The Drosophila developmental gene fat facets has a human homologue in Xp11.4 which escapes X-inactivation and has related sequences on Yq11.2
The Drosophila developmental gene fat facets has a human homologue in Xp11.4 which escapes X-inactivation and has related sequences on Yq11.2Michael H. Jones+,§, Robert A. Furlong+, Heather Burkin, I. Jennifer Chalmers, Graeme M. Brown, Omar Khwaja and Nabeel A. Affara*
Human Molecular Genetics Group, University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK
Received June 7, 1996;Revised and Accepted August 22, 1996DDBJ/EMBL/GenBank accession no. X98296
EST 221 derived from human adult testis detects homology to the Drosophila fat facets gene (faf) and has related sequences on both the X and Y chromosomes mapping to Xp11.4 and Yq11.2 respectively. These two loci have been termed DFFRX and DFFRY for Drosophila fat facets related X and Y. The major transcript detected by EST 221 is ~8 kb in size and is expressed widely in a range of 16 human adult tissues. RT-PCR analysis of 13 different human embryonic tissues with primers specific for the X and Y sequences demonstrates that both loci are expressed in developing tissues and quantitative RT-PCR of lymphoblastoid cell lines carrying different numbers of X chromosomes reveals that the X-linked gene escapes X-inactivation. The amino acid sequence (2547 residues) of the complete open reading frame of the X gene has 44% identity and 88% similarity to the Drosophila sequence and contains the conserved Cys and His domains characteristic of deubiquitinating enzymes, suggesting its biochemical function may be the hydrolysis of ubiquitin from protein-ubiquitin conjugates. The requirement of faf for normal oocyte development in Drosophila combined with the map location and escape from X-inactivation of DFFRX raises the possibility that the human homologue plays a role in the defects of oocyte proliferation and subsequent gonadal degeneration found in Turner syndrome.
Systematic sequence analysis of cDNA clones to generate ESTs has proved to be an effective means of identifying human transcripts that share homology with genes described in other species (1 ). This can give an indication of function and when combined with mapping to a particular chromosome region, it is possible to identify ESTs that are candidates for particular disease loci (2 ,3 ). Analysis of cDNA clones from a human adult testis cDNA library has identified EST 221 which detects similarity to the Drosophila developmental gene fat facets (faf) (4 ). In Drosophila, mutations of the faf gene are associated with two phenotypes. First, the gene is essential for normal oogenesis and, second, the gene product influences the fate of cells destined to become photoreceptors in the developing compound eye (5 ). Recently, it has been shown that faf is a member of a family of deubiquitinating genes whose products remove ubiquitin from protein-ubiquitin conjugates (6 ). Thus the gene may play an important regulatory role at the level of protein degradation by preserving proteins marked by conjugation with ubiquitin for digestion by the proteasome.
This paper describes the analysis of the human X-linked homologue of the faf gene. The gene is X-Y homologous, mapping to Xp11.4 and Yq11.2 and these have been named DFFRX and DFFRY. Transcription occurs from the X and Y loci in both human adult and embryonic tissues and the gene on the X has been shown to escape X-inactivation. The location of DFFRX in proximal Xp (Xp11.4), its escape from X-inactivation and the importance of faf for oocyte development in Drosophila raises the intriguing possibility that DFFRX is a candidate for the gonadal degeneration observed in Turner syndrome provoked by a failure of oocytes to proliferate and develop (7 ,8 ).
