Yeast Artificial Chromosome Transgene Research Articles

Gata3 Hypomorphic Mutant Mice Rescued with a Yeast Artificial Chromosome Transgene Suffer a Glomerular Mesangial Cell Defect

GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generated Gata3 hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb Gata3 yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb Gata3 YAC transgene recapitulated Gata3 expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease.

The LRF/ZBTB7A Transcription Factor Is a BCL11A-Independent Repressor of Fetal Hemoglobin

Induction of fetal-type hemoglobin (HbF: α2γ2) is a promising means to treat hemoglobinopathies; however, precisely how HbF expression is silenced in adult erythroid cells is not fully understood. Such knowledge is essential to develop mechanism-based, targeted approaches to reactivate HbF production. Here, we show that Leukemia/lymphoma Related Factor (LRF), encoded by the ZBTB7A gene, is a novel and potent repressor of HbF production.To assess the effects of LRF loss on the mouse erythroid transcriptome, we performed RNA-Seq analysis using splenic erythroblasts from control and LRF conditional knockout (Zbtb7aF/F Mx1-Cre+) mice. LRF-deficient adult erythroblasts showed significant induction of Hbb-bh1, but not Hbb-y. The results were validated at the protein levels via isoelectric focusing of peripheral blood (PB) hemolysates and MALDI-TOFMS analysis. LRF loss also reactivated human fetal globin expression in vivo in LRF conditional KO mice harboring the human β-globin gene cluster as a yeast artificial chromosome transgene (β-YAC).To determine whether LRF loss could induce HbF in human erythroid cells, we employed human CD34+ hematopoietic stem and progenitor (HSPC)-derived primary erythroblasts and determined γ-globin expression levels upon shRNA-mediated LRF knockdown (KD). HbF levels in LRF KD cells (49-70%) were much greater than those seen in parental or scrambled-shRNA transduced cells. We next employed a novel human immortalized erythroid line (HUDEP-2). This line possesses an advantage over lines currently used for globin switching studies because it expresses predominantly adult hemoglobin (HbA: α2β2), with very low background HbF expression. Using CRISPR/cas9 gene modification, we knocked out ZBTB7A in HUDEP-2 cells and performed RNA-Seq analysis. As expected, γ-globin (HBG1 and HBG2) transcripts, but not those of embryonic ε-globin (HBE1), were markedly induced in ZBTB7A KO (ZBTB7AΔ/Δ) HUDEP-2 cells. ZBTB7AΔ/Δ cells exhibited HbF levels greater than 60%, while that of parental cells was less than 3%. Notably, the HbF reactivation occurred without changes in levels of transcripts encoding known HbF repressors, including BCL11A, the principal known switching factor.We next performed chromatin-immunoprecipitation and sequencing (ChIP-Seq) with an anti-LRF antibody using HSPC-derived proerythroblasts and HUDEP-2 cells. The most enriched motif identified in either was concordant with that previously identified in vitro using CAST analysis (Maeda et. al. Nature 2005), confirming antibody specificity. Supporting a direct role of LRF at the β-globin cluster, we observed several significant enrichment of LRF-ChIP binding signals at adult (HBB), fetal (HBG1) globin loci and the upstream hypersensitivity (HS) sites within the locus control region (LCR). ATAC-Seq (for assay for transposase-accessible chromatin with high-throughput sequencing) analysis revealed strong chromatin accessibility at the γ-globin locus in ZBTB7AΔ/Δ cells. Strikingly, differential enrichment of ATAC-signals in ZBTB7AΔ/Δ cells was evident only at the γ-locus. Thus, while LRF binds to the HBB locus and HS sites as well as to the HBG1 locus, LRF depletion specifically opens chromatin at the γ-globin locus.Finally, to determine whether LRF and BCL11A suppress γ-globin expression via distinct mechanisms, we established LRF/BCL11A double knockout HUDEP-2 cells. Strikingly, HUDEP-2 lines lacking both LRF and BCL11A exhibited almost a complete switch in expression from adult- to fetal-type globin, suggesting that these two factors cumulatively represent the near entirety of γ-globin repressive activity in adult erythroid cells. Our findings reveal a novel molecular mechanism regulating γ-globin silencing and may open a new window for therapeutic targeting in the treatment of hemoglobinopathies. DisclosuresBauer:Biogen: Research Funding; Editas Medicine: Consultancy. Orkin:Editas Inc.: Consultancy.

