associates with two human polypeptides homologous to Drosophila HP1, a heterochromatin protein involved in position-effect variegation. LBR fusion protein binds to HP1 proteins present in cell lysates and synthesized by in vitro translation. Antibodies against LBR also co-immunoprecipitate HP1 proteins from cell extracts. LBR can interact with chromodomain proteins that are highly conserved in eukaryotic species and may function in the attachment of heterochromatin to the inner nuclear membrane in cells (Ye, 1996).

To study the dynamics of mammalian HP1 proteins recombinant forms of mHP1alpha, M31 and M32 have been microinjected into the cytoplasm of living cells. The three fusion proteins were efficiently transported into the nucleus and targeted specific chromatin areas. However, before incorporation into these areas the exogenous proteins accumulate in a peripheral zone and associate closely with the nuclear envelope. This transient association does not occur when the cells are treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibit saturable binding to purified nuclear envelopes and stain the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allows mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His6-tagged peptide representing this region inhibits recruitment of LAP2beta and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly (Kourmouli, 2000).

HP1 and telomeres

Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. In human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsalpha) or HP1(Hsbeta), but not HP1(Hsgamma), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsalpha) or HP1(Hsbeta) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-DeltaHP1(Hsalpha), GFP-DeltaHP1(Hsbeta), or GFP-DeltaHP1(Hsgamma) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsalpha) or HP1(Hsbeta). In contrast, cells expressing mutant GFP-DeltaHP1(Hsalpha) or GFP-DeltaHP1(Hsbeta) showed a decrease in population doubling time and decreased sensitivity to IR when compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsalpha) or HP1(Hsbeta) suppressed tumorigenicity, whereas expression of mutant HP1(Hsalpha) or HP1(Hsbeta) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsalpha) or HP1(Hsbeta) present, because their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state (Sharma, 2003).

Aurora-B/AIM-1 regulates the dynamic behavior of HP1alpha at the G2-M transition

Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation and undergoes large-scale, progressive dissociation from heterochromatin in prophase cells. However, the mechanisms regulating the dynamic behavior of HP1 are poorly understood. This study investigated the role of Aurora-B with respect to the dynamic behavior of HP1alpha. Mammalian Aurora-B (see Drosophila Aurora B), AIM-1, colocalizes with HP1alpha to the heterochromatin in G2. Depletion of Aurora-B/AIM-1 inhibits dissociation of HP1alpha from the chromosome arms at the G2-M transition. In addition, depletion of INCENP (see Drosophila Inner centromere protein) leads to aberrant cellular localization of Aurora-B/AIM-1, but it does not affect heterochromatin targeting of HP1alpha. It has been proposed in the binary switch hypothesis that phosphorylation of histone H3 at Ser-10 negatively regulates the binding of HP1alpha to the adjacent methylated Lys-9. However, Aurora-B/AIM-1-mediated phosphorylation of H3 induces dissociation of the HP1alpha chromodomain but not of the intact protein in vitro, indicating that the center and/or C-terminal domain of HP1alpha interferes with the effect of H3 phosphorylation on HP1alpha dissociation. Interestingly, Lys-9 methyltransferase SUV39H1 is abnormally localized together along the metaphase chromosome arms in Aurora-B/AIM-1-depleted cells. In conclusion, these results showed that Aurora-B/AIM-1 is necessary for regulated histone modifications involved in binding of HP1alpha by the N terminus of histone H3 during mitosis (Terada, 2006).

A chromatin code for alternative splicing involving a putative association between CTCF and HP1alpha proteins

Alternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII). Recent experiments have suggested a new complex network of splicing regulation involving chromatin, transcription and multiple protein factors. In particular, the CCCTC-binding factor (CTCF), the Argonaute protein AGO1, and members of heterochromatin protein 1 (HP1) family have been implicated in the regulation of splicing associated to chromatin and the elongation of RNAPII. These results raise the question of whether these proteins may associate at the chromatin level to modulate alternative splicing. Using ChIP-Seq data for CTCF, AGO1, HP1alpha, H3K27me3, H3K9me2, H3K36me3, RNAPII, total H3 and 5metC and alternative splicing arrays from two cell lines, this study analyzed the combinatorial code of their binding to chromatin in relation to the alternative splicing patterns between two mammalian cell lines, MCF7 and MCF10. Using Machine Learning techniques, the changes were obtained in chromatin signals that are most significantly associated to splicing regulation between these two cell lines. Moreover, a map was built of the chromatin signals on the pre-mRNA, i.e., a chromatin-based RNA-map, which can explain 606 (68.55%) of the regulated events between MCF7 and MCF10. This chromatin code involves the presence of HP1alpha, CTCF, AGO1, RNAPII and histone marks around regulated exons and can differentiate patterns of skipping and inclusion. Additionally, a significant association of HP1alpha and CTCF activities was found around the regulated exons and a putative DNA binding site for HP1alpha. These results show that a considerable number of alternative splicing events could have a chromatin-dependent regulation involving the association of HP1alpha and CTCF near regulated exons. Additionally, further evidence was found for the involvement of HP1alpha and AGO1 in chromatin-related splicing regulation (Agirre, 2015).

Alternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII). Recent experiments have suggested a new complex network of splicing regulation involving chromatin, transcription and multiple protein factors. In particular, the CCCTC-binding factor (CTCF), the Argonaute protein AGO1, and members of heterochromatin protein 1 (HP1) family have been implicated in the regulation of splicing associated to chromatin and the elongation of RNAPII. These results raise the question of whether these proteins may associate at the chromatin level to modulate alternative splicing (Agirre, 2015).

Using ChIP-Seq data for CTCF, AGO1, HP1α, H3K27me3, H3K9me2, H3K36me3, RNAPII, total H3 and 5metC and alternative splicing arrays from two cell lines, an analysis was carried out of the combinatorial code of their binding to chromatin in relation to the alternative splicing patterns between two cell lines, MCF7 and MCF10. Using Machine Learning techniques, the changes in chromatin signals were obtained that are most significantly associated to splicing regulation between these two cell lines. Moreover, a map was built of the chromatin signals on the pre-mRNA, i.e., a chromatin-based RNA-map, which can explain 606 (68.55%) of the regulated events between MCF7 and MCF10. This chromatin code involves the presence of HP1α, CTCF, AGO1, RNAPII and histone marks around regulated exons and can differentiate patterns of skipping and inclusion. Additionally, a significant association was found of HP1α and CTCF activities around the regulated exons and a putative DNA binding site for HP1α. These results show that a considerable number of alternative splicing events could have a chromatin-dependent regulation involving the association of HP1α and CTCF near regulated exons. Additionally, further evidence was found for the involvement of HP1α and AGO1 in chromatin-related splicing regulation (Agirre, 2015).

This work has derived a chromatin code for splicing that involves binding signals for HP1α and CTCF, as well as AGO1, RNAPII and histone marks, activity around regulated exons. Feature selection and cross-validation shows that this regulatory code is predictive for nearly 70% of the alternative splicing events regulated between two cell lines, MCF7 and MCF10, providing further evidence for a role of chromatin in the regulation of alternative splicing. This code also provides evidence for specific associations of various factors in relation to splicing differences between the two studied cell lines. This model shows that AGO1 activity downstream of alternative exon correlates with splicing changes in the direction of skipping in MCF7 compared to MCF10A, providing further indication that AGO1 association to chromatin could be implicated in splicing regulation. The previously described increased binding of CTCF binding downstream of inclusion events was also uncovered. Additionally, the density of RNAPII downstream of regulated exons, which tends to co-occur with CTCF and HP1α is an informative attribute to predict splicing change; and a relative increase in the region flanking the exon correlates with exon skipping in MCF7 compared to MCF10A. The association of the RNAPII density related to exon definition has been observed before and there is plenty of evidence supporting a regulation of alternative splicing associated with RNAPII elongation rates. These results corroborate the importance of RNAPII occupancy in the exon inclusion or skipping, and provide directionality in the relation between density changes and the pattern of differential splicing between cell lines (Agirre, 2015).

H3K36me3 also appeared as a relevant mark for splicing decisions in the current model. Several reports have described H3K36me3 as an exon marker and there is evidence of higher densities of H3K36me3 at constitutive exons compared to alternative exons. However, the opposite pattern has also been described, as for specific genes an increased density of H3K36me3 has been related to exon skipping, which agrees with the current study. Since since this study only analyzed splicing events in genes that do not change expression, the results imply that the observed changes in H3K36me3 signal near exon boundaries were not a consequence of gene expression, and could indeed correspond to a role in splicing (Agirre, 2015).

