Wu Lab

Research

The presence of homology can have far-reaching consequences for gene structure and function, ranging from gene activation or repression to changes in DNA sequence, methylation, and chromatin structure1-3. Good examples of the impact of homology can be found among mammals, plants, insects, ciliates, and fungi, and includes the phenomena of transvection2,4-7, X-inactivation8-13, parental imprinting14-15, RIP16, paramutation17, and many other forms of gene silencing. Together, these and other fascinating outcomes of the presence of homology define the rapidly growing field of homology effects. Importantly, the critical roles played by homology effects in gene regulation and inheritance make them key players in human development. Therefore, it is no surprise that the medical sciences are now looking to homology effects for both the causes of a variety of diseases18-22 as well as potential therapies2; for example, homology-based medical research is addressing the imprinting-related Prader-Willi, Angelman, and Beckwith-Wiedemann Syndromes21, Rett Syndrome22, autism22, as well as diseases that are influenced by X-inactivation20. With an eye toward the implications of homology effects on human health, we are pursuing the following topics:

Transvection

Polycomb Group (PcG) genes

X-inactivation

Ultraconserved Elements

Personal genetics education project (PGED)

A long-standing focus of our laboratory has concerned the manner in which genes are regulated. In particular, we have been studying the phenomenon of transvection, which causes genes to respond to the proximity of a homologue2,4-7. As elegantly demonstrated by Ed Lewis4, transvection encompasses mechanisms by which a gene can directly affect the expression of a paired homologue lying on a separate chromosome. This is in contrast to those forms of regulation arising from the cis-action of regulatory elements on their target transcription unit lying nearby on the same chromosome. Transvection and transvection-related phenomena occur in wide range of species, including humans, but the most well-studied examples are found in Drosophila, where homologous chromosomes are paired in all somatic cells2,4-7. Many of our studies focus on the yellow locus, where we have enjoyed years of collaboration with the laboratory of Pamela Geyer (University of Iowa). Transvection at yellow can occur by the enhancers of one allele acting in trans on the promoter of a paired homologue (Fig. 1)23-25. We have also obtained data suggesting a surprising, second mechanism of yellow transvection, called insulator bypass, in which close pairing of an internally deleted yellow allele with an allele bearing an insulator is believed to cause changes in gene topology that are severe enough to allow an enhancer to bypass the insulator (Fig. 2)24,25. These forms of gene regulation alert us to the importance of considering trans influences when studying the regulation of gene expression in any organism, including humans.

How does transvection work? Of the two forms of yellow transvection, we have learned the most about enhancer action in trans. Here, we find that the choice of an enhancer to act in cis or trans is controlled by the cis-linked promoter23,25-27. Specifically, using genetic and molecular biological approaches, we have shown that yellow enhancers exhibit strong preference for the cis-linked promoter, choosing trans action only when the cis-linked promoter is compromised. We have further demonstrated that the feature of the cis-linked promoter that guides enhancer choice is independent of the identity of that promoter and are now determining the relative importance of transcriptional competence and promoter integrity27. (See research page of Anne Lee.) In addition to exploring cis-trans choice, we are also addressing the generality of transvection. Will trans action characterize all enhancers and promoters? (See research pages of Jack Bateman and Anne Lee.) A universal capacity of genetic elements for trans action could have considerable implications for gene expression in the densely packed nucleus.

How widespread is transvection? There is evidence for transvection and transvection-related pairing-mediated processes outside of Drosophila1,2,12,13,16-18,22,28-34. Concomitant with these data is the growing number of all homology effects, be they dependent on pairing or not. The implications are significant; a capacity to sense homology may be a common attribute of genes, and homologue pairing and pairing-mediated gene regulation may be more prevalent than once believed. Furthermore, if genetic elements, such as enhancers and promoters, are able to act in trans between paired homologous genes, might they also be mediating potent interactions between nonhomologous genes? For example, recent studies have revealed that such interactions may well be playing key roles in T-cell development35.

Homologue pairing is critical not only for pairing-mediated forms of gene regulation, but also for recombination, DNA repair, and gene replacement strategies. These mechanisms and technologies are important for both basic research as well as medical therapies. Therefore, much of our efforts are devoted to advancing the understanding of mechanisms that allow chromosomes and genes to find and then pair with their homologues. Our strategies involve FISH and RNAi analyses in cell culture as well as genetic, molecular biological, and cytological studies in the whole organism.

