1. Volume 15, Issue 3, Pages 467-476 (August 2004)
HP1 Controls Telomere Capping, Telomere Elongation, and Telomere Silencing by Two Different Mechanisms in Drosophila 
Barbara Perrini, Lucia Piacentini, Laura Fanti, Fabio Altieri, Silvia Chichiarelli, Maria Berloco, Carlo Turano, Anna Ferraro, Sergio Pimpinelli 
Molecular Cell 
Volume 15, Issue 3, Pages (August 2004)
DOI: /j.molcel
Copyright © 2004 Cell Press Terms and Conditions

2. Figure 1
HP1 Is Involved in the Control of Telomeric Sequence Transcription and Telomere Elongation
(A) A polytene chromosome spread from salivary glands of larvae from crosses between Su(var)2-504/Cy females and wild-type males. Telomeres from the Su(var)2-504/Cy strain are clearly elongated (arrows). An example of elongated telomeres from the Su(var)2-502/Cy strain (arrows) with respect to the wild-type telomeres (arrowheads) is shown in the insert.
(B–D) Examples of HeT-A (red) and TART (green) telomeric localization by in situ hybridization. Left: DAPI staining; middle: merged HeT-A and TART signals; right: merged image including DAPI. (B) Localization of HeT-A (red signals) and TART (green signals) on telomeres of wild-type larvae. HeT-A and TART sequences are distally and proximally, respectively, located on the paired telomeres of the left arm of the second chromosome (arrowheads). (C) Localization of HeT-A and TART on the telomeres of the left arm of the second chromosome from salivary glands of a larva from crosses between Su(var)2-504/Cy females and wild-type males. Compared to the wild-type telomere (arrowheads), the telomere from the Su(var)2-504/Cy strain shows an increase in HeT-A sequences (red arrows) and a terminal addition of TART sequences (arrows). (D) Another example of HeT-A and TART localization (from the same crosses as Figure 1C). In this case, the telomere elongation is due to telomeric rearrangements of the two transposons, rather than to a simple terminal addition. The telomere from the Su(var)2-504/Cy strain has in fact lost the proximal TART sequences (yellow arrows) that are present on the wild-type telomeres (arrowheads). In addition, the same telomere shows a terminal addition of TART sequences.
(E) HeT-A sequences are scarcely transcribed in wild-type, but they are clearly transcribed in heterozygous HP1 mutants and, more abundantly, in trans-heterozygous HP1 mutants with Su(var)2-504 (04/05) or the chromodomain Su(var)2-502 (02/05) mutations.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
HP1 and H3-Me3K9 Localization in Wild-Type Drosophila Polytene Chromosomes
(A) A polytene chromosome spread stained with DAPI.
(B) A merged image showing the immunopatterns of HP1 (red signals) and H3-Me3K9 (green signals) on the same preparation. The yellow/orange signals clearly show that HP1 and H3-Me3K9 colocalize at the chromocenter (chr), at the telomeres (white arrows), and at the euchromatic 31 region (white arrowhead). However, the separate green and red signals (examples at green and red arrows) show that several H3-Me3K9 sites and HP1 sites do not colocalize.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
The HP1 Chromodomain Is Not Required for HP1 Localization at the Telomeres or for Telomere Stability, but It Is Necessary for the Telomeric Localization of H3-Me3K9
(A) Polytene chromosomes of a Su(var)2-502/Su(var)2-505 (02/05) mutant larva stained with DAPI (red) and immunostained against HP1 (green). The presence of HP1 at all the telomeres (arrows) shows that the chromodomain is not required for its telomeric localization. chr = chromocenter.
(B) Particular of two telomeres of a polytene chromosome from a Su(var)2-502/Su(var)2-505 mutant larva, immunostained against H3-Me3K9. The absence of any immunosignal at the telomeres (arrows) shows that the HP1 chromodomain is required for the telomeric localization of H3-Me3K9.
(C) An example of neuroblast metaphase from Su(var)2-502/Su(var)2-505 mutant larvae (02/05). Note the absence of telomeric fusion.
(D) An example of neuroblast metaphase from a Su(var)2-504/Su(var)2-505 mutant larva lacking HP1 (04/05). There are clearly multiple telomeric fusions.
(E–G) Imaginal disks stained with acridine orange: (E) wild-type and (F) Su(var)2-502/Su(var)2-505 show the same phenotype, while (G) Su(var)2-504/Su(var)2-505 (04/05) shows an extensive apoptosis.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
The Telomeric Localizations of HP1 and H3-Me3K9 and the Transcription of Telomeric Sequences Do Not Depend on the Activity of the HMTases E(Z) and SU(VAR)3-9 or the Main Components of RNAi
(A) Simultaneous immunostainings against the E(Z) (insert) and HP1 proteins (right) of polytene chromosomes from a larva homozygous for the thermosensitive loss-of-function mutation E(z)61 (= E(z)S2) raised at the restrictive temperature of 29°C. While E(Z) immunostaining does not produce any signal, HP1 immunostaining produces a pattern similar to wild-type, including signals at all of the telomeres (arrows; chr = chromocenter).