Interestingly, this study found a strong association between CTCF and HP1α signals genome-wide and intragenically, and the activity of both factors correlate with exon inclusion. Besides acting as insulator, CTCF is involved in the splicing regulation of some exons as an antagonist of DNA methylation and also works as a barrier for spreading of heterochromatin, through which it can influence RNAPII elongation. These analyses show that HP1α-binding downstream of the cassette exons, with the co-localization of CTCF, affects alternative splicing. HP1α belongs to a family of non-histone chromosomal proteins and is a key player in the transcriptional gene silencing (TGS) pathway. HP1 proteins have already been linked before to the regulation of splicing by chromatin. In particular, a study published the conclusion of this work also describes a positional effect on splicing for HP 1 proteins, providing further evidence of the relevance of the HP1 family in linking chromatin with RNA processing and giving support to the curren model. The same study found that HP1 proteins could act as mediators between DNA methylation and splicing for a subset of the regulated events. Although there have been previous reports of a relation between DNA methylation and alternative splicing, this study did not find it to be a strong determinant of the splicing changes between MCF7 and MCF10 cells, indicating that the HP1-dependent code that this study describes is related to a DNA-methylation independent effect that may be more prevalent in the investigated cell types (Agirre, 2015).

Even though there is only limited evidence of direct DNA-binding for HP1α, this study found a consensus motif associated to the significant HP1α-ChIP-Seq signals, which is highly specific to the significant HP1αChIP-Seq signals and non-overlapping with the motifs for CTCF, AGO1 or H3K9me2. HP1 proteins generally consist of two highly conserved domains. While one of the domains is known to bind H3K9me, the other one acts as the interaction interface with other proteins. The two domains are separated by a hinge region of variable length, which has been related to DNA and RNA binding. The found motif may be related to a sequence-specific interaction of this protein region with DNA, which may act as a modulator of the interaction of HP1 with H3K9 methylation. Recent analyses also provide evidence of HP1 proteins interacting with RNA binding proteins, highlighting their plasticity and central role in RNA processing regulation linked to chromatin (Agirre, 2015).

This study also found a frequent overlap of AGO1 with CTCF and HP1α-clusters, but not the other way around. Moreover, HP1α was found in the same downstream region as AGO1 but in the direction of inclusion, and regulating a distinct set of events. Depletion of AGO1 expression can induce splicing changes in both directions but generally decreases splicing efficiency. These analyses show that AGO1 and the co-localized CTCF and HP1α produce splicing changes in opposite directions. Despite the co-localization of AGO1 with CTCF and HP1α binding sites, this study found a weak but independent binding motif for AGO1. Recent analyses have produced candidate binding motifs for Drosophila and mouse Argonaute proteins. However, the motif from the current study does not resemble any of these motifs, suggesting a DNA- independent association of AGO1 to chromatin (Agirre, 2015).

Different predictive models to predict the splicing outcome, also called splicing codes, have been proposed before, but these did not include chromatin marks or proteins that interact with chromatin, like HP1, AGO1, CTCF and RNAPII, as described in this study. These analyses thus complement these previous descriptions by incorporating these new determinants of alternative splicing regulation. Although, motifs in the pre-mRNA sequence remain the main determinants of splicing regulation, this analysis indicates that a considerable fraction may be influenced by the properties of chromatin. There have been previous attempts to establish a general relation between histone marks and splicing regulation. However, only in one case a predictive model was proposed. Additionally, these approaches analyzed the relation between chromatin and splicing looking at one single condition at the time, rather than comparing two conditions, and exons were classified as constitutive or alternative based on RNA data from one single condition, rather than distinguishing those that are regulated from non-regulated ones between two conditions. The current approach has the advantage that, by comparing two conditions locally, it circumvents the caveats of comparing genomic regions with different sequence and structural properties. Moreover, the current approach relates changes of the chromatin signal between two conditions to the splicing changes of exons between the same two conditions, which provides a better descriptor of the association between chromatin changes and splicing regulation. In summary, this study has shown that a chromatin code for splicing can be defined involving HP1α, CTCF, RNAPII, various histone marks and AGO1, which can differentiate patterns of skipping, inclusion and non-regulated exons between two conditions. Additionally, the conserved motif found for HP1α and the presence of HP1α and AGO1 in the described splicing code provides further support for their involvement in chromatin-related splicing regulation (Agirre, 2015).

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

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.