Carolyn A. Craig

Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin

Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.

Immunofluorescent Staining of Polytene Chromosomes: Exploiting Genetic Tools

Publisher Summary This chapter describes a method for determining the in vivo distribution of chromosomal proteins on Drosophila polytene chromosomes. By combining genetic, biochemical, and molecular biology techniques with the cytological approach, a greater understanding of the molecular mechanisms of gene regulation can be gained. Few other systems offer the well-developed genetic tools in combination with the ability to perform cytological studies as Drosophila does. Immunostaining of Drosophila polytene chromosomes is a powerful tool for investigating the components of chromatin on a genome-wide scale. Many of the nuclei of Drosophila undergo multiple rounds of DNA replication without cell division during the larval stages of development, a process known as endoreduplication. While the euchromatic regions are copied ca. 10 times, the pericentric heterochromatin undergoes only a few rounds of replication, and centromeric satellite DNA and the Y chromosome are not amplified due to their heterochromatic nature. Immunological methods for studying the association of proteins with polytene chromosomes have been used to address a variety of biological questions. In wild-type flies, immunostaining has been used to determine the global distribution of one or more proteins; colocalization studies have been done to determine if a protein of interest might be in close association or part of a multiprotein complex with other proteins.

Interaction of heterochromatin protein 2 with HP1 defines a novel HP1-binding domain

Heterochromatin Protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster localized principally in the pericentric heterochromatin, telomeres, and fourth chromosome, all regions associated with HP1. Mutations in HP2 can suppress position effect variegation, indicating a role in gene silencing and heterochromatin formation [Shaffer, C. D. et al. (2002) Proc. Natl. Acad. Sci.U.S.A. 99, 14332-14337]. In vitro coimmunoprecipitation experiments with various peptides from HP2 have identified a single HP1-binding domain. Conserved domains in HP2, including those within the HP1-binding region, have been identified by recovering and sequencing Su(var)2-HP2 from D. willistoni and D. virilis, as well as examining available sequence data from D. pseudoobscura. A PxVxL motif, shown to be an HP1-binding domain in many HP1-interacting proteins, is observed but is not well-conserved in location and sequence and does not mediate HP2 binding to HP1. The sole HP1-binding domain is composed of two conserved regions of 12 and 16 amino acids separated by 19 amino acids. Site-directed mutagenesis within the two conserved regions has shown that the 16 amino acid domain is critical for HP1 binding. This constitutes a novel domain for HP1 interaction, providing a critical link for heterochromatin formation in Drosophila.

Chapter 8 Distribution of Chromosomal Proteins in Polytene Chromosomes of Drosophila

Publisher Summary This chapter discusses the distribution of chromosomal proteins in polytene chromosomes of Drosophila . The gene regulation is facilitated by the identification and characterization of proteins that interact with DNA. The chapter identifies and characterizes proteins specifically associated with either euchromatin or heterochromatin. Techniques that allow establishing the pattern of interaction between a given protein and the genome as a whole are used. In Drosophila , many of the chromosomal proteins are studied by using the technique described in the chapter, that of immunolocalization of proteins on polytene chromosomes. The chapter presents a technique for determining the in situ distribution of chromosomal proteins in polytene chromosomes with the emphasis on a procedure using formaldehyde fixation followed by squashing in acetic acid. The chapter also discusses the characteristics for ideal polytene chromosomes for immunofluorescent microscopy. To localize the positions of chromosomal proteins on the polytene chromosomes, the chromosome spreads are reacted with a primary antibody against a particular protein, followed by reaction with a fluorescent or other secondary antibody against the primary antibody.