Genome-Wide Control of the Distribution of Meiotic Recombination

Meiotic recombination events are not randomly distributed in the genome but occur in specific regions called recombination hotspots. Hotspots are predicted to be preferred sites for the initiation of meiotic recombination and their positions and activities are regulated by yet-unknown controls. The activity of the Psmb9 hotspot on mouse Chromosome 17 (Chr 17) varies according to genetic background. It is active in strains carrying a recombinant Chr 17 where the proximal third is derived from Mus musculus molossinus. We have identified the genetic locus required for Psmb9 activity, named Dsbc1 for Double-strand break control 1, and mapped this locus within a 6.7-Mb region on Chr 17. Based on cytological analysis of meiotic DNA double-strand breaks (DSB) and crossovers (COs), we show that Dsbc1 influences DSB and CO, not only at Psmb9, but in several other regions of Chr 17. We further show that CO distribution is also influenced by Dsbc1 on Chrs 15 and 18. Finally, we provide direct molecular evidence for the regulation in trans mediated by Dsbc1, by showing that it controls the CO activity at the Hlx1 hotspot on Chr 1. We thus propose that Dsbc1 encodes for a trans-acting factor involved in the specification of initiation sites of meiotic recombination genome wide in mice.     

Trans-Regulation of Mouse Meiotic Recombination Hotspots by Rcr1

Meiotic recombination is required for the orderly segregation of chromosomes during meiosis and for providing genetic diversity among offspring. Among mammals, as well as yeast and higher plants, recombination preferentially occurs at highly delimited chromosomal sites 1–2 kb long known as hotspots. Although considerable progress has been made in understanding the roles various proteins play in carrying out the molecular events of the recombination process, relatively little is understood about the factors controlling the location and relative activity of mammalian recombination hotspots. To search for trans-acting factors controlling the positioning of recombination events, we compared the locations of crossovers arising in an 8-Mb segment of a 100-Mb region of mouse Chromosome 1 (Chr 1) when the longer region was heterozygous C57BL/6J (B6) × CAST/EiJ (CAST) and the remainder of the genome was either similarly heterozygous or entirely homozygous B6. The lack of CAST alleles in the remainder of the genome resulted in profound changes in hotspot activity in both females and males. Recombination activity was lost at several hotspots; new, previously undetected hotspots appeared; and still other hotspots remained unaffected, indicating the presence of distant trans-acting gene(s) whose CAST allele(s) activate or suppress the activity of specific hotspots. Testing the activity of three activated hotspots in sperm samples from individual male progeny of two genetic crosses, we identified a single trans-acting regulator of hotspot activity, designated Rcr1, that is located in a 5.30-Mb interval (11.74–17.04 Mb) on Chr 17. Using an Escherichia coli cloning assay to characterize the molecular products of recombination at two of these hotspots, we found that Rcr1 controls the appearance of both crossover and noncrossover gene conversion events, indicating that it likely controls the sites of the double-strand DNA breaks that initiate the recombination process.     

Transmission Distortion Affecting Human Noncrossover but Not Crossover Recombination: A Hidden Source of Meiotic Drive

Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.     

The Bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage DNA replication

Mycobacteriophage Bxb1 encodes a serine-integrase that catalyzes both integrative and excisive site-specific recombination. However, excision requires a second phage-encoded protein, gp47, which serves as a recombination directionality factor (RDF). The viability of a Bxb1 mutant containing an S153A substitution in gp47 that eliminates the RDF activity of Bxb1 gp47 shows that excision is not required for Bxb1 lytic growth. However, the inability to construct a Δ47 deletion mutant of Bxb1 suggests that gp47 provides a second function that is required for lytic growth, although the possibility of an essential cis-acting site cannot be excluded. Characterization of a mutant prophage of mycobacteriophage L5 in which gene 54 - a homologue of Bxb1 gene 47 - is deleted shows that it also is defective in induced lytic growth, and exhibits a strong defect in DNA replication. Bxb1 gp47 and its relatives are also unusual in containing conserved motifs associated with a phosphoesterase function, although we have not been able to show robust phosphoesterase activity of the proteins, and amino acid substitutions with the conserved motifs do not interfere with RDF activity. We therefore propose that Bxb1 gp47 and its relatives provide an important function in phage DNA replication that has been co-opted by the integration machinery of the serine-integrases to control the directionality of recombination.     

