Cooperative and antagonistic contributions of two heterochromatin proteins to transcriptional regulation of the Drosophila sex determination decision.

Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. Here we report a similar phenotype for the telomeric partner of HP1, HOAP, and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, Sxl(Pe), is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (Sxl(Pm)) in both sexes. HOAP mutants show inappropriate Sxl(Pe) firing in males and the concomitant inappropriate splicing of Sxl(Pm)-derived transcripts, while females show premature firing of Sxl(Pe). HP1 mutants, by contrast, display Sxl(Pm) splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with Sxl(Pe) sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to Sxl(Pe) are severely compromised. Our genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at Sxl(Pe).

N terminus of calpain 1 is a mitochondrial targeting sequence.

The ubiquitous m- and mu-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, mu-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of mu-calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form mu-calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic alpha-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of mu-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.

Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance.

In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. We discover in Drosophila that the two proteins control partially redundant pathways for telomere protection: the loss of ATM leads to the fusion of some telomeres, whereas the loss of both ATM and ATR renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. We provide evidence suggesting that both the loading of telomere capping proteins and normal telomeric silencing requires ATM and ATR in Drosophila and propose that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins.

Mutations in the heterochromatin protein 1 (HP1) hinge domain affect HP1 protein interactions and chromosomal distribution.

Heterochromatin Protein 1 (HP1) is a conserved component of the highly compact chromatin found at centromeres and telomeres. A conserved feature of the protein is multiple phosphorylation. Hyper-phosphorylation of HP1 accompanies the assembly of cytologically distinct heterochromatin during early embryogenesis. Hypo-phosphorylated HP1 is associated with the DNA-binding activities of the origin recognition complex (ORC) and an HMG-like HP1/ORC-Associated Protein (HOAP). Perturbations in HP1 localization in pericentric and telomeric heterochromatin in mutants for Drosophila ORC2 and HOAP, respectively, indicate roles for these HP1 phosphoisoforms in heterochromatin assembly also. To elucidate the roles of hypo- and hyper-phosphophorylated HP1 in heterochromatin assembly, we have mutated consensus Protein Kinase-A phosphorylation sites in the HP1 hinge domain and examined the mutant proteins for distinct in vitro and in vivo activities. Mutations designed to mimic hyper-phosphorylation render the protein incapable of binding HOAP and the DmORC1 subunit but confer enhanced homo-dimerization and lysine 9-methylated histone H3-binding to the protein. Mutations rendering the protein unphosphorylatable, by contrast, do not affect homo-dimerization or binding to lysine 9-di-methylated histone H3, HOAP, or DmORC1 but do confer novel DmORC2-binding activity to the protein. This mutant protein is ectopically localized throughout the chromosomes when overexpressed in vivo in the presence of a full dose of DmORC2. This ectopic targeting is accompanied by ectopic targeting of lysine 9 tri-methylated histone H3. The distinct activities of these mutant proteins could reflect distinct roles for HP1 phosphoisoforms in heterochromatin structure and function.

Novel Drosophila heterochromatin protein 1 (HP1)/origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations.

Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin.

HP1/ORC complex and heterochromatin assembly.

We have used the highly conserved heterochromatin component, heterochromatin protein 1 (HP1), as a molecular tag for purifying other protein components of Drosophila heterochromatin. A complex of HP1 associated with the origin recognition complex (ORC) and an HP1/ORC-associated protein (HOAP) was purified from the maternally loaded cytoplasm of early Drosophila embryo. We propose that the DNA-binding activities of ORC and HOAP function to recruit underphosphorylatedisoforms of HP1 to sites of heterochromatin nucleation. The roles of highly phosphorylated HP1, other DNA-binding proteins known to interact with HP1, and histone modifying activities in heterochromatin assembly are also addressed.