.hiphop is a gTLD proposed in ICANN's New gTLD Program and currently run by Dot Hip Hop, LLC, led by Monte Cahn, Jeff Neuman, and Scott Pruitt. The 2012 applicant was Registry Operator Uniregistry (UNR). Their application succeeded and was delegated to the Root Zone on 15 May 2014.
Despite the .hiphop domain originally being advertised as part of UNR's April auction, Dot Hip Hop LLC (DHH) separately negotiated the purchase of the TLD in an arms-length transaction. UNR submitted the assignment request for .hiphop to ICANN in August 2021. The assignment was delayed for the same reasons as the other, auctioned TLDs.
In December, DHH submitted an urgent reconsideration request regarding ICANN's inaction on the assignment request for the .hiphop TLD. In its request, DHH noted that it had spent considerable time explaining, and re-explaining, its position on the NFT associated with .hiphop, which was transferred as part of DHH's purchase agreement with UNR. The Board Accountability Mechanisms Committee denied the request for urgent action, because only Board inaction could be subject to such a request. ICANN posted a blog in January 2022 regarding the .hiphop situation and the Uniregistry assignments more broadly.
During our studies of the function of HipHop, we observed that putting a 3XHA tag on the N-terminus of Hiphop generated a mutant allele, and named it hiphopHA (Fig 1A). Previously we showed that hiphop deletion mutations are homozygous lethal at an early larval stage . hiphopHA homozygotes (genotype: hiphopHA/HA), on the other hand, survived at Mendelian ratio (see S1 Table for progeny counts), with no visible phenotypes except that females had reduced fertility (Fig 1B), while male fertility is not affected (n>200). In addition, hiphopHA hemizygous females are sterile (Fig 1B). These females carry hiphopHA on one chromosome 3 and a deficiency (df) of the hiphop region on the homologous chromosome (genotype: hiphopHA/df). Moreover, females trans-heterozygous for hiphopHA and hiphopL14 (an hiphop deletion mutation ) were also sterile. The fertility of hiphopHA homozygotes or hemizygotes was not significantly affected by the maternal age as shown in Fig 1B and 1C. We observed a drop in the number of embryos laid by hiphopHA/HA mothers. We believe that this might have been caused by second-site mutations in the stock as the hemizygotes have wildtype level of fecundity (Fig 1C). Therefore, hiphopHA is a recessive mutation that affects female fertility in a dosage dependent manner. To further confirm that the sterility was indeed due to a loss of HipHop function, we introduced a transgene carrying a wildtype copy of hiphop and rescued female sterility (Fig 1B).
A. hiphop alleles and constructs. At the top is the wildtype (wt) hiphop locus with the coding region shown as a black rectangle. The approximate position of the 3xHA tag is indicated for the hiphopHA allele. The approximate position of the EGFP tag is indicated for the hiphop genomic construct used in the rescue experiment. The approximate position of the region deleted in the hiphopL41 allele is indicated by a parenthesis. B. The number of progenies produced in relation with maternal ages. The four genotypes tested are shown at the top right. Maternal ages are grouped into three categories and shown as the X axis. The number of females tested (n) is shown for each genotype. For hiphopHA/df mothers, the progeny counts were zero regardless of maternal age. ns: not significant; **: p
The female specific sterility of hiphopHA mutants suggests defects during oogenesis that might be unrelated to telomere capping. Transposons in Drosophila, including those at the telomeres, are silenced in the female germline. We therefore investigated whether telomeric elements are properly silenced in the mutant ovaries.
We performed whole genome RNA sequencing using ovarian RNA from 15-day old hiphopHA/+ and both hiphopHA/HA and hiphopHA/df females. As shown in Fig 2A, RNA seq identified a defect in transposon silencing that is limited to TEs at telomeres: HeT-A, TART, TAHRE. An expression analysis of other TEs in the genome is shown in S2 Table. To facilitate further investigation into the potential mechanisms underlying this de-repression, we employed a qPCR assay in subsequent analyses with primers previously used for the detection of specific TEs [33,34]. For HeT-A elements, our qPCR primers anneal to two separate regions. To control for possible age-related effects, we used age-matched samples from the three genotypes of interest (hiphopHA/+; hiphopHA/HA; and hiphopHA/df). As shown in Fig 2B, the mutant germline shows a gradual increase of HeT-A transcripts when compared with the heterozygous control, resulting in cumulative ~40-180-fold increase at a maternal age of about 35 days. On the other hands, the I element, a non-telomeric non-LTR retrotransposon in the Drosophila genome , is not de-repressed regardless of the age of the females. Based on these preliminary results, we used RNA samples from 15-day old ovaries in subsequent characterizations. As shown in Fig 2C, although de-repression of HeT-A could be demonstrated most consistently, transcription of TART or TAHRE showed more variable results using the method of qPCR. We suggest that these variations are due to the fact that both TART and TAHRE are minor elements, and that their sequence variations might be less compatible with our primers. Nevertheless, the non-telomeric I element consistently showed a lack of silencing defects in these independent trials. These different responses from telomeric versus non-telomeric elements to the hiphop mutation are in sharp contrast to the situation in piRNA-mutant germlines. In aub and spnE mutant ovaries, all tested transposons are de-repressed (Fig 2D). In addition, the telomeric elements are de-silenced to a greater extent in the piRNA mutant than in hiphop mutant ovaries (compare Fig 2C with 2D, note the log scale of the y-axis). Therefore, the hiphopHA mutation leads to a specific de-silencing of telomere retrotransposons in the germline. Interestingly, the level of telomeric transcript is also elevated in the embryos produced by hiphop mutant females suggesting that they are maternally deposited (Fig 2C).