Genome-wide loss of 5-hmC is a novel epigenetic feature of Huntington's disease

5-Hydroxymethylcytosine (5-hmC) may represent a new epigenetic modification of cytosine. While the dynamics of 5-hmC during neurodevelopment have recently been reported, little is known about its genomic distribution and function(s) in neurodegenerative diseases such as Huntington's disease (HD). We here observed a marked reduction of the 5-hmC signal in YAC128 (yeast artificial chromosome transgene with 128 CAG repeats) HD mouse brain tissues when compared with age-matched wild-type (WT) mice, suggesting a deficiency of 5-hmC reconstruction in HD brains during postnatal development. Genome-wide distribution analysis of 5-hmC further confirmed the diminishment of the 5-hmC signal in striatum and cortex in YAC128 HD mice. General genomic features of 5-hmC are highly conserved, not being affected by either disease or brain regions. Intriguingly, we have identified disease-specific (YAC128 versus WT) differentially hydroxymethylated regions (DhMRs), and found that acquisition of DhmRs in gene body is a positive epigenetic regulator for gene expression. Ingenuity pathway analysis (IPA) of genotype-specific DhMR-annotated genes revealed that alternation of a number of canonical pathways involving neuronal development/differentiation (Wnt/β-catenin/Sox pathway, axonal guidance signaling pathway) and neuronal function/survival (glutamate receptor/calcium/CREB, GABA receptor signaling, dopamine-DARPP32 feedback pathway, etc.) could be important for the onset of HD. Our results indicate that loss of the 5-hmC marker is a novel epigenetic feature in HD, and that this aberrant epigenetic regulation may impair the neurogenesis, neuronal function and survival in HD brain. Our study also opens a new avenue for HD treatment; re-establishing the native 5-hmC landscape may have the potential to slow/halt the progression of HD.

A Wt1-Dmrt1 Transgene Restores DMRT1 to Sertoli Cells of Dmrt1−/− Testes: A Novel Model of DMRT1-Deficient Germ Cells1

DMRT1 is an evolutionarily conserved transcriptional factor expressed only in the postnatal testis, where it is produced in Sertoli cells and germ cells. While deletion of Dmrt1 in mice demonstrated it is required for postnatal testis development and fertility, much is still unknown about its temporal- and cell-specific functions. This study characterized a novel mouse model of DMRT1-deficient germ cells that was generated by breeding Dmrt1-null (Dmrt1(-/-)) mice with Wt1-Dmrt1 transgenic (Dmrt1(+/-;tg)) mice, which express a rat Dmrt1 cDNA in gonadal supporting cells by directing it from the Wilms tumor 1 locus in a yeast artificial chromosome transgene. Like Dmrt1(-/-) mice, male Dmrt1(-/-) transgenic mice (Dmrt1(-/-;tg)) were infertile, while female mice were fertile. Immunohistochemistry and Western blot analysis showed transgenic DMRT1 expressed in supporting cells of the newborn gonads of both sex and in Sertoli cells of the testis afterbirth. Sertoli cells were evaluated by electron microscopy, revealing that maturation of Dmrt1(-/-;tg) Sertoli cells was incomplete. Morphological analysis of testes from 42-day-old mice showed that, compared to Dmrt1(-/-) mice, Dmrt1(-/-;tg) mice have improved seminiferous tubule structure, with lumens present in many. Immunohistochemistry of the polarity markers ESPIN and NECTIN-2 showed that DMRT1 in Sertoli cells is required for NECTIN-2 expression and influences organization of ectoplasmic specializations. Further functional analyses of the transgene on a Dmrt1(-/-) background showed that it did not rescue the decrease in Dmrt1(-/-) testis size, but when expressed on a wild-type background, exogenous DMRT1 prevented the normal age-related decline in testis size and enhanced sperm progressive motility. The studies suggest that DMRT1 in Sertoli cells regulates tubule morphology, spermatogenesis, and sperm function via its effects on Sertoli cell maturation and polarity. Furthermore, expression and function of transgenic DMRT1 in Sertoli cells establishes a novel mouse model of DMRT1-deficient germ cells generated by breeding Dmrt1-null mice with Wt1-Dmrt1 transgenic mice (rescue; Dmrt1(-/-;tg)).

