Domains of Heterochromatin Protein 1 Required forDrosophila melanogasterHeterochromatin Spreading

Abstract

Centric regions of eukaryotic genomes are packaged into heterochromatin, which possesses the ability to spread along the chromosome and silence gene expression. The process of spreading has been challenging to study at the molecular level due to repetitious sequences within centric regions. A heterochromatin protein 1 (HP1) tethering system was developed that generates ''ectopic heterochromatin'' at sites within euchromatic regions of the Drosophila melanogaster genome. Using this system, we show that HP1 dimerization and the PxVxL interaction platform formed by dimerization of the HP1 chromo shadow domain are necessary for spreading to a downstream reporter gene located 3.7 kb away. Surprisingly, either the HP1 chromo domain or the chromo shadow domain alone is sufficient for spreading and silencing at a downstream reporter gene located 1.9 kb away. Spreading is dependent on at least two H3K9 methyltransferases, with SU(VAR)3-9 playing a greater role at the 3.7-kb reporter and dSETDB1 predominately acting at the 1.9 kb reporter. These data support a model whereby HP1 takes part in multiple mechanisms of silencing and spreading.H ETEROCHROMATIN protein 1 (HP1) was identified in Drosophila as a nonhistone chromosomal protein enriched in centric heterochromatin ( James and Elgin 1986;James et al. 1989). On polytene chromosomes, HP1 localizes near centromeres and telomeres, along the fourth chromosome and at $200 sites within the euchromatic arms ( James et al. 1989;Fanti et al. 2003). Heterochromatin has the ability to ''spread,'' or propagate in cis, along the chromosome (Weiler and Wakimoto 1995). Spreading is observed when a chromosomal rearrangement places a euchromatic domain next to a heterochromatic domain. Cytologically, spreading is visualized as densely compact chromatin that emanates from the chromocenter, the structure formed by the fusion of centromeres, and extends into the banded regions of polytene chromosomes (Belyaeva and Zhimulev 1991). Euchromatic genes brought into juxtaposition with heterochromatin by chromosomal rearrangements exhibit gene silencing, termed position effect variegation (PEV) (Weiler and Wakimoto 1995). Mutations in Su(var)2-5, the gene encoding HP1, suppress silencing, suggesting HP1 plays a key role in spreading (Eissenberg et al. 1990). The molecular processes of spreading are not well understood.Repetitive sequences within heterochromatin make it difficult to study spreading at the molecular level. In addition, specific repetitive elements are thought to function as initiation sites for heterochromatin formation (Sun et al. 2004;Haynes et al. 2006), making it challenging to separate initiation from spreading. To overcome these problems, we generated a system that nucleates small domains (,20 kb) of repressive chromatin that share many properties with centric heterochromatin. Here we refer to these as ectopic heterochromatin domains. These domains are generated by expressing a fusion protein, consisting of the DNA binding domain of the Escherichia coli lac represso...

Histone Variants and Epigenetics

“…ATRX has a bifunctional histone tail recognition domain with high affinity for an H3 tail that is both unmethylated at K4 and trimethylated at K9 (Eustermann et al 2011), and so likely is recruited to telomeric sites that are enriched for H3K9me and lack H3K4me. Telomeres are also enriched for heterochromatin-associated protein 1 (HP1), which recruits the Su(var)3-9 H3K9 methyltransferase, and binds its H3K9 methylated product (Hines et al 2009), and so an enzyme that methylates the tails of replacement H3.3 at telomeres is present at a high local concentration in which new H3.3 is incorporated. This implies that all of the components necessary for maintaining H3K9 methylation are present at telomeres: the enzyme that performs the modification, the modification-specific-binding module on the machine that uses ATP to provide energy for the replacement process, and the fresh unmodified H3.3 substrate that becomes incorporated into the new nucleosome (Fig.…”

The epigenetic H3S10 phosphorylation mark is required for counteracting heterochromatic spreading and gene silencing inDrosophila melanogaster

“…The isolated DNA was used as a template for quantitative real-time (qRT) PCR performed with the Stratagene Mx4000 real-time cycler. The PCR mixture contained Brilliant II SYBR Green QPCR Master Mix (Stratagene) as well as the corresponding primers: hsp70-whiteforward 59-GCAACCAAGTAAATCAACTGC-39, hsp70-white-reverse 59-GTT-TTGGCACAGCACTTTGTG-39, which amplify region +149 to +250 (Hines et al, 2009). Cycling parameters were 10 minutes at 95˚C, followed by 40 cycles of 30 seconds at 95˚C, 30 seconds at 55˚C and 30 seconds at 72˚C.…”

“…Chromatin was immunoprecipitated from the salivary glands of 'JIL-1 transgene'/+; JIL-1 z2 /JIL-1 z2 da-GAL4; 118E-10/+ larvae using one of the following antibodies: a rabbit antibody against phosphorylated H3S10; a purified rabbit IgG antibody (negative control); or monoclonal antibodies against H3K9me2 or glutathione S-transferase (GST) as a negative control. Primers corresponding to the hsp70-white gene were used to amplify the precipitated material (Hines et al, 2009). Experiments were performed in duplicate, and relative enrichment of hsp70-white DNA from the phosphorylated H3S10 and H3K9me2 immunoprecipitates was normalized to the corresponding control antibody immunoprecipitates, which were performed in tandem for each experimental sample.…”

Trans-inactivation: Repression in a wrong place

“…The JIL-1 kinase and the H3S10 histone modification counteract heterochromatin spreading. [4][5][6][7][8][9][10] According to the current model of heterochromatin formation, Su(var)3-9 HKMT methylates the lysine 9 residue of histone H3. HP1a binds to H3K9me and then recruits another molecule of Su(var)3-9 for further methylation of H3 in the adjacent nucleosome.…”

“…HP1a binds to H3K9me and then recruits another molecule of Su(var)3-9 for further methylation of H3 in the adjacent nucleosome. 10,11 This methylation/HP1a binding loop repeats until it reaches a boundary element 12 or ceases due to the action of histone code modifiers like JIL-1 kinase 9 . This model of heterochromatin propagation assumes the assembly of protein complexes via the short-range interactions between protein domains along the chromatin fiber, thus providing a molecular basis for the classic concept of linear heterochromatin spreading.…”