NRF2 mediates γ-globin gene regulation through epigenetic modifications in a ß-YAC transgenic mouse model.

IMPACT STATEMENT: Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human ß-like globin gene locus of ß-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform.

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.

Genetic Assays to Study Repeat Fragility in Saccharomyces cerevisiae.

Trinucleotide repeats are common in the human genome and can undergo changes in repeat number and cause length-dependent chromosome fragility. Expanded CAG repeats have been linked to over 14 human diseases and are considered hotspots for breakage and genomic rearrangement. Here we describe two Saccharomyces cerevisiae based assays that evaluate the rate of chromosome breakage that occurs within a repeat tract (fragility), with variations that allow the role of transcription to be evaluated. The first fragility assay utilizes end-loss and subsequent telomere addition as the main mode of repair of a yeast artificial chromosome (YAC). The second fragility assay relies on the fact that a chromosomal break stimulates recombination-mediated repair. A PCR-based assay can be used to evaluate instability of the repeat in the same conditions used to measure repeat fragility. These assays have contributed to understanding the genetic mechanisms that cause chromosome breaks and tract-length changes at unstable trinucleotide repeats.

Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering in Saccharomyces cerevisiae.

Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected-a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.

From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.

Triplex-forming peptide nucleic acids induce heritable elevations in gamma-globin expression in hematopoietic progenitor cells.

Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the ß-globin subunit cause hemoglobinopathies such as sickle cell disease and ß-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including ß-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes.

Mi2ß is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in ß-YAC transgenic mice.

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (A)γ-globin gene. We show that Mi2ß is essential for γ-globin gene silencing using Mi2ß conditional knockout ß-YAC transgenic mice. In addition, increased expression of (A)γ-globin was detected in adult blood from ß-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type ß-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2ß knockout deletion mutation or the cis-acting -566 (A)γ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

Recombinant protein expression in milk of livestock species.

Producing complex recombinant proteins in the milk of transgenic animals offers several advantages: large amounts of proteins can be obtained, and in most cases, these proteins are properly folded, assembled, cleaved, and glycosylated. The level of expression of foreign genes in the mammalian gland cannot be predicted in all cases, and appropriate vectors must be used. The main elements of these vectors are as follows: a well-characterized specific promoter, the coding region of the gene of interest, preferably with a homologous or heterologous intron, to improve transcription efficiency, and an insulator or boundary element to counteract the chromosomal position effects at the integration site. Once high expression levels are achieved, and the recombinant protein is purified, an essential step in the analysis of the final product is determining its degree of glycosylation. This is an important readout because it can affect among other parameters the stability and immunogenicity of the recombinant protein.

RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability.

Genome instability, a hallmark of cancer progression, is thought to arise through DNA double strand breaks (DSBs). Studies in yeast and mammalian cells have shown that DSBs and instability can occur through RNA:DNA hybrids generated by defects in RNA elongation and splicing. We report that in yeast hybrids naturally form at many loci in wild-type cells, likely due to transcriptional errors, but are removed by two evolutionarily conserved RNase H enzymes. Mutants defective in transcriptional repression, RNA export and RNA degradation show increased hybrid formation and associated genome instability. One mutant, sin3Δ, changes the genome profile of hybrids, enhancing formation at ribosomal DNA. Hybrids likely induce damage in G1, S and G2/M as assayed by Rad52 foci. In summary, RNA:DNA hybrids are a potent source for changing genome structure. By preventing their formation and accumulation, multiple RNA biogenesis factors and RNase H act as guardians of the genome.

Interaction of postsynaptic density protein-95 with NMDA receptors influences excitotoxicity in the yeast artificial chromosome mouse model of Huntington's disease.