EST 221 was isolated from an adult human testis cDNA library and mapped to the X and Y chromosomes by Southern blot analysis of DNA from a monochromosome somatic cell hybrid panel (Fig. 1 ). Further mapping on the Y chromosome was achieved by Southern analysis of DNA from individuals with deletions of the Y long arm. This placed the Y locus proximal to the KALp gene in Yq11.2, a result that was confirmed by PCR analysis with EST 221 primers of CEPH mega YAC clones forming a contig across this region of the Y chromosome (9 ). The CEPH YACs 711F4 and 900E7 were shown to contain sequences homologous to EST 221 (using primers Y221D and Y221J marked in Fig. 2 ) thus placing the locus within an interval of ~500-1000 kb, flanked by the markers DYS11 and DYS246 (see Y consensus map, ref. 10 ). Mapping of the X locus to proximal Xp (Xp21.1-Xcen) was determined by analysis of X somatic cell hybrids carrying deletions of Xp and confirmed by screening the CEPH mega YAC library with the EST 221 primers X221R and X221K (also marked in Fig. 2 ). This identified three different YAC clones (892D10, 782F4 and 781F4) containing the marker DXS993 which has been mapped to Xp11.4 in an interval defined by the markers DXS556 and DXS77. This interval covers ~1.2 Mb (see X chromosome workshop report by Nelson et al., ref. 11 ).
EST 221 was used to screen a testis cDNA library to initiate a cDNA walk to derive clones covering the complete open reading frame of DFFRX. Foetal brain and retinal cDNA libraries were also used to yield clones contributing to the cDNA walk. During this walk both X and Y derived cDNA clones were isolated and assigned to either sex chromosome using PCR primers based on sequence differences between the clones. The isolation of X and Y specific cDNA clones indicates that both loci are transcribed in adult tissues. The sequence of the transcript defining the open reading frame of DFFRX is shown in Figure 2 (accession number X98296).
Over the two regions (5' and 3') of DFFRY that have been isolated as cDNA clones (corresponding to nucleotides 6-1901 and nucleotides 5815-7907 of the DFFRX nucleotide sequence), the X and Y sequences share 91% and 88% identity, respectively. Interestingly the position of the proposed initiating ATG codon which is preceded by in-frame stop codons is the same for both DFFRX and DFFRY (not shown). Both sequences have a purine (A) in the -3 position conforming to the consensus initiation sequence of Kozak (12 ). Analysis of the currently available 5' sequence of DFFRY (~2 kb) shows the presence of an open reading frame encoding for 644 amino acids that remains open at the 3' end of the sequence. However, the more 3' segment of DFFRY contains premature multiple stop codons in all three reading frames, suggesting that the Y locus may either encode a truncated protein or represent a non-functional pseudogene. The arrows in Figure 2 highlight the position and orientation of PCR primers used for expression analysis and mapping studies.
Alignment of the putative amino acid residues of Drosophila faf and human DFFRX genes shows 44% identity and 88% similarity (not shown). Both putative gene products contain conserved Cys and His domains that have been described in a number of gene products that in vitro and also in vivo have been shown to function as ubiquitin C-terminal hydrolases. These comprise the yeast UBP1-UBP4 (13 -15 ), the human TRE-2 (15 ) and the Drosophila faf (6 ) gene products. Figure 3 shows a comparison of the conserved Cys and His domains of the putative protein products of 11 genes that may function as deubiquitinating enzymes (see Fig. 3 legend for references). In addition to the residues that are absolutely conserved in the domains of these proteins, DFFRX and faf also have a high number of residues in common particularly at the C-terminal end of the His domain.
Northern blot analysis of a range of 16 adult human tissues has shown that sequences detected by EST 221 are widely expressed (Fig. 4 A). The expression in a range of embryonic human tissues was determined by RT-PCR using primers specific for the X and Y genes (these are Y221D and Y221J for the Y and X221R and X221K for the X; marked in Fig. 2 ). Y221D and Y221J had three and four mismatched bases to the corresponding X sequence respectively. The primers were designed to amplify across introns whose positions were determined by PCR analysis of genomic DNA. This provides an internal control for genomic DNA contamination of cDNA used in RT-PCR experiments. The genomic fragments generated by the X and Y specific primers are 2.2 and 1.2 kb, whereas the corresponding cDNA PCR products are 401 and 304 bp, respectively. Figure 4 C shows the analysis of 13 different human embryonic tissues and demonstrates that transcripts from both loci are present in a wide range of tissues. The sex of the embryonic tissues is unknown, and it is likely that failure to see a Y product in some tracks reflects a female origin of the tissue. The strength of the PCR product obtained from the Y specific primers may in part reflect the more efficient amplification of smaller PCR fragments. As an internal control, the same samples were analysed using primers from the ubiquitously expressed gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This is found expressed in all the embryonic tissues.