Control of Hemoglobin Switching by BCL11A.

Abstract 5All vertebrates switch expression of globin chains during development. In humans b-like globins switch from embryonic to fetal to adult, whereas in the mouse a single switch from embryonic to adult occurs. The switch from human fetal (g) to adult (b) expression is especially critical in the b-hemoglobin disorders, such as sickle cell anemia and the b-thalassemias. Delay of the switch or reactivation of the fetal gene in the adult stage greatly ameliorates clinical severity. Despite intensive molecular studies of the human b-globin cluster over more than two decades, the proteins regulating the switch, and the mechanisms controlling the process, have been largely elusive. Recently, genome-wide association studies identified genetic variation at a chromosome 2 locus that correlates with the level of HbF in different populations. The most highly associated single nucleotide polymorphisms (SNPs) reside in an intron of the BCL11A gene, which encodes a zinc-finger repressor protein. Previously we showed that shRNA-mediated ex vivo knockdown of BCL11A in cultured human CD34-derived erythroid precursors leads to robust HbF expression, consistent with a role for BCL11A in maintaining g-genes in a silenced state in adult cells. To address in vivo roles of BCL11A either in development or in globin gene silencing in an intact individual, we have employed stringent genetic tests of function in mice that carry a complete human b-globin gene cluster as a yeast artificial chromosome transgene (b-locus mice). Knockout of BCL11A in mice leads to failure to silence the endogenous b-like embryonic genes in adult erythroid cells of the fetal liver (>2500-fold derepression). The ratio of human g to b globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that g constitutes >90% of the b-like human expression at embryonic day (E)14.5 and >75% at E18.5. These quantitatively striking findings indicate that BCL11A controls developmental silencing of g-globin gene expression. To address by formal genetics the contribution of BCL11A to g silencing in adult animals we have employed conditional inactivation of BCL11A through hematopoietic- and erythroid-specific Cre-alleles. These experiments reveal that BCL11A is also required in vivo for g-gene silencing in adults. We observed that human g-globin expression is persistently derepressed >2000-fold (as compared to littermate controls) in bone marrow erythroblasts of 15-20 week old b-locus mice upon erythroid-specific deletion of BCL11A. Taken together, these findings establish BCL11A as the first genetically validated transcriptional regulator of both developmental control of globin switching and silencing of g-globin expression in adults. The recognition of these roles for BCL11A now permits focused mechanistic studies of the switch. In human erythroid cells, BCL11A physically interacts with at least two corepressor complexes, Mi-2/NuRD and LSD1/CoREST, as well as the erythroid transcription factor GATA-1 and the HMG-box protein SOX6. Rather than binding to the promoters of the g- or b-globin genes as do these latter factors, BCL11A protein occupies the upstream locus control and g-d-intergenic regions of the b-globin cluster (as determined by high resolution ChIP-Chip analysis), suggesting that BCL11A mediates long-range interactions and/or reconfigures the locus during different stages. An in-depth mechanistic understanding of globin switching offers the prospect for design of target-based activation of HbF in adult erythroid cells of patients with hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

X-Inactivation: Close Encounters of the X Kind

X chromosome inactivation ensures equal dosage of X-linked genes between male and female mammals. Two new studies have shown that the initiation of inactivation is preceded by X chromosome pairing; their results implicate this pairing in the choice and counting functions of X chromosome inactivation.

