Drosophila rhino Encodes a Female-Specific Chromo-domain Protein That Affects Chromosome Structure and Egg Polarity

Abstract

Here we describe our analyses of Rhino, a novel member of the Heterochromatin Protein 1(HP1) subfamily of chromo box proteins. rhino (rhi) is expressed only in females and chiefly in the germline, thus providing a new tool to dissect the role of chromo-domain proteins in development. Mutations in rhi disrupt eggshell and embryonic patterning and arrest nurse cell nuclei during a stage-specific reorganization of their polyploid chromosomes, a mitotic-like state called the “five-blob” stage. These visible alterations in chromosome structure do not affect polarity by altering transcription of key patterning genes. Expression levels of gurken (grk), oskar (osk), bicoid (bcd), and decapentaplegic (dpp) transcripts are normal, with a slight delay in the appearance of bcd and dpp mRNAs. Mislocalization of grk and osk transcripts, however, suggests a defect in the microtubule reorganization that occurs during the middle stages of oogenesis and determines axial polarity. This defect likely results from aberrant Grk/Egfr signaling at earlier stages, since rhi mutations delay synthesis of Grk protein in germaria and early egg chambers. In addition, Grk protein accumulates in large, actin-caged vesicles near the endoplasmic reticulum of stages 6–10 egg chambers. We propose two hypotheses to explain these results. First, Rhi may play dual roles in oogenesis, independently regulating chromosome compaction in nurse cells at the end of the unique endoreplication cycle 5 and repressing transcription of genes that inhibit Grk synthesis. Thus, loss-of-function mutations arrest nurse cell chromosome reorganization at the five-blob stage and delay production or processing of Grk protein, leading to axial patterning defects. Second, Rhi may regulate chromosome compaction in both nurse cells and oocyte. Loss-of-function mutations block nurse cell nuclear transitions at the five-blob stage and activate checkpoint controls in the oocyte that arrest Grk synthesis and/or inhibit cytoskeletal functions. These functions may involve direct binding of Rhi to chromosomes or may involve indirect effects on pathways controlling these processes.

The Cutoff protein regulates piRNA cluster expression and piRNA production in theDrosophilagermline

“…The role of Cuff in the expression of piRNA clusters and its localization within nurse cell nuclei prompted us to investigate whether this protein is found in a complex with Rhi (Volpe et al , 2001; Klattenhoff et al , 2009). This HP1 variant was recently shown to bind specific regions in the cluster 1/42AB and promote the transcription of dual‐strand piRNA clusters (Klattenhoff et al , 2009).…”

“…The mechanism that regulates the expression of the piRNA clusters and the proteins involved in this nuclear process remain largely elusive. It has recently been shown that a germline‐specific HP1 variant encoded by the rhino ( rhi ) gene is essential for the regulation of the dual‐strand clusters (Volpe et al , 2001; Klattenhoff et al , 2009). Accordingly, mutations in rhi abolish the expression of these loci, which results in a general depletion of the corresponding piRNA population (Klattenhoff et al , 2009).…”

An introduction to PIWI-interacting RNAs (piRNAs) in the context of metazoan small RNA silencing pathways

“…The latter hypothesis is supported by an additional signature that was first observed in rhino (rhi) mutant ovaries in Drosophila [ 62 ]. Rhino (HP1d) is paralog of the Heterochromatin Protein 1 (HP1) and required for transcription of bidirectional piRNA clusters [ 55 , 102 , 103 ]. In the absence of rhino, secondary piRNAs aberrantly cleave mRNAs and induce a trail of phased piRNAs from the target mRNA (JB Sci15).…”

Functions of HP1 proteins in transcriptional regulation

“…It was identified by producing monoclonal antibodies against proteins isolated from fractionated nuclei originating from Drosophila embryos [ 31 ] and subsequently characterized by its molecular weight, localization to heterochromatin, and cDNA sequence. Following the completion of the first D. melanogaster reference genome assembly, HP1b and HP1c were discovered through sequence similarity searches in 2001 [ 32 ], and two germline-specific HP1 proteins, Rhino (also known as HP1d) and HP1e were described shortly thereafter [ 33 , 34 ]. Later comparative phylogenomic analyses showed that Dipteran HP1 genes evolve rapidly and arise de novo from duplication events [ 11 , 17 ].…”

“…HP1d, also known as Rhino (Rhi), is a critical factor for the transcription of piRNA precursors from piRNA clusters [ 107 ]. Unlike Drosophila HP1a, HP1b, and HP1c which are expressed ubiquitously in somatic cells, Rhi is expressed in the female germline [ 33 ]. Rhi has a history of positive selection [ 34 ], and it localizes to piRNA clusters, targeted by a mechanism independent of piRNA production [ 107 , 108 ].…”