The X-inactivation status of DFFRX was determined through the use of quantitative RT-PCR with the same X-specific primers used to analyse expression of embryonic tissues. As an internal control, the same mRNA samples and cDNA populations were analysed with primers that amplify a fragment from exons of the X-linked HPRT (hypoxanthine phosphoribosyl transferase) gene, a gene known to undergo X-inactivation. Figure 5 demonstrates that the expected DFFRX (401 bp) and HPRT (462 bp) fragments can be detected by RT-PCR of mRNA isolated from lymphoblastoid cell lines containing a single X chromosome, two X chromosomes and four X chromosomes. Densitometric measurements were taken of the RT-PCR fragments produced from each cell line for the two genes. The ratios of 2X:1X and 4X:1X are 1.7 and 4.4 for DFFRX and 1.1 and 1.4 for HPRT. From this, it can be seen that irrespective of the number of X chromosomes present in the cell, the level of expression of HPRT detected by RT-PCR remains relatively constant. In contrast, the level of DFFRX expression rises as the copy number of X chromosomes increases, indicating that DFFRX escapes X-inactivation.
The conserved Cys and His domains of DFFRXare characteristic of deubiquitinating enzymes found in yeast (13 -15 ) and suggest that the biochemical function of the protein may be to hydrolyze ubiquitin from protein-ubiquitin conjugates. It has been demonstrated biochemically that faf (which has Cys and His conserved domains that are almost identical to DFFRX) has C-terminal ubiquitin hydrolase activity (6 ). Such activity protects proteins from being degraded by the proteasome and thus provides a mechanism for regulation at the protein level that can modify cellular phenotype.
Both DFFRX and DFFRY are expressed widely indicating that the gene may not have a specific phenotypic effect in a particular tissue or cell type. However, the Drosophila gene has been shown to be subject to alternative splicing and it is possible that differing isoforms of DFFRX have modified ligand specificity in different cell types. This is supported by preliminary evidence for alternative splicing of DFFRX (N.Affara, unpublished data).
The high degree of conservation between the Drosphila faf gene and the DFFRX sequence implies an important function for the locus in humans. The phenotypes observed in flies mutant for the faf gene flag two possible functions. First, the gene is important for normal eye development where it influences the fate of cells within the ommatidia destined to become photoreceptors. In mutant flies there is disorganized ectopic differentiation of photoreceptors cells in the facets of the compound eye (5 ). The chromosomal location of DFFRX in Xp11.4 [also confirmed by the mapping of a faf-related EST by Banfi et al. (16 )] is significant in this respect as several retinal disorders map to this region; retinitis pigmentosum 2 and 3 (RP2, RP3), cone dystrophy 1 (COD 1), Aland Island Eye Disease (AIED) and congenital stationary night blindness 1 (CSNB 1) (11 ). However, it seems improbable that DFFRX is involved in most of these disorders. The gene for RP3 has been isolated recently (17 ) and codes for a protein showing homology to the guanine-nucleotide-exchange factor RCC1. The fine mapping to CEPH YACs has provided precise localisations in relation to these disease loci and probably excludes DFFRX as a candidate for RP2, CSNB1 and AIED. At the genomic level the gene would have to cover a minimum distance of 2 Mb proximally to begin to overlap with the 7 Mb interval (DXS7-DXS255) into which RP2, AIED and CNSB1 have been mapped. COD 1 has been mapped into a 7.3 Mb interval that overlaps the DFFRX interval and thus the gene is a possible candidate for this retinal disease. For DFFRX to be considered as a candidate gene for COD1 it is assumed that DFFRY would have to be functional. An inactive Y-gene with an X homologue that escapes X-inactivation would lead to an effective hemizygous state in normal males for a gene which may be required in two functional copies in females to permit development of photoreceptors in the eye.