Evidence suggests that NMDA-type glutamate receptors contribute to degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). Previously, we demonstrated that NMDA receptor (NMDAR)-mediated current and/or toxicity is increased in MSNs from the yeast artificial chromosome (YAC) transgenic mouse model expressing polyglutamine (polyQ)-expanded (mutant) full-length human huntingtin (htt). Others have shown that membrane-associated guanylate kinases (MAGUKs), such as PSD-95 and SAP102, modulate NMDAR surface expression and excitotoxicity in hippocampal and cortical neurons and that htt interacts with PSD-95. Here, we tested the hypothesis that an altered association between MAGUKs and NMDARs in mutant huntingtin-expressing cells contributes to increased susceptibility to excitotoxicity. We show that htt coimmunoprecipitated with SAP102 in HEK293T cells and striatal tissue from wild-type and YAC transgenic mice; however, the association of SAP102 with htt or the NMDAR NR2B subunit was unaffected by htt polyQ length, whereas association of PSD-95 with NR2B in striatal tissue was enhanced by increased htt polyQ length. Treatment of cultured MSNs with Tat-NR2B9c peptide blocked binding of NR2B with SAP102 and PSD-95 and reduced NMDAR surface expression by 20% in both YAC transgenic and wild-type MSNs, and also restored susceptibility to NMDAR excitoxicity in YAC HD MSNs to levels observed in wild-type MSNs; a similar effect on excitotoxicity was observed after knockdown of PSD-95 by small interfering RNA. Unlike previous findings in cortical and hippocampal neurons, rescue of NMDA toxicity by Tat-NR2B9c occurred independently of any effect on neuronal nitric oxide synthase activity. Our results elucidate further the mechanisms underlying enhanced excitotoxicity in HD.

Transgenic rescue of SF-1-null mice.

Steroidogenic factor 1 (SF-1, Nr5a1, and Ad4bp) is an orphan nuclear receptor required for adrenal and gonad development and endocrine regulation. To extend our understanding of SF-1 function and the mechanisms controlling its expression, a transgenic rescue strategy was employed to locate important transcriptional control regions and to reveal functional roles of the protein. A rat yeast artificial chromosome containing Ftz-F1, the gene encoding SF-1, was used to generate mice with different transgenes that varied in size. Rat SF-1 mRNA expression was assayed to assess each transgene's targeting ability. SF-1-deficient/transgene-positive (SF-1(-/-); tg/+) "rescue" mice were then generated and the animals' developmental and reproductive status was evaluated. The results identified differences in expression patterns and rescue abilities that provided insight into SF-1 transcriptional control and function. Comparing transgene maps and mRNA profiles placed critical transcriptional elements for pituitary and hypothalamic expression to a region 3' to intron 4, whereas examination of rescued mice revealed that an approximately 153-kb region of the Ftz-F1 locus recapitulates most or all activity ascribed to the endogenous allele. A second line of rescued mice was hypomorphic, with males showing defects in androgen-dependent tissues due to abnormal Leydig cell differentiation. Histological analysis of embryonic (e14.5) and adult testes from these mice implicated SF-1 in roles that are distinct in fetal and adult Leydig cells.

The role of p22 NF-E4 in human globin gene switching.

The human stage selector protein, a complex containing the ubiquitous transcription factor CP2 and the erythroid-specific factor p22 NF-E4, facilitates the interaction of the gamma-globin genes with the locus control region in fetal erythroid cells. Enforced expression of p22 NF-E4 in K562 cells and human cord blood progenitors increases fetal globin gene expression, and in progenitors, reduces beta-globin expression. To examine the role of NF-E4 in an in vivo model of hemoglobin switching, we enforced the expression of p22 NF-E4 in transgenic mice carrying the human beta-globin locus yeast artificial chromosome. Although murine erythropoiesis and globin gene expression is unaffected in these mice, the expression profile of the human globin genes is altered. All three transgenic lines displayed an increased gamma:beta-globin ratio in E12.5-14.5 fetal liver, resulting in a delay in the fetal/adult switch. At E12.5, this is primarily due to a reduction of beta-gene expression, whereas at E14.5, the increased gamma:beta ratio is due to enhanced gamma-gene expression. Despite this, the switch in globin subtype is fully completed in the adult bone marrow. These findings indicate that p22 NF-E4 is capable of influencing human globin gene expression in vivo but is incapable of overriding the intrinsic mechanisms governing gamma-gene silencing in this context.