Studies of pairing in cell culture: We are well on our way to carrying out a genome-wide RNAi-based screen in cell culture36 for genes that are important for pairing. Applying 3D FISH analyses to several Drosophila cell lines, we have found that approximately 70% or more of cells give a single dot of signal for any of several unique sequence probes, indicating that these lines support a high degree of homologue pairing. Furthermore, inspection of mitotic spreads has yielded images of homologues intertwined long their lengths, confirming a report many years ago by Halfer and Barigozzi37. When optimal cell lines and protocols have been found, we will scale up our efforts and carry out a genome-wide screen for genes involved in pairing. (See research pages of Benjamin Williams and Jack Bateman.) Then, using the information from Drosophila cell culture, we will initiate studies to determine whether pairing can be induced in mammalian cells.

Figure 3. Pairing. in cell culture.An example of four loci pairing into 1 or 2 foci signal (yellow) in nuclei (blue)of cultured cells. Figure 4. Even during mitosis, homologous chromosomes appear to be intertwined.

Studies of pairing in the fly: We have found the overall level of pairing to be sufficient throughout the Drosophila genome to support yellow enhancer action in trans; pairs of allelically placed transgenes inserted randomly in the genome permit transvection38. Furthermore, our analysis of chromosome rearrangements indicates that productive pairing of yellow alleles does not appear to depend on discrete pairing elements. Rather, yellow pairing may require only ~700 kbp of uninterrupted flanking homology (See research page of Sharon Ou).

Does somatic pairing occur in humans? There is evidence for low, but significant, levels of somatic homologue pairing in humans. For example, cytological studies have revealed centromeric pairing of human chromosomes 1 and 17 in brain tissues 39,40 as well as homologous pairing of the pericentric regions in human lymphocytes41. Of particular interest are reports of chromosomal proximity in the region associated with the imprinted Prader-Willi and Angelman Syndromes, the latter of which may be, in some cases, phenotypically and genetically related to autism and Rett Syndrome18,22. Furthermore, altered levels of the methyl CpG binding protein 2 in human cells has recently been correlated with changes in the propensity of the Prader-Willi and Angelman Syndrome regions to become juxtaposed in the nucleus22. Interestingly, observations of imprinted loci have been also been reported in mice42. Additional arguments for the ability of human cells to support homologue pairing include the co-localization of plasmids bearing ß-globin sequences with the endogenous ß-globin region31, occurrence of mitotic recombination and its role in cancer43,44, and, most recently, somatic pairing of X chromosomes via their X inactivation centers68,69. In sum, somatic homologue pairing may well be occurring in human cells. Our goal is to determine the manner in which this pairing can be induced such that it can be harnessed for medical research and therapies.

Homologous recombination lies at the foundation of many protocols in both the basic as well as medical sciences, yet we are unable to harness it effectively for gene disruption and gene replacement except in a handful of experimental systems, the vast majority of these being fungal or prokaryotic. It is the cornerstone of genetic studies and gene therapies, but the low frequency with which it occurs is discouraging, and its cost and labor-intensive nature can be prohibitive, especially in mammalian and medical systems. In general, three strategies have been taken to improve the landscape: modification of the targeting vector, modification of the target itself, or use of powerful selective markers (for example, refs. 45, 46). Here, we focus on a fourth avenue for technology development — the enhancement of homologue pairing. To this end, we will use the outcome of our screen for the factors that drive homologue pairing in Drosophila (see previous section) to guide us in the design of protocols for promoting homologue pairing in mammalian (human) cells. Such protocols should contribute to the development of general sequence-nonspecific technologies for homologous recombination.

Our genetic studies led us to discover that some genes of the Polycomb group (PcG) can be mutated to modify a transvection-associated phenotype. This observation provided a striking genetic link between the PcG genes and pairing-related events47. PcG genes are now known to encode chromatin proteins and, excitingly, the targets of their action include pairing-sensitive genetic elements48-50. Currently, we are looking most closely at Psc and Su(z)2, two adjacent PcG genes51, the first of which is a key member of the Polycomb repressive complex 1 (PRC1) and the only one of this complex that can, on its own, significantly inhibit chromatin remodeling and transcription in vitro52,53. Through collaborative studies with the laboratory of Robert Kingston, we have found distinct functional domains within the Psc protein51,54 and are now expanding this study to the homologous Su(z)2 gene. (See research page of Richard Emmons.) We anticipate that an understanding of the functional and structural features of Psc and Su(z)2 will elucidate the molecular basis for how homologue pairing contributes to gene regulation.