The second function relates to the role that faf plays in oocyte formation. In Drosophila,mutations in the gene lead to a failure of normal oocyte development and the inability of the fertilized egg to undergo normal embryogenesis (5 ). This is a maternal effect requiring the product of faf to be layed down in the oocyte. The map location of DFFRX coincides with the region of the X defined by partial deletions in females (resulting in monosomy for part of proximal Xp) as being critical for the major stigmata associated with the Turner phenotype (18 ). Particularly noteworthy in this context is the failure of oocytes to pass through the first meiotic prophase in Turner females, with massive oocyte loss leading to the degeneration of the developing ovary into a streak gonad (7 ,8 ). This is clearly different to the Drosophila phenotype where an egg capable of fertilization is produced. Nevertheless, in both cases there is defective oocyte development raising the possibility that DFFRX is important in human oogenesis. It has been proposed by Ferguson-Smith (19 ,20 ) and Zinn et al. (21 ) that the gene or genes that underlie the Turner phenotype should escape X-inactivation in the female, thus providing the necessary diploid dose of gene product. In males, the X-linked genes should possess a functional Y-linked homologue unless the stigma relates to a female specific function such as oocyte development. Thus a role of DFFRX in oogenesis is only consistent with the Y-linked gene being non-functional or having a modified male-specific function.
At present it remains open as to whether DFFRY encodes a functional, albeit truncated protein, or represents a non-functional transcript. Alignment of the available DFFRX and DFFRY sequences shows that the premature stop codon identified in DFFRY lies 3' to the conserved Cys and His domains of DFFRX (R.A.Furlong, unpublished data), thus a de-ubiquitinating function of DFFRY remains a possibility. An assessment of the degree of conservation of these domains in DFFRY, through sequence analysis of the Y transcript, is currently in progress and should help to resolve the issue of whether DFFRX/Y are plausible candidates for COD 1 or the gonadal phenotype observed in Turner syndrome.
Southern analysis was carried out using standard procedures and DNA prepared as described in Maniatis et al. (22 ). Monochromosome somatic cell hybrids were obtained from the NIGMS Corriel cell repository and the X somatic cell hybrid lines carrying deletions of Xp were those described in Sargent et al. (2 ). Mapping of DFFRX and DFFRY to CEPH mega YAC clones was performed by PCR screening of the CEPH Mega YAC library and subsequent purification of clones as described in Jones et al. (9 ). All PCR reactions were carried out in a buffer containing 25 mM TAPS pH 9.3, 50 mM KCl, 1 mM DTT, 2 mM MgCl2 and 0.05% W1 detergent. All primers were designed to operate in the following conditions: Anneal 58oC for 1 min; extension 72oC for 1 min; denaturation 95oC for 1 min. PCR was generally for 30 cycles. Each reaction contained 20 pmol of each primer.
Nested PCR from a retina cDNA library (Clontech) using DFFRX 5'-directed primers (primary PCR primer DFFRXcs2 (5'-GTG ATC GAG GAT CTG GAG AAC G-3'); secondary PCR primer DFFRXcs3 (5'-GAG GAA ACT GAT TCA CAT GGA CG-3') and a vector primer ([lambda]gt11F) gave several discrete bands retb1-retb5. Sequence analysis of retb4 and retb5 showed that these overlapped with DFFRX. Retb4 was then used to screen a retina cDNA library and five single-plaque positives were isolated. Inserts were amplified using NM1149F (5'-CCT TTG AGC AAG TTC AGC CT-3') and NM1149R (5'-AGA GGT GGC TTA TGA GTA TTT CTT-3') primers and sequenced using NM1149Fcs (5'-CAG CCT GGT TAA GTC CAA G-3') or NM1149Rcs (5'-CCA GGG TAA AAA GCA AAA G-3'). Sequence analysis showed that two clones (R64.1.3, R66.2.1) were similar, but not identical to the existing DFFRX sequence and to the other three clones. These two clones were from the DFFRY gene; the remainder (R65.1, R67.1.1 and R69.1.3) were from DFFRX.
