Analysis of chromatin structure of genes silenced by heterochromatin in trans

While heterochromatic gene silencing in cis is often accompanied by nucleosomal compaction, characteristic histone modifications, and recruitment of heterochromatin proteins, little is known concerning genes silenced by heterochromatin in trans. An insertion of heterochromatic satellite DNA in the euchromatic brown (bw) gene of Drosophila melanogaster results in bwDominant (bwD), which can inactivate loci on the homolog by relocation near the centric heterochromatin (trans-inactivation). Nucleosomal compaction was found to accompany trans-inactivation, but stereotypical heterochromatic histone modifications were mostly absent on silenced reporter genes. HP1 was enriched on trans-inactivated reporter constructs and this enrichment was more pronounced on adult chromatin than on larval chromatin. Interestingly, this HP1 enrichment in trans was unaccompanied by an increase in the 2MeH3K9 mark, which is generally thought to be the docking site for HP1 in heterochromatin. However, a substantial increase in the 2MeH3K9 mark was found on or near the bwD satellite insertion in cis, but did not spread further. These observations suggest that the interaction of HP1 with chromatin in cis is fundamentally different from that in trans. Our molecular data agree well with the differential phenotypic effect on bwD trans-inactivation of various genes known to be involved in histone modification and cis gene silencing.     

Sequence elements in cis influence heterochromatic silencing in trans

The brown(Dominant) (bw(D)) allele contains a large insertion of heterochromatin, which causes the locus to aberrantly associate with heterochromatin in interphase nuclei and silences the wild-type allele in heterozygotes. Transgenes placed near the bw(+) locus, in trans to bw(D), can also be silenced. The strength of silencing (called trans inactivation) varies with the regulatory sequences of the transgene and its distance away from the bw(D) insertion site in trans. In this study, we examine endogenous sequences in cis that influence susceptibility of a reporter gene to trans inactivation. Flanking deletions were induced in two parental lines containing P-element transgenes showing trans inactivation of the mini-white reporter. These new lines, which have mini-white under the influence of different endogenous sequence elements, now show varied ability to be silenced by bw(D). Determination of the deleted regions and the levels of mini-white expression and trans inactivation has allowed us to explore the correlation between cis sequence elements and susceptibility to trans inactivation and to identify a 301-bp sequence that acts as an enhancer of trans inactivation. Intriguingly, this region encompasses the upstream regions of two divergently transcribed genes and contains a sequence motif that may bind BEAF, a protein involved in delimiting chromatin boundaries.     

Comparative analysis of position-effect variegation mutations in Drosophila melanogaster delineates the targets of modifiers.

In Drosophila melanogaster, heterochromatin-induced silencing or position-effect variegation (PEV) of a reporter gene has provided insights into the properties of heterochromatin. Class I modifiers suppress PEV, and class II modifiers enhance PEV when the modifier gene is present in fewer than two doses. We have examined the effects of both class I and class II modifiers on four PEV mutations. These mutations include the inversions In(1)w(m4) and In(2R)bw(VDe2), which are classical chromosomal rearrangements that typify PEV mutations. The other mutations are a derivative of brown(Dominant), in which brown+ reporters are inactivated by a large block of heterochromatin, and a P[white+] transposon insertion associated with second chromosome heterochromatin. In general, we find that class I modifiers affect both classical and nonclassical PEV mutations, whereas class II modifiers affect only classical PEV mutations. We suggest that class II modifiers affect chromatin architecture in the vicinity of reporter genes, and only class I modifiers identify proteins that are potentially involved in heterochromatin formation or maintenance. In addition, our observations support a model in which there are different constraints on the process of heterochromatin-induced silencing in classical vs. nonclassical PEV mutations.     

Intercalary heterochromatin in polytene chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Intercalary heterochromatin consists of extended chromosomal domains which are interspersed throughout the euchromatin and contain silent genetic material. These domains comprise either clusters of functionally unrelated genes or tandem gene duplications and possibly stretches of noncoding sequences. Strong repression of genetic activity means that intercalary heterochromatin displays properties that are normally attributable to classic pericentric heterochromatin: high compaction, late replication and underreplication in polytene chromosomes, and the presence of heterochromatin-specific proteins. Late replication and underreplication occurs when the suppressor of underreplication protein is present in intercalary heterochromatic regions. Intercalary heterochromatin underreplication in polytene chromosomes results in free double-stranded ends of DNA molecules; ligation of these free ends is the most likely mechanism for ectopic pairing between intercalary heterochromatic and pericentric heterochromatic regions. No support has been found for the view that the frequency of chromosome aberrations is elevated in intercalary heterochromatin.     