Wild-Type Huntingtin Reduces the Cellular Toxicity of Mutant Huntingtin In Vivo

We have developed yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 or YAC72) human huntingtin (htt), in a developmental- and tissue-specific manner, that is identical to endogenous htt. YAC72 mice develop selective degeneration of medium spiny projection neurons in the lateral striatum, similar to what is observed in Huntington disease. Mutant human htt expressed by YAC transgenes can compensate for the absence of endogenous htt and can rescue the embryonic lethality that characterizes mice homozygous for targeted disruption of the endogenous Hdh gene (-/-). YAC72 mice lacking endogenous htt (YAC72 -/-) manifest a novel phenotype characterized by infertility, testicular atrophy, aspermia, and massive apoptotic cell death in the testes. The testicular cell death in YAC72 -/- mice can be markedly reduced by increasing endogenous htt levels. YAC72 mice with equivalent levels of both wild-type and mutant htt (YAC72 +/+) breed normally and have no evidence of increased testicular cell death. Similar findings are seen in YAC46 -/- mice compared with YAC46 +/+ mice, in which wild-type htt can completely counteract the proapoptotic effects of mutant htt. YAC18 -/- mice display no evidence of increased cellular apoptosis, even in the complete absence of endogenous htt, demonstrating that the massive cellular apoptosis observed in YAC46 -/- mice and YAC72 -/- mice is polyglutamine-mediated toxicity from the mutant transgene. These data provide the first direct in vivo evidence of a role for wild-type htt in decreasing the cellular toxicity of mutant htt.

A human YAC transgene rescues craniofacial and neural tube development in PDGFRalpha knockout mice and uncovers a role for PDGFRalpha in prenatal lung growth.

The platelet-derived growth factor alpha-receptor (PDGFRalpha) plays a vital role in the development of vertebrate embryos, since mice lacking PDGFRalpha die in mid-gestation. PDGFRalpha is expressed in several types of migratory progenitor cells in the embryo including cranial neural crest cells, lung smooth muscle progenitors and oligodendrocyte progenitors. To study PDGFRalpha gene regulation and function during development, we generated transgenic mice by pronuclear injection of a 380 kb yeast artificial chromosome (YAC) containing the human PDGFRalpha gene. The YAC transgene was expressed in neural crest cells, rescued the profound craniofacial abnormalities and spina bifida observed in PDGFRalpha knockout mice and prolonged survival until birth. The ultimate cause of death was respiratory failure due to a defect in lung growth, stemming from failure of the transgene to be expressed correctly in lung smooth muscle progenitors. However, the YAC transgene was expressed faithfully in oligodendrocyte progenitors, which was not previously observed with plasmid-based transgenes containing only upstream PDGFRalpha control sequences. Our data illustrate the complexity of PDGFRalpha genetic control, provide clues to the location of critical regulatory elements and reveal a requirement for PDGF signalling in prenatal lung growth, which is distinct from the known requirement in postnatal alveogenesis. In addition, we found that the YAC transgene did not prolong survival of Patch mutant mice, indicating that genetic defects outside the PDGFRalpha locus contribute to the early embryonic lethality of Patch mice.

(Over)correction of FMR1 deficiency with YAC transgenics: behavioral and physical features.

