Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press LETTER Structural Organization of the WD Repeat Protein-encoding Gene HIRA in the DiGeorge Syndrome Critical Region of Human Chromosome 22 St~phanie Lorain, 1 Suzanne Demczuk, 2 Val~rie Lamour, 1 Steve Toth, 3 Alain Aurias, 2 Bruce A. Roe, 3 and Marc Lipinski ~'4 1Laboratoire de Biologie des Tumeurs Humaines, Centre National de la Recherche Scientifique URA 1156, Institut Gustave Roussy, 94805 Villejuif CEDEX, France; 2Laboratoire de G~n~tique des Tumeurs, Institut National de la Sant~ et de la Recherche M~dicale U 434, Institut Curie, 75231 Paris CEDEX 05, France; 3Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 78019-0370 The human gene HIRA lies within the smallest critical region for the DiGeorge syndrome [DGS], a haploinsufficiency developmental disorder associated with instertitial deletions in most patients in a juxtacentromeric region of chromosome 22. The HIRA protein sequence can be aligned over its entire length with Hirl and Hir2, two yeast proteins with a regulatory function in chromatin assembly. The HIRA transcription unit was found to spread over -I00 kb of the DGS critical region. The human transcript is encoded from 25 exons between 59 and 861 bp in size. Domains of highest conservation with Hirl and Hir2 are encoded from exons I-I1 and 13-25, respectively. The amino- and carboxy-terminal regions of homology are separated from each other by a domain unique to HIRA that is encoded from a single exon. Seven WD repeats are conserved between yeast and man in the amino-terminal region of the HIR proteins. Individual repeats were found to be encoded from one, two, or three exons of the HIRA gene. End sequences have been obtained for all 24 introns, opening the way to PCR amplification of the entire coding sequence starting from genomic DNA. Point mutations can also be sought in 16 of the 24 introns that are readily PCR-amplifiable. The DiGeorge syndrome (DGS), the Shprintzen, ders, have indicated that a reduction to hemizy- or velo-cardio-facial, syndrome (VCFS), and cases gosity for a juxtacentromeric region on the long of conotruncal and heart abnormalities represent arm of chromosome 22 could be responsible for developmental disorders whose clinical features these various conditions (Scambler et al. 1991; overlap partially. In accordance, it has been pro- Carey et al. 1992; Driscoll et al. 1992a,b; Burn et posed that these disorders be grouped under the al. 1993; Desmaze et al. 1993; Morrow et al. CATCH22 (.cardiac defect, abnormal facies, thy- 1995). DGS, VCFS, and related abnormalities mic hypoplasia, cleft palate, h_ypocalcemia, chro- thus contribute examples of haploinsufficiency mosome 22qll deletion) acronym (Wilson et al. diseases, a recently recognized group of congen- 1993). Despite differences in the phenotypes ob- ital abnormalities that also includes entities such served in the patients, it appears that tissues af- as the Greig syndrome, the Langer-Giedion syn- fected mostly correspond to derivatives of the drome, Hirschprung's disease, the Smith- third and fourth pharyngeal pouches (Lammer Magenis syndrome, and the Miller-Dieker syn- and Opitz 1986). Cytogenetic analysis and mo- drome (Fisher and Scambler 1994). lecular genetic studies performed on sporadic pa- The region of overlap between the chromo- tients and, in rare instances, in individuals from somal fragments deleted in different patients de- families with a history of DGS or related disor- fines a critical region for DGS whose centromeric and telomeric boundaries are currently provided by the chromosome 22 breakpoints carried in the 4Corresponding author. E-MAIL [email protected]; FAX 33 1 45 59 64 94. X/22-33-11TG somatic cell hybrid, and in the 6:43-50 ©1996 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/96 $5.00 GENOME RESEARCH~43 Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press LORAIN ET AL. GM5878 cell line, respectively (Desmaze et al. pose that the gene be named HIRA (Lamour et al. 1993). Because the size of the smallest deleted 1995), because it encodes a likely homolog of region appears to be consistently several hundred Hirl and Hir2, two polypeptides with nuclear lo- kilobases long, CATCH22 disorders could in fact calization signals but no DNA-binding site that point to a contiguous gene syndrome, with sev- appear to function within a multiprotein com- eral genes located within the critical region being plex negatively regulating the transcription of collectively responsible for the variability in the core histone genes in the yeast Saccharomyces cer- phenotypes observed in these patients. Major ef- evisiae (Sherwood et al. 1993). In contrast with its forts have been devoted to the identification