The effect of JIL-1 on position-effect variegation is proportional to the total amount of heterochromatin in the genome

In this study we have taken advantage of recent whole genome sequencing studies that have determined the DNA content in the heterochromatic regions of each Drosophila chromosome to directly correlate the effect on position-effect variegation of a pericentric insertion reporter line, 118E-10 with the total amount of heterochromatic DNA. Heterochromatic DNA levels were manipulated by adding or subtracting a Y chromosome as well as by the difference in the amount of pericentric heterochromatin between the X and Y chromosome. The results showed a direct, linear relationship between the amount of heterochromatic DNA in the genome and the expression of the w marker gene in the 118E-10 pericentric reporter line and that increasing amounts of heterochromatic DNA resulted in increasing amounts of pigment/gene activity. In Drosophila heterochromatic spreading and gene silencing is counteracted by H3S10 phosphorylation by the JIL-1 kinase, and we further demonstrate that the haplo-enhancer effect of JIL-1 is proportional to the amount of total heterochomatin, suggesting that JIL-1's activity is dynamically modulated to achieve a more or less constant balance depending on the levels of heterochromatic factors present.     

Intercalary heterochromatin in Drosophila melanogaster polytene chromosomes and the problem of genetic silencing

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.     

Mechanisms of HP1-mediated gene silencing in Drosophila

Heterochromatin Protein 1 (HP1) is a structural component of silent chromatin at telomeres and centromeres. Euchromatic genes repositioned near heterochromatin by chromosomal rearrangements are typically silenced in an HP1-dependent manner. Silencing is thought to involve the spreading of heterochromatin proteins over the rearranged genes. HP1 associates with centric heterochromatin through an interaction with methylated lysine 9 of histone H3, a modification generated by SU(VAR)3-9. The current model for spreading of silent chromatin involves HP1-dependent recruitment of SU(VAR)3-9, resulting in the methylation of adjacent nucleosomes and association of HP1 along the chromatin fiber. To address mechanisms of silent chromatin formation and spreading, HP1 was fused to the DNA-binding domain of the E. coli lacI repressor and expressed in Drosophila melanogaster stocks carrying heat shock reporter genes positioned 1.9 and 3.7 kb downstream of lac operator repeats. Association of lacI-HP1 with the repeats resulted in silencing of both reporter genes and correlated with a closed chromatin structure consisting of regularly spaced nucleosomes, similar to that observed in centric heterochromatin. Chromatin immunoprecipitation experiments demonstrated that HP1 spread bi-directionally from the tethering site and associated with the silenced reporter transgenes. To examine mechanisms of spreading, the effects of a mutation in Su(var)3-9 were investigated. Silencing was minimally affected at 1.9 kb, but eliminated at 3.7 kb, suggesting that HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner.     

Synapsis of a chromosomal pair heterozygous for a pericentric inversion and the presence of a heterochromatic short arm

Analysis of meiotic pairing configurations in a deer mouse heterozygous for both a pericentric inversion and the presence of a heterochromatic short arm at chromosome 15 revealed straight-paired synaptonemal complexes with equal axial lengths in a majority of the pachytene nuclei. Nonhomologous pairing in this bivalent occurs by direct heterosynapsis of the inverted segments followed by synaptic adjustment of the heterochromatin heteromorphism.     

Cell-by-cell dissection of gene expression and chromosomal interactions reveals consequences of nuclear reorganization

The functional consequences of long-range nuclear reorganization were studied in a cell-by-cell analysis of gene expression and long-range chromosomal interactions in the Drosophila eye and eye imaginal disk. Position-effect variegation was used to stochastically perturb gene expression and probe nuclear reorganization. Variegating genes on rearrangements of Chromosomes X, 2, and 3 were probed for long-range interactions with heterochromatin. Studies were conducted only in tissues known to express the variegating genes. Nuclear structure was revealed by fluorescence in situ hybridization with probes to the variegating gene and heterochromatin. Gene expression was determined alternately by immunofluorescence against specific proteins and by eye pigment autofluorescence. This allowed cell-by-cell comparisons of nuclear architecture between cells in which the variegating gene was either expressed or silenced. Very strong correlations between heterochromatic association and silencing were found. Expressing cells showed a broad distribution of distances between variegating genes and their own centromeric heterochromatin, while silenced cells showed a very tight distribution centered around very short distances, consistent with interaction between the silenced genes and heterochromatin. Spatial and temporal analysis of interactions with heterochromatin indicated that variegating genes primarily associate with heterochromatin in cells that have exited the cell cycle. Differentiation was not a requirement for association, and no differences in association were observed between cell types. Thus, long-range interactions between distal chromosome regions and their own heterochromatin have functional consequences for the organism.     

