Cloning of medaka glial cell-derived neurotrophic factor (GDNF) and its receptor GFR alpha 1

Master'sMASTER OF SCIENC

Autoregulation of the Drosophila Noncoding roX1 RNA Gene

Most genes along the male single X chromosome in Drosophila are hypertranscribed about two-fold relative to each of the two female X chromosomes. This is accomplished by the MSL (male-specific lethal) complex that acetylates histone H4 at lysine 16. The MSL complex contains two large noncoding RNAs, roX1 (RNA on X) and roX2, that help target chromatin modifying enzymes to the X. The roX RNAs are functionally redundant but differ in size, sequence, and transcriptional control. We wanted to find out how roX1 production is regulated. Ectopic DC can be induced in wild-type (roX1+ roX2+) females if we provide a heterologous source of MSL2. However, in the absence of roX2, we found that roX1 expression failed to come on reliably. Using an in situ hybridization probe that is specific only to endogenous roX1, we found that expression was restored if we introduced either roX2 or a truncated but functional version of roX1. This shows that pre-existing roX RNA is required to positively autoregulate roX1 expression. We also observed massive cis spreading of the MSL complex from the site of roX1 transcription at its endogenous location on the X chromosome. We propose that retention of newly assembled MSL complex around the roX gene is needed to drive sustained transcription and that spreading into flanking chromatin contributes to the X chromosome targeting specificity. Finally, we found that the gene encoding the key male-limited protein subunit, msl2, is transcribed predominantly during DNA replication. This suggests that new MSL complex is made as the chromatin template doubles. We offer a model describing how the production of roX1 and msl2, two key components of the MSL complex, are coordinated to meet the dosage compensation demands of the male cell

The <i>Drosophila Over Compensating Males</i> Gene Genetically Inhibits Dosage Compensation in Males

<div><p>Male <i>Drosophila</i> are monosomic for the X chromosome, but survive due to dosage compensation. They use the Male Specific Lethal (MSL) complex composed of noncoding <i>roX</i> RNA and histone modifying enzymes to hypertranscribe most genes along the X ∼1.6–1.8 fold relative to each female allele. It is not known how the MSL complex achieves this precise adjustment to a large and diverse set of target genes. We carried out a genetic screen searching for novel factors that regulate dosage compensation in flies. This strategy generated thirty alleles in a previously uncharacterized gene, <i>over compensating males</i> (<i>ocm</i>) that antagonizes some aspect of MSL activity. The mutations were initially recovered because they derepressed an MSL-dependent eye color reporter. Null <i>ocm</i> mutations are lethal to both sexes early in development revealing an essential function. Combinations of hypomorphic <i>ocm</i> alleles display a male specific lethality similar to mutations in the classic <i>msl</i> genes, but <i>ocm</i> males die due to excessive, rather than lack of dosage compensation. Males that die due to very low MSL activity can be partially rescued by <i>ocm</i> mutations. Likewise, males that would die from <i>ocm</i> mutations can be rescued by reducing the dose of various <i>msl</i> and <i>roX</i> genes. <i>ocm</i> encodes a large nuclear protein that shares a novel cysteine rich motif with known transcription factors.</p> </div

Null alleles of <i>ocm</i> results in decreased cell numbers in the fly eyes.

<p>Different <i>ocm</i> alleles were assayed for cell viability in <i>Drosophila</i> eye. (A-B) <i>ocm^S1590F</i>, a weak hypomorphic allele of <i>ocm</i> does not affect cell growth. Other hypomorphic alleles, <i>ocm^G1646E</i> and <i>ocm^V1286D</i> were also tested but they are indistinguishable to <i>ocm^S1590F</i> (data not shown). Increasingly severe alleles (C–D) <i>ocm^V1334D</i>, (E–F) <i>ocm¹⁶⁶</i>^{Δ<i>11 bp</i>} (null allele) result in fly eyes dramatically reduced in size.</p

Identification of <i>CG3363</i> as <i>ocm</i>.

<p>A. Genomic region near 60C showing predicted genes and five BACs used to rescue <i>ocm</i>. The BACs shown in black failed to rescue <i>ocm</i> mutations, but 117N13 (yellow) rescued viability and fertility of multiple <i>ocm</i> alleles. B. Conceptual translation of <i>ocm</i> reveals a protein with distinct motifs conserved between other Drosophila species (colored boxes) separated by diverged regions (thin line). The hatched box indicates the segment of OCM used to raise antibodies. C. Similar coding regions are found in the genomes of some other insects. The Bombyx alignment is taken from genomic DNA lacking cDNA support, so the exact alignment is uncertain. D. The Cys431 region (red) is also found in chordate Mga. E. Several Daphnia proteins contain Cys431 motifs. A second cys-rich region (orange) is also present in two of the Daphnia proteins.</p

Antibodies to OCM recognize a 250 kD nuclear protein.