Combinatorial Regulation of Meiotic Holliday Junction Resolution in C. elegans by HIM-6 (BLM) Helicase,SLX-4, and the SLX-1, MUS-81 and XPF-1 Nucleases

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of “univalents” linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process.     

Mutations at the human minisatellite MS32 integrated in yeast occur with high frequency in meiosis and involve complex recombination events

Minisatellites are composed of tandem repetitive DNA sequences and are present at many positions in the human genome. They frequently mutate to new length alleles in the germline, by complex and incompletely understood recombination mechanisms which may operate during meiosis. In several minisatellites the mutation events are restricted to one end of the repeat array, indicating a possible association with elements that act in cis. Mutant alleles do not show exchange of flanking regions. To construct a model system suitable for further investigations of the mutation process, we have integrated the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot upstream of the LEU2 locus in the yeast Saccharomyces cerevisiae. Here we provide direct evidence for a meiotic origin of MS32 mutations. Mutation events were polarised towards both ends of the minisatellite and varied from simple duplications and deletions to complex intra- and interallelic events. Interallelic events were frequently accompanied by exchange of regions flanking the minisatellite. The results also support the notion that cis-acting elements are involved in the mutational process. The fact that MS32 mutant structures are similar in yeast and human shows that meiotic recombination plays a crucial role in both organisms and emphasises the usefulness of yeast strains harbouring minisatellites as a model system for the study of minisatellite mutation. Received: 1 March 1997 / Accepted: 16 May 1997     

Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis

The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein–DNA complexes that support this process have mainly been studied using RAG–RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's ‘12/23 rule’ and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the postcleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination.     

Formation of the P1.1 pseudoknot is critical for both the cleavage activity and substrate specificity of an antigenomic trans-acting hepatitis delta ribozyme

Hepatitis delta virus RNAs possess self-cleavage activities that produce 2′,3′-cyclic phosphate and 5′-hydroxyl termini (i.e. cis-acting delta ribozyme). Trans-acting delta ribozymes have been engineered by removing a junction from the cis version, thereby producing one molecule possessing the substrate sequence and the other the catalytic domain. According to the pseudoknot model, the secondary structure of the delta ribozyme includes a pseudoknot (i.e. P1.1 stem) formed by two base pairs from residues of the L3 loop and J1/4 junction. A collection of 48 P1.1 stem mutants was synthesized in order to provide an original characterization of both the importance and the structure of this pseudoknot in a trans-acting version of the ribozyme. Several structural differences were noted compared to the results reported for cis-acting ribozymes. For example, a combination of two stable Watson–Crick base pairs composing the essential P1.1 stem was demonstrated to be crucial for a significant level of activity, while the cis version required only one base pair. In addition, we present the first physical evidences revealing that the composition of the P1.1 stem affects the substrate specificity for ribozyme cleavage. Depending on the residues forming the J1/4 junction, non-productive ribozyme–substrate complexes can be observed. This phenomenon is proposed to be important for further development of a gene-inactivation system based on delta ribozyme.     

DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination.     

Contrasted Patterns of Crossover and Non-crossover at Arabidopsis thaliana Meiotic Recombination Hotspots

The vast majority of meiotic recombination events (crossovers (COs) and non-crossovers (NCOs)) cluster in narrow hotspots surrounded by large regions devoid of recombinational activity. Here, using a new molecular approach in plants, called “pollen-typing”, we detected and characterized hundreds of CO and NCO molecules in two different hotspot regions in Arabidopsis thaliana. This analysis revealed that COs are concentrated in regions of a few kilobases where their rates reach up to 50 times the genome average. The hotspots themselves tend to cluster in regions less than 8 kilobases in size with overlapping CO distribution. Non-crossover (NCO) events also occurred in the two hotspots but at very different levels (local CO/NCO ratios of 1/1 and 30/1) and their track lengths were quite small (a few hundred base pairs). We also showed that the ZMM protein MSH4 plays a role in CO formation and somewhat unexpectedly we also found that it is involved in the generation of NCOs but with a different level of effect. Finally, factors acting in cis and in trans appear to shape the rate and distribution of COs at meiotic recombination hotspots.     

