Investigation Of X Chromosome Recognition: The Role Of Small Rna In Drosophila Dosage Compensation

In humans and flies, females have two X chromosomes but males have one X chromosome and one Y chromosome. This leads to a fatal imbalance in X-linked gene expression in one sex. In mammals and in the fruit fly Drosophila, modulation of X chromosome expression is critical for survival. This process is termed dosage compensation. Flies increase expression from the male X chromosome two-fold. This is achieved by the Male Specific Lethal (MSL) complex, which consists of two large, non-coding RNA on the X transcripts (roX1 and roX2) and five proteins. The roX RNAs have a critical role in complex localization to the X chromosome. Simultaneous mutation of roX1 and roX2 reduces X localization of the MSL proteins, lowers X-linked expression and reduces male survival. Using roX1 roX2 mutants, we performed genetic studies to identify modifiers of X chromosome recognition. In spite of a lack of expression in somatic tissues, the Y chromosome is a potent modifier of the roX1 roX2 phenotype. I postulated that the Y chromosome could affect dosage compensation through a small RNA-dependent pathway, and performed a screen of RNAi mutations. This screen identified four siRNA genes that, when mutated, enhance roX1 roX2 male lethality and disrupt MSL localization to the X chromosome. The role of the siRNA pathway in dosage compensation prompted an investigation of potential sources of siRNA. A class of 1.688g/cm3 satellite-related repeats is exclusive to the X chromosome (1.688X). These are transcribed, and thus capable of generating siRNA in animals. Ectopic expression of long single stranded 1.688X RNA reduced roX1 roX2 male survival. In contrast, expression of double stranded 1.688X hairpin RNA produced high levels of corresponding small RNA and dramatically rescued roX1 roX2 male survival. MSL localization to the X chromosome was partially restored in flies expressing 1.688X hairpin RNA. Rescue of roX1 roX2 males was dependent upon the siRNA genes Dcr2 and Ago2. These studies reveal that small RNA from X-linked repeats acts through the siRNA pathway to promote X chromosome recognition. I postulate that the 1.688X RNA repeats underline X chromosome identity. Future studies exploring this process will help us to understand the molecular basis for exclusive modification of the X chromosome

The Mouse INO80 Chromatin-Remodeling Complex Is an Essential Meiotic Factor for Spermatogenesis1

The ability to faithfully transmit genetic information across generations via the germ cells is a critical aspect of mammalian reproduction. The process of germ cell development requires a number of large-scale modulations of chromatin within the nucleus. One such occasion arises during meiotic recombination, when hundreds of DNA double-strand breaks are induced and subsequently repaired, enabling the transfer of genetic information between homologous chromosomes. The inability to properly repair DNA damage is known to lead to an arrest in the developing germ cells and sterility within the animal. Chromatin-remodeling activity, and in particular the BRG1 subunit of the SWI/SNF complex, has been shown to be required for successful completion of meiosis. In contrast, remodeling complexes of the ISWI and CHD families are required for postmeiotic processes. Little is known regarding the contribution of the INO80 family of chromatin-remodeling complexes, which is a particularly interesting candidate due to its well described functions during DNA double-strand break repair. Here we show that INO80 is expressed in developing spermatocytes during the early stages of meiotic prophase I. Based on this information, we used a conditional allele to delete the INO80 core ATPase subunit, thereby eliminating INO80 chromatin-remodeling activity in this lineage. The loss of INO80 resulted in an arrest during meiosis associated with a failure to repair DNA damage during meiotic recombination

Mammalian SWI/SNF Chromatin Remodeler is Essential for Reductional Meiosis in Males

The mammalian SWI/SNF nucleosome remodeler is required for spermatogenesis. Here, the authors show that PBAF is essential for meiotic cell division in males and required to activate the expression of critical genes involved in spindle assembly and nuclear division in spermatocytes

Key mediators of somatic ATR signaling localize to unpaired chromosomes in spermatocytes

Meiotic silencing of unpaired chromatin (MSUC) occurs during the first meiotic prophase, as chromosomes that fail to pair are sequestered into a transcriptionally repressive nuclear domain. This phenomenon is exemplified by the heterologous sex chromosomes of male mammals, where the ATR DNA damage response kinase is crucial for this silencing event. However, the mechanisms underlying the initiation of MSUC remain unknown. Here, we show that essential components of ATR signaling in murine somatic cells are spatially confined to unpaired chromosomes in spermatocytes, including the ATR-dependent phosphorylation of the single-stranded DNA (ssDNA)-binding complex replication protein A (RPA) and the checkpoint kinase CHK1. These observations support a model in which ssDNA plays a central role in the recruitment of ATR during MSUC, and provide a link to meiotic progression through activation of CHK1