A novel mechanism of intragenic complementation between Phe to Ala calmodulin mutations.

Calmodulin (CaM) performs essential functions in cell proliferation in Saccharomyces cerevisiae. Previously, we isolated fourteen temperature-sensitive Phe-to-Ala mutations of the CaM-encoding gene CMD1. These mutations were classified into four intragenic complementation groups, suggesting that each group represents a loss of CaM interaction with its specific essential target protein. Nuf1p/Spc110p, one of the essential targets, is a spindle pole body component that is required for proper mitosis. We investigated which intragenic complementation group of CaM represents the malfunction of Nuf1p. Immunoprecipitation analysis showed that two cmd1 mutations belonging to two distinct intragenic complementation groups had the most severely impaired complex formation with Nuf1p at the restrictive temperature. The temperature-sensitive growth of these cmd1 mutants was suppressed by a CaM-independent dominant allele of NUF1. Additionally, these mutants displayed characteristic mitotic defects: an increased ratio of artificial chromosome loss, which could be suppressed by the CaM-independent dominant allele of NUF1, and aberrant microtubule structures. These results indicate that these cmd1 mutants display the temperature-sensitive growth due to the compromised interaction with Nuf1p. However, the interaction was restored in a heterozygous diploid of the two cmd1 alleles, suggesting that intragenic complementation between these cmd1 alleles occurs by a novel mechanism, whereby co-presence of both mutant proteins rescues the interaction with Nuf1p.

Potentiation of NMDA receptor-mediated excitotoxicity linked with intrinsic apoptotic pathway in YAC transgenic mouse model of Huntington's disease.

Evidence suggests N-methyl-D-aspartate receptor (NMDAR) activation is involved in the degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). We tested the hypothesis that enhanced NMDAR-mediated excitotoxicity is mediated by the mitochondrial-associated apoptotic pathway in cultured MSNs from YAC transgenic mice expressing full-length huntingtin (htt) with a polyglutamine (polyQ) expansion of 46 or 72 (YAC46 or YAC72). NMDAR-mediated Ca(2+) transients and mitochondrial membrane depolarization were significantly increased in YAC compared to wild-type mice MSNs. Inhibitors of the mitochondrial permeability transition (mPT), cyclosporin A and bongkrekic acid, and coenzyme Q10 (an anti-oxidant involved in bioenergetic metabolism) dramatically diminished NMDA-induced cell death and eliminated genotypic differences. In YAC46 MSNs, NMDA stimulated significantly higher activation of caspase-3 and caspase-9 but not caspase-8, and NMDA-induced caspase-3 and -9 activation was markedly attenuated by cyclosporin A. Agents that improve mitochondrial function or inhibit the permeability transition may eliminate increased caspase activation and cell death associated with enhanced NMDAR activity in HD.

Epigenetic assembly of centromeric chromatin at ectopic alpha-satellite sites on human chromosomes.

To investigate the mechanism of chromatin assembly at human centromeres, we isolated cultured human cell lines in which a transfected alpha-satellite (alphoid) YAC was integrated ectopically into the terminal region of host chromosome 16, where it was stably maintained. Centromere activity of the alphoid YAC was suppressed at ectopic locations on the host chromosome, as indicated by the absent or reduced assembly of CENP-A and -C. However, long-term culture in selective medium, or short-term treatment with the histone deacetylase inhibitor Trichostatin A (TSA), promoted the re-assembly of CENPA, -B and -C at the YAC site and the release of minichromosomes containing the YAC integration site. Chromatin immunoprecipitation analyses of the re-formed minichromosome and the alphoid YAC-based stable human artificial chromosome both indicated that CENP-A and CENP-B assembled only on the inserted alphoid array but not on the YAC arms. On the YAC arms at the alphoid YAC integration sites, TSA treatment increased both the acetylation level of histone H3 and the transcriptional level of a marker gene. An increase in the level of transcription was also observed after long-term culture in selective medium. These activities, which are associated with changes in chromatin structure, might reverse the suppressed chromatin state of the YAC at ectopic loci, and thus might be involved in the epigenetic change of silent centromeres on ectopic alphoid loci.

Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington's disease (HD) transcripts and is activated in HD tissue.

Fourteen neurological diseases have been associated with the expansion of trinucleotide repeat regions. These diseases have been categorized into those that give rise to the translation of toxic polyglutamine proteins and those that are untranslated. Thus far, compelling evidence has not surfaced for the inclusion of a model in which a common mechanism may participate in the pathobiology of both translated and untranslated trinucleotide diseases. In these studies we show that a double-stranded RNA-binding protein, PKR, which has previously been linked to virally-induced and stress-mediated apoptosis, preferentially binds mutant huntingtin RNA transcripts immobilized on streptavidin columns that have been incubated with human brain extracts. These studies also show, by immunodetection in tissue slices, that PKR is present in its activated form in both human Huntington autopsy material and brain tissue derived from Huntington yeast artificial chromosome transgenic mice. The increased immunolocalization of the activated kinase is more pronounced in areas most affected by the disease and, coupled with the RNA binding results, suggests a role for PKR activation in the disease process.

Comparative mapping of BTA15 and HSA11 including a region containing a QTL for meat tenderness.

The starting point of the present study was the reported identification of a chromosomal region on bovine Chromosome (Chr) 15 (BTA15) carrying loci affecting meat tenderness. A comparative linkage map of BTA15 and human Chr 11 (HSA11) was constructed to identify potential positional candidate genes and to provide a resource of genetic markers to support marker-assisted selection (MAS). Relative rearrangements between the bovine and human genomes for these chromosomes are the most complex observed in comparative mapping between the two species, with nine alternating blocks of conserved synteny between HSA11 and bovine Chrs 15 and 29. The results of this study were the addition of nine genes to the HSA11/BTA15 comparative linkage map, and development of five microsatellite markers within the quantitative trait locus (QTL) interval. One gene with known effects on muscle development (MYOD1) was mapped to the interval. A second gene (CALCA) involved in regulation of calcium levels, a key factor in postmortem tenderization, also mapped within the interval. Refinement of the comparative map and QTL position will reduce the interval on the human transcription map to be scanned in search of candidates, reducing the effort and resources required to identify the allelic variation responsible for the genetic effect.

Improvement of FISH mapping resolution on combed DNA molecules by iterative constrained deconvolution: a quantitative study.

Image restoration approaches, such as digital deconvolution, are becoming widely used for improving the quality of microscopic images. However, no quantification of the gain in resolution of fluorescence images is available. We show that, after iterative constrained deconvolution, fluorescent cosmid signals appear to be 25% smaller, and 1.2-kb fragment signals on combed molecules faithfully display the expected length.

Expression of the human CFTR gene from episomal oriP-EBNA1-YACs in mouse cells.

Plasmids carrying the origin of plasmid replication ( oriP ) and expressing the EBNA-1 protein from the Epstein-Barr virus replicate and segregate in human cells and are thus potentially useful vectors for gene therapy. As very large circular molecules, up to 660 kb in size, can be maintained episomally using this system, it is possible to include intact human genes with all their long-range controlling elements which might give high levels of tissue-specific and controlled gene expression. We have shown previously that a 320 kb yeast artificial chromosome (YAC) carrying the intact human CFTR gene can complement the Cambridge null cystic fibrosis mice as a transgene. We have now modified this YAC to a circular molecule carrying both oriP and the EBNA-1 gene. We show that this oriP-EBNA1-YAC can be stably maintained as unrearranged episomes in mouse LA-9 cells, which do not express endogenous cftr, and in mouse CMT-93 cells, which do express endogenous cftr. The human CFTR gene is expressed in some of the cell lines, but the level of expression is very variable between cell lines and is not related to the copy number of the elements.