<p>A. Anti-OCM western with size standard shown left. Lane 1, wild type embryos. Lane 2, embryos from <i>Df ocm</i>/+ mothers. Lane 3, wild type adults. Lane 4, <i>Df ocm</i>/+ adults. Hemizygous tissue gives a weaker OCM band. Loading control (LC) is mitochondrial complex V. B-E. Third instar imaginal eye disc showing an <i>ocm</i>/<i>ocm</i> clone surrounded by <i>ocm</i>/+ cells.</p

MSL proteins alone cannot drive <i>roX1</i> expression late in development.

<p>A) 4 day old larvae were heatshocked to induce expression of Flp, resulting in the removal of the blocking sequence from GAL4 and subsequent expression of both MSL2 and GFP. MSL2 is expected to initiate <i>roX</i> transcription and MSL complex assembly. (B) GFP+ clones mark imaginal disc cells that have successfully removed the blocking sequences from <i>GAL4</i> (B′–B″). Induction of MSL2 results in punctate subnuclear foci in imaginal disc cells. (C) MSL2 immunostaining of polytene chromosome shows late MSL2 paints the entire X chromosome. (D) Whole salivary gland showing GFP induced in some cells. (E–E′) <i>roX1</i> FISH of whole mount of similar GFP+ salivary glands or (F) polytene squashes shows successful induction of <i>roX1</i> expression in a subset of cells. (G) <i>roX1</i> FISH of wildtype males (H) The same experiment was repeated in <i>roX1⁺roX2⁻</i> larvae. However, in the absence of <i>roX2</i>, MSL2 fails to drive <i>roX1</i> expression. (I) Despite the presence of GFP+ (late MSL2 expressing) cells, MSL2 is not detectable over the X in (I′–I″) imaginal disc cells or (J) polytene chromosomes. (K) Whole salivary gland showing successful GFP expression in <i>roX1⁺roX2⁻</i> larvae. (L–L′) Expression of <i>roX1</i> is never observed painting the X or as nascent transcripts at band 3F in separately processed GFP+ glands or on (M) polytene squashes.</p

Late induction of <i>roX1</i> expression requires <i>roX2</i> RNA.

<p>In nuclei where dosage compensation was successfully turned on after late <i>msl2</i> induction, extensive <i>roX2</i> was observed painting the entire X chromosome. (A) However, only 1% of the chromosomes showed extensive <i>roX1</i> painting. 34% and 59% of chromosomes showed <i>roX1</i> expression confined to several Mbp around (B) or just at the endogenous <i>roX1</i> locus (C), respectively. The remaining chromosomes (6%) had no <i>roX1</i> expression despite the presence of <i>roX2</i> (data not shown). <i>roX1</i> and <i>roX2</i> were detected by biotin (green, A–C) and digoxigenin (red, A′–C′) labeled antisense riboprobes, respectively. The merged figure is shown in A″–C″. White arrows denote the endogenous <i>roX1</i> locus at band 3F.</p

Autoregulation model.

<p>The earliest <i>roX1</i> transcripts (red) made at blastoderm originate from an uncharacterized MSL-independent promoter. This RNA may assemble with MSL protein subunits to produce the first functional MSL complexes needed to bind the internal DHS enhancer that drives sustained transcription (blue) from the male-specific promoters. When present, <i>roX2</i> RNA can also drive <i>roX1</i> transcription. Components of the replication pre-initiation complex also bind the DHS sequence in male cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002564#pgen.1002564.s008" target="_blank">Figure S8A</a>). The <i>msl2</i> transcripts are made predominantly during replication and new MSL2 protein is needed to assemble and stabilize newly made <i>roX1</i> RNA.</p

Mutations in <i>ocm</i> increase MSL activity around mosaic <i>roX1</i> transgenes.

<p>A and B. The <i>GMroX1-75C</i> reporter shows mosaic eye pigmentation in males, but females have pure white eyes. C and D. Reducing <i>ocm</i> activity increases eye pigmentation in males (more MSL activity) but has no effect on females who lack dosage compensation. E and F. The increased eye pigmentation seen in <i>ocm</i> males requires full MSL1 activity. G-J. A different <i>roX1</i> mosiac reporter displays the same male-specific <i>ocm</i> phenotype. K-N. Position effect variegation as measured by <i>In(1) w</i>^m4 is not affected by <i>ocm</i> mutations. The exact phenotypes of the flies are in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060450#s4" target="_blank">methods</a> section. O. Several hypomorphic <i>ocm</i> allelic combinations produce abundant females, but few or no males. X axis is viability. Allelic designations indicate codons affected. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060450#pone.0060450.s006" target="_blank">Table S2</a> for details.</p