The HeT-A element encodes a single Orf1p protein. In Western blots using total extracts from ovaries (one shown in Fig 2E), we detected a marked increase of Orf1p level in both of the hiphop mutant backgrounds using an anti-Orf1p generated previously . The piRNA-defective ovary displays an even higher increase of Orf1p level (Fig 2E), similar to qPCR results measuring transcript levels. Therefore, at least some of the transcripts generated as a result of de-silencing are capable of being translated.
The extent of telomere de-silencing in hiphopHA mutants is remarkable if one considers that it happens under the background of an intact piRNA pathway. To help identify potential mechanisms, we first addressed the question of whether HipHop-mediated silencing is specific to the retrotransposon per se, regardless of where the elements are positioned in the genome. This is relevant because we and others have shown that transcripts generated from transgenes under the regulatory control of HeT-A were greatly elevated in a piRNA defective germline, even when the transgenes are not inserted at a telomeric position [36,37]. If such a transgene were similarly de-repressed under the hiphopHA mutant background as its endogenous counterparts at the telomeres, we would conclude that HipHop acts on HeT-A specifically. As shown in the qPCR results in Fig 3A, this does not appear to be the case. The endogenous HeT-A elements are de-repressed in either a hiphop or a piRNA defective background (spnE-), while a gfp-marked HeT-A transgene is only de-silenced under the latter since the slight elevation of HeT-AGFP expression under the hiphopHA/df background did not reach statistical significance (Fig 3A). This is shown for two independent HeT-AGFP insertions on chromosomes X and 3. Therefore, we suggest that HipHop exerts its silencing effect in cis.
A. TE levels in hiphop and piRNA mutants. Ovaries were taken from adults with a transgenic HeT-A element marked with orf1p-gfp inserted at non-telomeric positions on chromosome 3 (HeT-AGFP@III) or the X chromosome (HeT-AGFP@X). Transcript levels from four telomeric regions and two non-telomeric regions: I elements and the transgenic HeT-A (gfp) were measured and plotted for two mutants and their heterozygous siblings. Note that the gfp-marked elements were de-repressed in spnE but not hiphopHA/df mutant germlines. B. Genetic interaction between piRNA and hiphop mutants. Transcripts from hiphop mutants, and hiphop mutants that were also heterozygous for aub were measured in ovarian samples. C. Genetic interaction between hiphop mutants and mutants of HP1-like proteins. Transcripts from hiphop mutants, and hiphop mutants that were also heterozygous for either Su(var)205 (top) or rhino (bottom) were measured in ovarian samples. For Su(var)205, Df(mut) = Df(2L)BSC227; for rhino, Df(mut) = Df(2R)Exel7149. D. Chromatin immunoprecipitation of HipHop and HP1 at telomeric elements. Chromatin occupancy of HipHop and HP1 proteins was assayed by ChIP followed by qPCR on five regions on telomeric elements for two mutants and their heterozygous siblings. ns: not significant; *: p
The above results provide the first line of evidence suggesting that HipHop-mediated telomeric silencing in the germline is independent of the global piRNA pathway. We gathered additional evidence in support of this proposition from a genetic interaction study in which we compared transcript levels of telomeric elements under a hiphopHA/df background with those under the same hiphopHA/df background that was also heterozygous for an aub mutation. As shown in Fig 3B, this reduction of aub dosage has no effect on telomeric silencing brought about by the hiphop mutation. In contrast, when we conducted a similar genetic study with a reduction of either HP1 or its germline specific paralog Rhino , we observed a further enhancement of HeT-A de-silencing (Fig 3C). 2b1af7f3a8