Transgene studies are often plagued by position effects arising from the chromosomal regions flanking insertion sites. Not only do such position effects complicate the functional analysis of any single transgene, they preclude straightforward comparisons of transgenes which have inserted randomly into different genomic regions. The difficulty of inserting transgenes into specific chromosomal sites generates additional complexities for researchers studying transvection, where protocols often require interacting transgenes to be placed in allelic positions. To address these issues, we have made heavy use of targeted gene replacement26,27, as described by Keeler et al. 55; this technology minimizes concerns regarding position effects because it permits us to make changes to genes in their natural chromosomal position. In addition, we have developed a technique for the precise placement of transgenes into predetermined sites in the Drosophila genome.67 (See the RMCE User Website; Bateman, Lee, & Wu, 2006 (pdf). This method adapts the technology of recombinase-mediated cassette exchange (RMCE)56 to use the recombination machinery of the φC31 integrase57. Essentially, it allows researchers to exchange a target cassette located in the genome with a donor cassette carried on a plasmid by inducing φC31 integrase-mediated recombination on both sides of the aligned cassettes. Recently, two other variations on the RMCE technology, one using the cre recombinase58 and the other using the FLP recombinase59, have been described for Drosophila. The availability of RMCE with all three recombinase systems should facilitate genetic manipulations in Drosophila. (These studies to develop a new RMCE system in Drosophila are being carried out by Jack Bateman, Anne Lee, and Laura Stadelmann.)

Our broad interest in homology effects has led us to consider a number of phenomena in a diversity of species. We have been especially intrigued by X-inactivation8-13 and parental imprinting14-15,21 as well as homology-related mechanisms at the level of DNA, such as asymmetric DNA marking and strand segregation60-62. Considering these homology effects, we have reexamined current models for embryonic X-inactivation, which is widely believed to involve a random choice between the maternal and paternal X chromosomes. In particular, we have proposed two alternative models63. One suggests that choice is not random, while the other is consistent with random choice, but not one between two X chromosomes.

Two of the most surprising mysteries to emerge from the human genome project are ultraconserved elements (UCEs) and copy number variants (CNVs). UCEs are sequences that are ≥200 bp long and exhibit 100% identity across distantly related organisms and are believed to encode highly conserved functions involved in key aspects of gene regulation and expression64. CNVs, on the other hand, reveal the dynamic capacity of the genome to vary and include the vast number of deletions and duplications that are polymorphic among individuals65. We have discovered that UCEs are significantly depleted among copy number variants70.

Positions on chromosome 17 of three sets of UCEs, CNVs (including a set identified here as DEL), two sets of segmental duplications (SD), and genes (taken from ref. 70).

Our study began with the identification by Bejerano et al. (2004)64 of 481 UCEs conserved among humans, rats, and mice. These sequences proved to be distinct from each other and single copy in the human genome. Bejerano et al.64 also observed that the only two chromosomes from which they are absent in humans are the Y and chromosome 21. This finding was intriguing to us, as the Y is normally present in only one copy in males and trisomies of chromosome 21 constitute the most frequent viable whole autosomal aneuploidy (Down Syndrome). In fact, it suggested that UCEs and/or the regions containing them may be dosage-sensitive and that this sensitivity contributes to the integrity of the genome by ensuring the presence of UCE-containing regions in exactly two copies. Importantly, the broad distribution of UCEs implied a genome-wide process, while their ultraconservation and individual distinctiveness suggested a mechanism involving copy counting via the physical pairing of the maternal and paternal copies of each UCE.

In collaboration with Adnan Derti, Fritz Roth, and George Church, we have tested the potential of UCEs to participate as copy counters by determining whether three sets of UCEs, totaling 896 elements, are depleted among two sets of human segmental duplications (SDs) and seven sets of human CNVs. Remarkably, we observe a striking depletion of UCEs among both sets of SDs (P<10-8) and six of the seven sets of CNVs (P<10-4). This finding is consistent with our model and sets the stage for further study70.

Personal genetics education project (PGED)

Remarkable technological advances in the fields of genetics, epigenetics, and DNA sequencing are bringing researchers, physicians, and the public at large closer to understanding the full nuance of information encoded in our personal genomes. Consideration of personal genomes and their implications for personal genetics has also raised awareness of difficult ethical issues. These issues are ancient; personal genetics has been shaping history for thousands of years through societal pressures and, in some cases, tragically, in the name of eugenics. Balancing these complexities are the many benefits which can be derived from a greater understanding of personal genetics. Specifically, practical applications of personal genetics has the potential to improve health care while simultaneously reducing health care costs, bringing society closer to that time when access to medical coverage will no longer reflect economic, political, or cultural status. Our laboratory is working to expand consideration of the ethical, legal, and social issues (ELSI) of personal genetics by developing an educational and outreach program (See research page of Dana Waring and the Personal Genetics Education Project).

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