(B) Polytene chromosomes from an E(z)61 larva raised at 29°C and immunostained against H3-Me3K9. The absence of E(Z) does not affect the telomeric localization of H3-Me3K9 (arrows).
(C) Polytene chromosomes from a wild-type larva immunostained against H3-Me3K27. The immunosignals are at the chromocenter and at many euchromatic sites on all of the chromosomes, but they are virtually absent at the telomeres (arrows).
(D) Polytene chromosomes from an E(z)61 larva raised at 29°C and immunostained against H3-Me3K27. Note the absence of any immunosignal.
(E) Neuroblast metaphase from an E(z) mutant larva showing isochromatid breakages (arrows). Note the absence of telomeric fusions.
(F) RT-PCR analysis of HeT-A transcripts in wild-type and E(z)61/E(z)1902, Su(var)3-906/Su(var)3-906, AGO1k00208/AGO1k08121, spn-E1/spn-E1, piwi1/piwi2, armi1/armi72.1, and Su(var)2-502/Su(var)2-505 female mutant larvae. Only in HP1 mutants is the amount of HeT-A transcripts dramatically increased with respect to wild-type. We found the same results in Su(var)2-504/Su(var)2-505.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Evidence for the Ability of HP1 to Directly Bind DNA In Vitro and In Vivo
(A) Western blot of in vitro HP1-DNA crosslinking induced by cis-DDP. Left lane: purified recombinant HP1 (rHP1); middle lane: crosslinked rHP1 after elution by 1.5 M thiourea; right lane: non-crosslinked rHP1. In the left and middle lanes, both the HP1 monomer (arrow) and the HP1 dimer (arrowhead) are present. In the right lane, the dimer is not visible due to the scarcity of the non-crosslinked protein.
(B) SDS-gel electrophoresis and Western blot of the in vivo proteins crosslinked to DNA by cis-DDP. In the second lane, the total proteins isolated from the DNA-protein crosslinked complexes have been stained with Coomassie. The lanes marked HP1, PC (POLYCOMB), and SU(VAR)3-7 are Western blots probed with antibodies against the indicated proteins. The arrows indicate the HP1 and SU(VAR)3-7 immunosignals, and the arrowhead indicates the absence of POLYCOMB in the expected position.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Determination, by X-ChIP Assay, of Direct Binding of HP1 to Telomeric DNA in Both Normal Telomeres and in Stable Terminal Deletions Ending inside the yellow Gene
(A) PCR was performed on DNA of cis-DDP crosslinked complexes extracted from wild-type and yTdl4 larval nuclei and immunoprecipitated with HP1 monoclonal antibody. Primers from telomeric HeT-A sequences and from the yellow gene were used. The amplification products of each primer pair using genomic DNA (g), anti-HP1 immunoprecipitation (+), and mock immunoprecipitation (−) from Ore-R wild-type nuclei and nuclei from the yTdl4 strain are shown. Below, a PCR with the same primers on a dilution series (1 = 200 ng; 2 = 80 ng; 3 = 32 ng; 4 = 12 ng) of the input-DNA to assess the exponential phase is shown.
(B) Quantitative representation of the results. The relative amounts of the amplified DNA were measured by densitometry. Each value is normalized to the same amount of DNA subject to amplification (e.g., the HeT-A amplification in the yTdl4 strain is performed starting from 200 ng for genomic DNA and 0.5 ng for ChIP DNA and mock). The presence of the HeT-A signal in the yTdl4-positive control lane is probably from autosomal telomeres.
(C) Interaction of HP1 with telomeric dsDNA and ssDNA. The binding of purified recombinant HP1 to a 124 bp double-stranded telomeric sequence and its corresponding single-stranded sequence is analyzed by EMSA as a function of increasing amounts of added protein. Lane 1 corresponds to markers of known molecular mass (GIBCO-BRL); lane 2, dsDNA (50 ng); lane 6, ssDNA (50 ng); lanes 3–5 and lanes 7–9, dsDNA and ssDNA, respectively, incubated with different amounts of HP1.
(D) HP1 shows higher affinity for single-strand than double-strand HeT-A DNA. Lanes 2–8 show radioactively labeled HeT-A dsDNA (1 ng) incubated with the same amount of HP1 (200 ng). In lanes 3–5 and lanes 6–8, increasing doses (10 ng, 100 ng, 1000 ng) of, respectively, single- or double-strand, cold HeT-A DNA were added. The lower dose of ssDNA (lane 3) abolishes the gel shift produced by HP1 (lane 2), while, at the same concentration of double-strand DNA, the gel shift is still clearly present (lane 6).
(E) HP1 fragments, lacking the indicated segments, used in the gel shift assay.
(F) EMSA of radiolabeled HeT-A ssDNA using the different HP1 fragments indicated in (E). The absence of shift (lane c) using the HP1 fragment lacking the hinge portion strongly suggests that this part of the protein is responsible for the binding of HP1 to the telomeric DNA.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Proposed Model for HP1 Multiple Telomeric Functions
In the first step, HP1 binds directly to telomeric DNA for telomere capping. Then, HP1 recruits a putative HMTase that trimethylates H3-K9 histone, creating a binding site for the HP1 chromodomain. The reiteration of the second step then creates repressive telomeric chromatin.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2004 Cell Press Terms and Conditions