Fragile X syndrome is a common cause of mental retardation involving loss of expression of the FMR1 gene. The role of FMR1 remains undetermined but the protein appears to be involved in RNA metabolism. Fmr1 knockout mice exhibit a phenotype with some similarities to humans, such as macroorchidism and behavioral abnormalities. As a step toward understanding the function of FMR1 and the determination of the potential for therapeutic approaches to fragile X syndrome, yeast artificial chromosome (YAC) transgenic mice were generated in order to determine whether the Fmr1 knockout mouse phenotype could be rescued. Several transgenic lines were generated that carried the entire FMR1 locus with extensive amounts of flanking sequence. We observed that the YAC transgene supported production of the human protein (FMRP) which was present at levels 10 to 15 times that of endogenous protein and was expressed in a cell- and tissue-specific manner. Macro-orchidism was absent in knockout mice carrying the YAC transgene indicating functional rescue by the human protein. Given the complex behavioral phenotype in fragile X patients and the mild phenotype previously reported for the Fmr1 knockout mouse, we performed a more thorough evaluation of the Fmr1 knockout phenotype using additional behavioral assays that had not previously been reported for this animal model. The mouse displayed reduced anxiety-related responses with increased exploratory behavior. FMR1 YAC transgenic mice overexpressing the human protein did produce opposing behavioral responses and additional abnormal behaviors were also observed. These findings have significant implications for gene therapy for fragile X syndrome since overexpression of the gene may harbor its own phenotype.

Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice.

Expression of a construct integrated at different genomic locations often varies because of position effects that have been subcategorized as stable (decreased level of expression) and variegating (decreased proportion of expressing cells). It is well established that locus control regions (LCRs) generally overcome position effects in transgenes. However, whether stable and variegated position effects are equally overcome by an intact LCR has not been determined. We report that single-copy yeast artificial chromosome transgenes containing an unmodified human beta -globin locus were not subject to detectable stable position effects but did undergo mild to severe variegating position effects at three of the four non-centromeric integration sites tested. We also find that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhancement. DNase I hypersensitive site (HSS) formation varies with the proportion of expressing cells, not the level of gene expression, suggesting that silencing of the transgene is associated with a lack of HSS formation in the LCR region. We conclude that transcriptional enhancement and variegating position effects are caused by fundamentally different but inter-dependent mechanisms.

Analysis of a DNase I hypersensitive site located -20.9 kb upstream of the CFTR gene.

The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a tightly regulated pattern of expression with spatial and temporal control. The regulatory elements achieving this appear to lie outside the basal promoter of the gene. We previously identified DNase I hypersensitive sites (DHSs) at -79.5 kb and -20.5 kb with respect to the CFTR translational start site which may contain important regulatory elements. We have now investigated further the DHS at -20.5 kb to evaluate its potential function in the regulation of CFTR expression. Finer mapping revealed that the DHS lies at -20.9 kb. Deletion of the DHS from a 310-kb yeast artificial chromosome (YAC) containing the human CFTR gene has shown that this site may be responsible for about 60% of wild-type levels of transcription from the YAC transgene when expressed in Caco2 cells. DNase I footprinting showed several regions of protection within the -20.9 kb region with nuclear extracts from Caco2 cells, but not with extracts from lymphoblastoid cells, which do not show the DHS. Matches to several transcription factor-binding sites were found, but supershift analysis with specific antibodies did not identify the transcription factors involved. Two purine/pyrimidine mirror repeat elements within the -20.9-kb DHS were shown not to adopt non-B-DNA conformations. Thus, we provide evidence for a role for the -20.9 kb DHS in the transcriptional regulation of the CFTR gene, although the mechanisms mediating this effect remain unclear.

Functional analysis of the DXPas34 locus, a 3' regulator of Xist expression.

X inactivation in female mammals is controlled by a key locus on the X chromosome, the X-inactivation center (Xic). The Xic controls the initiation and propagation of inactivation in cis. It also ensures that the correct number of X chromosomes undergo inactivation (counting) and determines which X chromosome becomes inactivated (choice). The Xist gene maps to the Xic region and is essential for the initiation of X inactivation in cis. Regulatory elements of X inactivation have been proposed to lie 3' to Xist. One such element, lying 15 kb downstream of Xist, is the DXPas34 locus, which was first identified as a result of its hypermethylation on the active X chromosome and the correlation of its methylation level with allelism at the X-controlling element (Xce), a locus known to affect choice. In this study, we have tested the potential function of the DXPas34 locus in Xist regulation and X-inactivation initiation by deleting it in the context of large Xist-containing yeast artificial chromosome transgenes. Deletion of DXPas34 eliminates both Xist expression and antisense transcription present in this region in undifferentiated ES cells. It also leads to nonrandom inactivation of the deleted transgene upon differentiation. DXPas34 thus appears to be a critical regulator of Xist activity and X inactivation. The expression pattern of DXPas34 during early embryonic development, which we report here, further suggests that it could be implicated in the regulation of imprinted Xist expression.