of carboxy-terminal region, which highly resembles possible DGS/VCFS gene candidates. An interest- that in Hir2 and similarly lacks any identifiable ing chromosomal rearrangement, a balanced protein motif, the amino-terminal region of t(2;22)(q14;q11) translocation, providing a con- HIRA consists of seven WD repeats, also present venient landmark within the region, was origi- in Hirl, and suggests possible interactions with nally detected in a patient (ADU) with a partial other regulatory proteins (Neer et al. 1994). We DGS phenotype (Augusseau et al. 1986). This have now elucidated the genomic structure of the translocation had been inherited from the human gene, which was found to consist of 25 proband's mother who was found to exhibit exons spread on a chromosomal segment -100 milder symptoms also compatible with a DGS/ kb in length. A relationship is demonstrated be- VCFS diagnosis (Demczuk et al. 1995). The chro- tween the exon arrangement in the gene and the mosome 22 breakpoint in the translocation prop- two distinct regions of similarity to the yeast ho- agated in this unique family and in the ADU cell mologs. In contrast, the seven WD repeats were line lies within the DGS smallest critical region. found to be encoded from one, two, or three ex- In the last several months, three different groups ons. have identified transcripts encoded from the im- mediate vicinity of the chromosome 22 break- point. A ubiquitous 4.4-kb messenger RNA, en- RESULTS AND DISCUSSION coding a protein with putative adhesive proper- HIRA Complete cDNA and Genomic ties, is transcribed from a region immediately Organization telomeric to the ADU breakpoint, but the gene, variously referred to as DGCR2 (Demczuk et al. The HIRA cDNA sequence originally reported 1995), IDD (Wadey et al. 1995), or LAN (Budarf et (EMBL accession no. X81844) contained 61 nu- al. 1995), does not appear to be rearranged by the cleotides upstream of the translation initiation translocation. Using a combination of proce- codon (Lamour et al. 1995). The sequence has dures including cDNA selection, exon trapping, now been extended further 5' (accession no. and GRAIL exon prediction from genomic se- X89887) by incorporation of 159 additional nu- quences, one of these groups has identified puta- cleotides present farther upstream in the trun- tive coding sequences on each DNA strand in the cated C5 clone (Halford et al. 1993). RNase pro- immediate vicinity of the translocation site in tection experiments (data not shown) indicate the ADU cell line (Budarf et al. 1995). One of that the 5' end of the cDNA sequence corre- these proposed exons would encompass the sponds to the main transcription initiation site. chromosomal breakpoint, but the corresponding The full-length HIRA transcript therefore consists transcript has not been cloned (Budarf et al. of 4018 nucleotides and a poly(A) tail. 1995). Part of the HIRA gene was known to be con- A third gene, also in the smallest DGS critical tained within the DAC30 cosmid (Larnour et al. region, lies farther telomeric to the ADU break- 1993). Examination and hybridization of EcoRI point (Halford et al. 1993; Lamour et al. 1995). restriction fragments, along with FISH data colo- The name Tuple I had been proposed for the gene calizing cosmids DAC30 and 48F8 within the because of moderate similarity between the DGS smallest critical region, demonstrated the amino acid sequence predicted from a partial inclusion of DAC30 into a previously reported cDNA clone and the products of the yeast TUP1 cosmid contig (Desmaze et al. 1993), which is gene and the fruit fly Enhancer of split locus (Hal- represented schematically in Figure 1A. Restric- ford et al. 1993). The protein alignments we have tion fragments were hybridized using portions of performed using the amino acid sequence de- the HIRA cDNA as probes. The 5" and 3' ends of duced from a complete cDNA have led us to pro- the HIRA cDNA were found to map to the 11-kb 44 ~ GENOME RESEARCH Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press HIRA GENE STRUCTURE A GM5878 ADU 33-11TG t(10;22) t(2;22) t(X;22) 49G 130G 49C 10 kb 113A J / B 1 2 3 4 5 6 7 8 9 1011 12 13 14 15 16 17 18 192021 22 23 24 25 13' I'-4 ATG Stop 100 bp WD repeats i ii lU iv v Vl Vll Figure 1 Genomic organization of the HIRA gene in the juxtacentromeric region of chromosome 22q. (A) A cosmid contig assembled starting from cosmids DAC30 and 48F8 maps to the region of chromosome 22 that is flanked by the breakpoints in the GM5878 and ADU cell lines. The corresponding EcoRl restriction map is shown below. Numbers correspond to the sizes (in kb) of the restriction fragments. (B) The gene is transcribed from 25 exons. The translation initiation and stop codons are indicated.