Nested PCR products from a foetal brain cDNA library (Clontech) using DFFRX 5'-directed primers (primary PCR primer DFFRXcs3; secondary 5'-modified PCR primer DFFRXcs4 5'-CUA CUA CUA CUA ACC TAT GAA TTT CAA ACT TCC AGC-3' with the 5-modified PCR primer [lambda]gt11R (5'-CAU CAU CAU CAU GGC CTG CCC GGT TAT TAT TAT-3') were size-selected and cloned into pAMP1 using the Cloneamp kit (GIBCO-BRL). Inserts were amplified with pSPORTF (5'-GTA AAA CGA CGG CCA GTG AA-3') and pSPORTR (5'-CTA TGA CCA TGA TTA CGC CAA G-3') primers and sequenced using pSPORTFcs (5'-GCG TAC GTA AGC TTG GAT C-3') or pSPORTRcs (5'-TAG GGA AAG CTG GTA CGC-3') primers.
Northern blot analysis. Analysis was performed using Northern blots obtained from Clontech. These have a range of poly A+ RNAs from 16 human adult tissues. The blots were hybridized (in the buffer and under the conditions recommended in the Clontech manual) with the probe EST 221 labelled with [32P]dCTP by random oligonucleotide priming and washed in 1* SSC, 0.1% SDS at 65oC for 20 min. The membranes were exposed to Kodak XAR5 film at -70oC for 24 h.RT-PCR analysis. RNA was prepared from cell lines and tissues using the Molecular Research Center Inc. Tri-Reagent kit using the recommended protocols. Total RNA (1 [mu]g) was used for reverse transcription using the Promega reverse transcription system under the recommended protocols. Primers [X221R, X221K, Y221D and Y221J for DFFRX and DFFRY respectively; HPRTCA (5'-CCT GGC GTC GTG ATT AGT GA-3') and HPRTCB (5'-TGC GAC CTT GAC CAT CTT TG-3') for HPRT; GAPDHF (5'-GAC CCC TTC ATT GAC CTC AAC TAC A-3') and GAPDHR ( 5'-CTA AGC AGT TGG TGG TGC AGG A-3') for GAPDH] at a concentration of 50 ng/[mu]l were end-labelled using T4 polynucleotide kinase and [gamma]-labelled ATP (Amersham 5000 Ci/mmol) in a reaction containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine and 5 U kinase at 37oC for 30 min. The reaction was inactivated at 90oC for 10 min and 100 ng (2 [mu]l of the reaction) was used to amplify by PCR the cDNA populations derived from reverse transcription of RNA. PCR reactions were sampled at 15, 20, 25 and 30 cycles to ensure that the amplification was still in the linear phase of the reaction. Aliquots of each reaction (2 [mu]l) were loaded on to a 40 cm * 1 mm 5% acrylamide (19:1 acrylamide/bisacryalmide) gel in 0.5* TBE (Tris/borate/EDTA buffer) and electrophoresed at 3 W for 17 h. The gel was dried on to Whatman 3MM paper and exposed to Kodak XOMAT film at -70oC for between 4 and 16 h. Densitometric measurements of the bands were performed using the Applied Imaging Lynx system.
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*To whom correspondence should be addressed
+Both authors contributed equally to this work
§Present address: Chugai Institute for Molecular Medicine, 153-2 Nagai, Niihari Mura, Niihari-Gun, Ibaraki, 300-41, Japan
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