Xist yeast artificial chromosome transgenes function as X-inactivation centers only in multicopy arrays and not as single copies.

X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation in cis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The Xist gene maps to the Xic region and has been shown to be essential for inactivation in cis. Transgenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid Xist-containing transgenes have previously been reported to be capable of cis inactivation and counting, the transgenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transgenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of Xist, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.

Localization of distant urogenital system-, central nervous system-, and endocardium-specific transcriptional regulatory elements in the GATA-3 locus.

We found previously that neither a 6-kbp promoter fragment nor even a 120-kbp yeast artificial chromosome (YAC) containing the whole GATA-3 gene was sufficient to recapitulate its full transcription pattern during embryonic development in transgenic mice. In an attempt to further identify tissue-specific regulatory elements modulating the dynamic embryonic pattern of the GATA-3 gene, we have examined the expression of two much larger (540- and 625-kbp) GATA-3 YACs in transgenic animals. A lacZ reporter gene was first inserted into both large GATA-3 YACs. The transgenic YAC patterns were then compared to those of embryos bearing the identical lacZ insertion in the chromosomal GATA-3 locus (creating GATA-3/lacZ "knock-ins"). We found that most of the YAC expression sites and tissues are directly reflective of the endogenous pattern, and detailed examination of the integrated YAC transgenes allowed the general localization of a number of very distant transcriptional regulatory elements (putative central nervous system-, endocardium-, and urogenital system-specific enhancers). Remarkably, even the 625-kbp GATA-3 YAC, containing approximately 450 kbp and 150 kbp of 5' and 3' flanking sequences, respectively, does not contain the full transcriptional regulatory potential of the endogenous locus and is clearly missing regulatory elements that confer tissue-specific expression to GATA-3 in a subset of neural crest-derived cell lineages.

LCR-dependent gene expression in beta-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs.

Yeast artificial chromosome (YAC) transgenesis is associated with a high frequency of deletions in the integrated transgenes. To determine the impact of these rearrangements on the ability to derive structure-function relationships using YACs, transgenic mice were generated with 248 or 155 kb beta-globin locus YACs. The transgenics were examined for structural integrity of the YAC using an approach of structural analysis that unambiguously demonstrates intactness of YAC transgene copies. Globin gene expression per copy of each integrated transgene and the profiles of globin gene expression during development were determined. Diverse deletion patterns were observed in one or more integrated YACs in all the 248 and most of the 155 kb transgenic lines we analyzed. However, when the structure of the major regulatory element of the beta-globin locus, the locus control region, was preserved, the genes of the beta-globin locus functioned normally and globin transgenes of both the 248 and 155 kb beta-YACs were expressed in a position-independent, copy number-dependent manner. Furthermore, the globin genes of both beta-YACs displayed normal developmental regulation. We conclude that YACs can be used for analysis of structure-function relationships of large genes or multigene loci in spite of the tendency for rearrangements and deletions of the integrated transgenes. However, detailed structural evidence for integrity and continuity of locus sequences is required for correct interpretation of functional data.