Rett Syndrome: Crossing the Threshold to Clinical Translation

Lying at the intersection between neurobiology and epigenetics, Rett syndrome (RTT) has garnered intense interest in recent years, not only from a broad range of academic scientists, but also from the pharmaceutical and biotechnology industries. In addition to the critical need for treatments for this devastating disorder, optimism for developing RTT treatments derives from a unique convergence of factors, including a known monogenic cause, reversibility of symptoms in preclinical models, a strong clinical research infrastructure highlighted by an NIH-funded natural history study and well-established clinics with significant patient populations. Here, we review recent advances in understanding the biology of RTT, particularly promising preclinical findings, lessons from past clinical trials, and critical elements of trial design for rare disorders

The Drosophila homolog of the mammalian imprint regulator, CTCF, maintains the maternal genomic imprint in Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>CTCF is a versatile zinc finger DNA-binding protein that functions as a highly conserved epigenetic transcriptional regulator. CTCF is known to act as a chromosomal insulator, bind promoter regions, and facilitate long-range chromatin interactions. In mammals, CTCF is active in the regulatory regions of some genes that exhibit genomic imprinting, acting as insulator on only one parental allele to facilitate parent-specific expression. In <it>Drosophila</it>, CTCF acts as a chromatin insulator and is thought to be actively involved in the global organization of the genome.</p> <p>Results</p> <p>To determine whether CTCF regulates imprinting in <it>Drosophila</it>, we generated <it>CTCF </it>mutant alleles and assayed gene expression from the imprinted <it>Dp(1;f)LJ9 </it>mini-X chromosome in the presence of reduced <it>CTCF </it>expression. We observed disruption of the maternal imprint when <it>CTCF </it>levels were reduced, but no effect was observed on the paternal imprint. The effect was restricted to maintenance of the imprint and was specific for the <it>Dp(1;f)LJ9 </it>mini-X chromosome.</p> <p>Conclusions</p> <p>CTCF in <it>Drosophila </it>functions in maintaining parent-specific expression from an imprinted domain as it does in mammals. We propose that <it>Drosophila </it>CTCF maintains an insulator boundary on the maternal X chromosome, shielding genes from the imprint-induced silencing that occurs on the paternally inherited X chromosome.</p> <p>See commentary: <url>http://www.biomedcentral.com/1741-7007/8/104</url></p

Mammalian SWI/SNF chromatin remodeler is essential for reductional meiosis in males

AbstractBRG1, a catalytic subunit of the mammalian SWI/SNF nucleosome remodeler is essential for male meiosis1. In addition to BRG1, multiple subunits (~10-14) some of which are mutually exclusive, constitute biochemically distinct SWI/SNF subcomplexes, whose functions in gametogenesis remain unknown. Here, we identify a role for the PBAF (Polybromo - Brg1 Associated Factor) complex in the regulation of meiotic cell division. The germ cell-specific depletion of PBAF specific subunit, ARID2 resulted in a metaphase-I arrest. Arid2cKO metaphase-I spermatocytes displayed defects in chromosome organization and spindle assembly. Additionally, mutant centromeres were devoid of Polo-like kinase1 (PLK1), a known regulator of the spindle assembly checkpoint (SAC)2. The loss of PLK1 coincided with an abnormal chromosome-wide expansion of centromeric chromatin modifications such as Histone H3 threonine3 phosphorylation (H3T3P) and Histone H2A threonine120 phosphorylation (H2AT120P) that are critical for chromosome segregation3,4. Consistent with the known role of these histone modifications in chromosome passenger complex (CPC) recruitment, Arid2cKO metaphase-I chromosomes display defects in CPC association. We propose that ARID2 facilitates metaphase-I exit by regulating spindle assembly and centromeric chromatin.</jats:p

A Role for siRNA in X-Chromosome Dosage Compensation in <i>Drosophila melanogaster</i>

Abstract Sex-chromosome dosage compensation requires selective identification of X chromatin. How this occurs is not fully understood. We show that small interfering RNA (siRNA) mutations enhance the lethality of Drosophila males deficient in X recognition and partially rescue females that inappropriately dosage-compensate. Our findings are consistent with a role for siRNA in selective recognition of X chromatin.</jats:p