Phosphorylation-Independent Regulation of Atf1-Promoted Meiotic Recombination by Stress-Activated,p38 Kinase Spc1 of Fission Yeast

Background

Stress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26.

Methodology/Principal Findings

We sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination.

Conclusions/Significance

The Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1.     

Effects of the RAD52 Gene on Recombination in SACCHAROMYCES CEREVISIAE

Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, γ-ray-induced, UV-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the his1–1/his1–315 and trp5–2/trp5–48 heteroalleles. Gene-centromere recombination also was not observed in rad52/rad52 diploids. No γ-ray- or UV-induced intragenic mitotic recombination is seen in rad52/rad52 diploids. The rate of spontaneous mitotic recombination is lowered five-fold at the his1–1/his1–315 and leu1–c/leu1–12 heteroalleles. Spontaneous reversion rates of both his1–1 and his1–315 were elevated 10 to 20 fold in rad52/rad52 diploids.—The RAD52 gene function is required for spontaneous mitotic recombination, UV- and γ-ray-induced mitotic recombination and meiotic recombination.     

A Quality Control Mechanism Linking Meiotic Success to Release of Ascospores

Eukaryotic organisms employ a variety of mechanisms during meiosis to assess and ensure the quality of their gametes. Defects or delays in successful meiotic recombination activate conserved mechanisms to delay the meiotic divisions, but many multicellular eukaryotes also induce cell death programs to eliminate gametes deemed to have failed during meiosis. It is generally thought that yeasts lack such mechanisms. Here, we show that in the fission yeast Schizosaccharomyces pombe, defects in meiotic recombination lead to the activation of a checkpoint that is linked to ascus wall endolysis – the process by which spores are released in response to nutritional cues for subsequent germination. Defects in meiotic recombination are sensed as unrepaired DNA damage through the canonical ATM and ATR DNA damage response kinases, and this information is communicated to the machinery that stimulates ascus wall breakdown. Viability of spores that undergo endolysis spontaneously is significantly higher than that seen upon chemical endolysis, demonstrating that this checkpoint contributes to a selective mechanism for the germination of high quality progeny. These results provide the first evidence for the existence of a checkpoint linking germination to meiosis and suggest that analysis solely based on artificial, enzymatic endolysis bypasses an important quality control mechanism in this organism and potentially other ascomycota, which are models widely used to study meiosis.     

Distinct regions of ATF/CREB proteins Atf1 and Pcr1 control recombination hotspot ade6-M26 and the osmotic stress response

The Atf1 protein of Schizosaccharomyces pombe contains a bZIP (DNA-binding/protein dimerization) domain characteristic of ATF/CREB proteins, but no other functional domains or clear homologs have been reported. Atf1-containing, bZIP protein dimers bind to CRE-like DNA sites, regulate numerous stress responses, and activate meiotic recombination at hotspots like ade6–M26. We defined systematically the organization of Atf1 and its heterodimer partner Pcr1, which is required for a subset of Atf1-dependent functions. Surprisingly, only the bZIP domain of Pcr1 is required for hotspot activity and tethering of Atf1 to ade6 promotes recombination in the absence of its bZIP domain and the Pcr1 protein. Therefore the recombination–activation domain of Atf1-Pcr1 heterodimer resides exclusively in Atf1, and Pcr1 confers DNA-binding site specificity in vivo. Atf1 has a modular organization in which distinct regions affect differentially the osmotic stress response (OSA) and meiotic recombination (HRA, HRR). The HRA and HRR regions are necessary and sufficient to activate and repress recombination, respectively. Moreover, Atf1 defines a family of conserved proteins with discrete sequence motifs in the functional domains (OSA, HRA, HRR, bZIP). These findings reveal the functional organization of Atf1 and Pcr1, and illustrate several mechanisms by which bZIP proteins can regulate multiple, seemingly disparate activities.     