Partial Rescue of GATA-3 by Yeast Artificial Chromosome Transgenes

GATA-3 is essential for murine embryonic development, but elucidating the genetic controls over the complex temporal and tissue-specific transcriptional regulatory pattern of this transcription factor gene has been problematic. Here we report the isolation and characterization of two yeast artificial chromosomes (YACs) bearing the murine GATA-3 gene. Ordered deletions of both YACs show that they define a 1-megabase pair contig spanning the GATA-3 locus. We found that a 120-kb YAC transgene, including 35 kb of 5′ as well as 60 kb of 3′ flanking sequence, confers normal GATA-3 expression at sites not revealed previously through analysis of plasmid transgenic lines. However, even this 120-kb YAC does not contain sufficient information to recapitulate the complete GATA-3 expression program during embryogenesis. While not complete in its regulatory capacity, the YAC transgene is nonetheless able to complement several homozygous GATA-3 mutant phenotypes and thereby prolong embryonic life.

Rescue of the embryonic lethal hematopoietic defect reveals a critical role for GATA-2 in urogenital development.

Mutations resulting in embryonic or early postnatal lethality could mask the activities of any gene in unrelated and temporally distinct developmental pathways. Targeted inactivation of the transcription factor GATA-2 gene leads to mid-gestational death as a consequence of hematopoietic failure. We show here that a 250 kbp GATA-2 yeast artificial chromosome (YAC) is expressed strongly in both the primitive and definitive hematopoietic compartments, while two smaller YACs are not. This largest YAC also rescues hematopoiesis in vitro and in vivo, thereby localizing the hematopoietic regulatory cis element(s) to between 100 and 150 kbp 5' to the GATA-2 structural gene. Introducing the YAC transgene into the GATA-2(-/-) genetic background allows the embryos to complete gestation; however, newborn rescued pups quickly succumb to lethal hydroureternephrosis, and display a complex array of genitourinary abnormalities. These findings reveal that GATA-2 plays equally vital roles in urogenital and hematopoietic development.

Imprinting of the mouse Igf2r gene depends on an intronic CpG island

Gametic imprinting is a developmental process that uses cis-acting epigenetic mechanisms to induce parental-specific expression in autosomal and X-linked genes. From the limited knowledge we have of imprinted genes it seems clear that they play a role in regulating the growth and development of the placenta and embryo in utero, and also in the early post-natal phase. The biological function of imprinting in mammals is not yet understood but it may act to regulate the supply of nutrition to the embryo in order to maintain a balance between fetal demands and maternal resources. The molecular basis of imprinting any gene has not yet been disclosed. However, the current model proposes that single or clustered genes should be associated with a specific imprinting element which carries a reversible epigenetic imprinting signal, inherited from one parent and maintained on the same parental chromosome in diploid cells. A common imprinting recognition sequence has not been identified amongst the 20 known imprinted genes, but such an element is thought to exist from observations of mouse transgenes and transgenes of the imprinted H19 gene, which have shown that short DNA sequences can maintain imprinted expression at different chromosome locations. However, it is also clear from gene deletion experiments in the mouse and the analysis of deletions in the human Prader-Willi/Angelman syndromes that imprinted genes are also influenced by distant DNA sequences. We have earlier proposed that CG rich sequences resembling CpG islands, which are associated with many imprinted genes and often subject to parental-specific methylation, could act as a common imprinting element. The mouse insulin-like growth factor type 2 receptor ( Igf2r) gene contains a CpG island known as region2, in the second intron. Region2 was proposed as the imprinting element of this gene because it inherited a methylation imprint from the female gamete that was maintained only on the maternal chromosome in diploid cells. We have used YAC (yeast artificial chromosome) transgenes carrying the complete Igf2r locus, to test if imprinting and parental-specific methylation of the mouse Igf2r gene is maintained when transferred to other chromosomal locations and to test whether imprinting is dependent on the intronic CpG island proposed as the imprinting element for this gene. Gametic imprints are epigenetic modifications which are imposed onto the gametic chromosomes and cause parental-specific differences in the expression of a small number of genes in the embryo. As a consequence, correct imposition of the imprints in the parental germlines is a prerequisite for successful development of mammals and any anomaly in the expression of imprinted genes is often accompanied by aberration of embryonic growth. The link between imprinting and growth regulation is best exemplified by the Igf2 and Igf2r genes. Both genes show parental-specific expression patterns in the embryo. Igf2 is a general embryonic mitogen, and mice lacking Igf2 are markedly reduced in size (DeChiara, T.M., Robertson, E.J., Efstratiadis, A., 1991. Parental imprinting of the mouse insulin-like growth factor II gene. Cell 64, 849–859). Igf2r acts to fine tune the amount of growth factor, and embryos lacking this gene show overgrowth and die perinatally (Lau, M.M., Stewart, C.E., Liu, Z., Bhatt, H., Rotwein, P., Stewart, C.L., 1994. Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality. Genes Dev. 8, 2953–2963; Wang, Z.Q., Fung, M.R., Barlow, D.P., Wagner, E.F., 1994. Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene. Nature 372, 464–467). The phenomenon of gametic imprinting is predicted to arise by an unusual regulation of the imposition and erasure of epigenetic modifications. There are three events which are required for imprinting: firstly, the imprint must be imposed in one of the gametes before fertilisation. Secondly, the difference between the parental chromosomes must be maintained in early development despite the fact that the embryonic genome becomes epigenetically reprogrammed. Thirdly, the imprint must be erased in the germline before a new one is established (failure to erase the imprint would lead to a stably inherited epigenetic state over the generations). The focus of this review is the imprinted mouse Igf2r gene. The present understanding of the mechnism imprinting this locus is summarized, compared with other imprinted genes, and a model to explain the data is discussed.