The Polycomb Repressive Complex 1 Protein BMI1 Is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells

The polycomb repressive complex 1 (PRC1), containing the core BMI1 and RING1A/B proteins, mono-ubiquitinylates histone H2A (H2A^ub) and is associated with silenced developmental genes at facultative heterochromatin. It is, however, assumed that the PRC1 is excluded from constitutive heterochromatin in somatic cells based on work performed on mouse embryonic stem cells and oocytes. We show here that BMI1 is required for constitutive heterochromatin formation and silencing in human and mouse somatic cells. BMI1 was highly enriched at intergenic and pericentric heterochromatin, co-immunoprecipitated with the architectural heterochromatin proteins HP1, DEK1, and ATRx, and was required for their localization. In contrast, BRCA1 localization was BMI1-independent and partially redundant with that of BMI1 for H2A^ub deposition, constitutive heterochromatin formation, and silencing. These observations suggest a dynamic and developmentally regulated model of PRC1 occupancy at constitutive heterochromatin, and where BMI1 function in somatic cells is to stabilize the repetitive genome.     

Characterization of sequences associated with position-effect variegation at pericentric sites in Drosophila heterochromatin

In a variety of organisms, euchromatic genes brought into juxtaposition with pericentric heterochromatin show position-effect variegation (PEV), a silencing of gene expression in a subset of the cells in which the gene is normally expressed. Previously, a P-element mobilization screen identified transgenic Drosophila stocks showing PEV of an hsp70-white ⁺ reporter gene; transgenes in many of these stocks map to the chromocenter of polytene chromosome. A screen at an elevated temperature identified two stocks that under standard culture temperatures show complete repression of the hsp70-white ⁺ transgene. The transgenes in both cases map to the chromocenter of polytene chromosomes. Different types of middle repetitive elements are adjacent to seven pericentric transgenes; unique sequences are adjacent to two of the perimetric transgenes. All of the transgenes show suppression of PEV in response to a mutation in the gene encoding heterochromatin protein 1 (HP1). This suppression correlates with a more accessible chromatin structure. The results indicate that a pericentric transgene showing PEV can be associated with different types of DNA sequences, while maintaining a common association with the chromosomal protein HP1. Received: 15 January 1998; in revised form: 27 May 1998 / Accepted: 4 September 1998     

Copy Number and Orientation Determine the Susceptibility of a Gene to Silencing by Nearby Heterochromatin in Drosophila

The classical phenomenon of position-effect variegation (PEV) is the mosaic expression that occurs when a chromosomal rearrangement moves a euchromatic gene near heterochromatin. A striking feature of this phenomenon is that genes far away from the junction with heterochromatin can be affected, as if the heterochromatic state ``spreads.'''' We have investigated classical PEV of a Drosophila brown transgene affected by a heterochromatic junction ~60 kb away. PEV was enhanced when the transgene was locally duplicated using P transposase. Successive rounds of P transposase mutagenesis and phenotypic selection produced a series of PEV alleles with differences in phenotype that depended on transgene copy number and orientation. As for other examples of classical PEV, nearby heterochromatin was required for gene silencing. Modifications of classical PEV by alterations at a single site are unexpected, and these observations contradict models for spreading that invoke propagation of heterochromatin along the chromosome. Rather, our results support a model in which local alterations affect the affinity of a gene region for nearby heterochromatin via homology-based pairing, suggesting an alternative explanation for this 65-year-old phenomenon.     

A reexamination of spreading of position-effect variegation in the white-roughest region of Drosophila melanogaster

In Drosophila, heterochromatin causes mosaic silencing of euchromatic genes brought next to it by chromosomal rearrangements. Silencing has been observed to "spread": genes closer to the heterochromatic rearrangement breakpoint are silenced more frequently than genes farther away. We have examined silencing of the white and roughest genes in the variegating rearrangements In(1)w(m4), In(1)w(mMc), and In(1)w(m51b). Eleven stocks bearing these chromosomes differ widely in the strength of silencing of white and roughest. Stock-specific differences in the relative frequencies of inactivation of white and roughest were found that map to the white-roughest region or the adjacent heterochromatin. Most stock-specific differences did not correlate with gross differences in the heterochromatic content of the rearranged chromosomes; however, two stocks, In(1)w(m51b) and In(1)w(mMc), were found to have anomalous additional heterochromatin that may act in trans to suppress variegating alleles. In comparing different stocks, the frequency of silencing of the roughest gene, which is more distant from heterochromatin, does not correlate with the frequency of silencing of the more proximal white gene on the same chromosome, in contradiction to the expectation of models of continuous linear propagation of silencing. We frequently observed rough eye tissue that is pigmented, as though an active white gene is skipped.