DNA sequence-mediated, evolutionarily rapid redistribution of meiotic recombination hotspots

Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor.     

Caenorhabditis elegans HIM-18/SLX-4 Interacts with SLX-1 and XPF-1 and Maintains Genomic Integrity in the Germline by Processing Recombination Intermediates

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR–mediated repair at stalled replication forks. A reduction in crossover recombination frequencies—accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction—support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.     

Cis- and trans-acting elements regulate the mouse Psmb9 meiotic recombination hotspot

In most eukaryotes, the prophase of the first meiotic division is characterized by a high level of homologous recombination between homologous chromosomes. Recombination events are not distributed evenly within the genome, but vary both locally and at large scale. Locally, most recombination events are clustered in short intervals (a few kilobases) called hotspots, separated by large intervening regions with no or very little recombination. Despite the importance of regulating both the frequency and the distribution of recombination events, the genetic factors controlling the activity of the recombination hotspots in mammals are still poorly understood. We previously characterized a recombination hotspot located close to the Psmb9 gene in the mouse major histocompatibility complex by sperm typing, demonstrating that it is a site of recombination initiation. With the goal of uncovering some of the genetic factors controlling the activity of this initiation site, we analyzed this hotspot in both male and female germ lines and compared the level of recombination in different hybrid mice. We show that a haplotype-specific element acts at distance and in trans to activate about 2,000-fold the recombination activity at Psmb9. Another haplotype-specific element acts in cis to repress initiation of recombination, and we propose this control to be due to polymorphisms located within the initiation zone. In addition, we describe subtle variations in the frequency and distribution of recombination events related to strain and sex differences. These findings show that most regulations observed act at the level of initiation and provide the first analysis of the control of the activity of a meiotic recombination hotspot in the mouse genome that reveals the interactions of elements located both in and outside the hotspot.     

A Highlights from MBoC Selection: EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis protein REC8

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 Lys-4 and Lys-36 methyltransferase PRDM9 to ensure successful double-strand-break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse model to deplete EWSR1 before the onset of meiosis and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand-break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through its component cohesin pREC8.     

Definition of the minimal cis-acting sequences necessary for genome maturation of the herpesvirus murine cytomegalovirus

Herpesvirus DNA replication proceeds via concatemeric replicative intermediates that are comprised of head-to-tail-linked genomes. Genome maturation is carried out by the terminase, a protein complex that mediates both insertion of concatemer DNA into capsids and its subsequent cleavage to release genomes within these capsids. This cleavage is sequence specific, but the governing cis-acting DNA sequences are only partially characterized. Two highly conserved motifs called pac1 and pac2 lie near the ends of herpesvirus genomes and are known to be critical for genome maturation. However, the potential importance of other sequences has not been fully investigated. We have undertaken to define all of the sequences necessary for efficient genome maturation for a herpesvirus by inserting ectopic cleavage sites into the murine cytomegalovirus genome and assessing their ability to mediate genome maturation. A combination of deletion and substitution mutations revealed that the minimal cleavage site is large (~180 bp) and complex. Sequences distal of pac1 (relative to the point of cleavage) were dispensable, suggesting that pac1 may be the sole cis-acting element on this side of the cleavage site. In contrast, a region distal to pac2 up to 150 bp from the point of cleavage was essential. Scanning substitutions revealed that the pac2 side of the cleavage site is complex and may contain multiple cis-acting sequence elements in addition to pac2. These results should facilitate the identification of trans-acting factors that bind to these elements and the elucidation of their functions. Such information will be critical for understanding the molecular basis of this complex process.