Imprinted expression of the Igf2r gene depends on an intronic CpG island.

Gametic imprinting is a developmental process that induces parental-specific expression or repression of autosomal and X-chromosome-linked genes. The mouse Igf2r gene (encoding the receptor for insulin- like growth factor type-2) is imprinted and is expressed from the maternal allele after embryonic implantation. We previously proposed that methylation of region 2, a region rich in cytosine-guanine doublets (a 'CpG island') in the second intron of Igf2r, is the imprinting signal that maintains expression of the maternal allele. Here we use mouse transgenes to test the role of region 2 and the influence of chromosome location on Igf2r imprinting. Yeast artificial chromosome transgenes successfully reproduced the imprinted methylation and expression pattern of the endogenous Igf2r gene; deletion of region 2 from these transgenes caused a loss of imprinting and restored biallelic Igf2r expression. These results define a primary role for region 2 and a negligible role for chromosomal location in Igf2r imprinting; they also show that methylation imprints can maintain allelic expression. Short transgenes containing only region 2 and yeast artificial chromosome transgenes with an inactive Igf2r promoter do not attract parental-specific methylation. All transgenes showing paternal-specific repression of Igf2r produced an antisense RNA whose transcription was dependent on region 2. The production of an antisense RNA by the repressed parental allele is reminiscent of the imprinting of the Igf2/H19 gene pair and may indicate that expression competition could play a general role in imprinting.

The X-linked immunodeficiency defect in the mouse is corrected by expression of human Bruton's tyrosine kinase from a yeast artificial chromosome transgene.

Mutations in the gene for Bruton's tyrosine kinase result in the B cell differentiation defects X-linked agammaglobulinemia in man and X-linked immunodeficiency in mice. Here we describe the generation of two yeast artificial chromosome (YAC)-transgenic mouse strains in which high-level expression of human Btk is provided by endogenous regulatory cis-acting elements that are present on a 340-kb transgene, Yc340-hBtk. The expression pattern of the transgenic human Btk was found to parallel that of the endogenous murine gene. When the Yc340-hBtk-transgenic mice were mated onto a Btk-deficient background, the xid B cell defects were fully corrected: conventional and CD5+ B-1 B cells were present in normal numbers, serum IgM and IgG3 levels as well as responses to T cell-independent type II antigens were in the normal ranges. In vivo competition experiments in Btk+/- female mice demonstrated that in the conventional B cell population the Yc340-hBtk transgene could fully compensate the absence of expression of endogenous murine Btk. We conclude that in the YAC-transgenic mice Btk is appropriately expressed in the context of native regulatory sequences.