Papers by Silvia V . Diaz-Perez
Fungal Genetics and Biology, 1996
Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aM... more Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aMagnaporthe griseabacterial artificial chromosome library.Fungal Genet. Biol.20,280–288. A bacterial artificial chromosome (BAC) library ofMagnaporthe griseacontaining 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents ofM. griseaand has been demonstrated to be representative of the genome by
Water Environment Research, 2003
Microbial & Comparative Genomics, 1997
We have used a variety of methods to characterize the genome of the archaeon Methanosarcina therm... more We have used a variety of methods to characterize the genome of the archaeon Methanosarcina thermophila TM-1. Pulsed-field gel analysis indicates a genome size of 2.8 Mb. We have constructed a bacterial artificial chromosome (BAC) library of M. thermophila and have used it to generate physical maps for this organism. The library is made up of 384 clones with an average insert size of 58 kb representing 8.0 genome equivalents. The utility of the library for low-resolution physical mapping was shown by identifying NotI linking clones and using these to order the NotI macrorestriction fragments of M. thermophila into a 2.8 Mb map. Hybridization of nine single copy genes and a 16S rRNA sequence to these macrorestriction fragments forms the basis for the first genetic map in this organism. High-resolution physical maps, consisting of overlapping clones, have been created using HindIII fingerprints of BAC clones. In this way, we identified a minimal path of five clones that span a 270 kb NotI fragment. The ease of manipulating BAC clones makes the BAC system an excellent choice for the construction of low-resolution and high-resolution physical and genetic maps of archaeal genomes. It also provides a substrate for future genome-sequencing efforts.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2006
PLoS ONE, 2011
The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in... more The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit bright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit bright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pregonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit bright iPGCs. Taken together, the generation of Blimp1positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1positive stage of development.
Neuromuscular Disorders, 2010
Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating ... more Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating dysferlinopathy, a rare muscular dystrophy caused by loss of dysferlin. We sought to understand the molecular basis for this disparity by studying the effects of a glucocorticoid on differentiation of the myoblast cell line, C2C12, and dysferlin-deficient C2C12s. We found that pharmacologic doses of dexamethasone enhanced the myogenic fusion efficiency of C2C12s and increased the induction of dysferlin, along with specific myogenic transcription factors, sarcolemmal and structural proteins. In contrast, the dysferlin-deficient C2C12 cell line demonstrated a reduction in long myotubes and early induction of particular muscle differentiation proteins, most notably, myosin heavy chain. Dexamethasone partially reversed the defect in myogenic fusion in the dysferlin-deficient C2C12 cells. We hypothesize that a key therapeutic benefit of glucocorticoids may be the up-regulation of dysferlin as an important component of glucocorticoid-enhanced myogenic differentiation.
Human Molecular Genetics, 2012
Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of... more Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cellbased model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1 -UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.
Human Molecular Genetics, 2010
Human pluripotent stem cells (hPSCs) hold significant promise for use in regenerative medicine, o... more Human pluripotent stem cells (hPSCs) hold significant promise for use in regenerative medicine, or as a model to understand human embryo development. However, the basic mechanisms required for proliferation and self-renewal of hPSCs have not been fully uncovered. Proliferation in all eukaryotes is dependent upon highly regulated expression of the histone H3 variant Centromere protein A (CENP-A). In the current study, we demonstrate that hPSCs have a unique messenger ribonucleic acid (mRNA) reserve of CENP-A not found in somatic fibroblasts. Using short hairpin RNA technology to reduce but not ablate CENP-A, we show that CENP-A-depleted hPSCs are still capable of maintaining a functional centromeric mark, whereas fibroblasts are not. However, upon induction of differentiation or DNA damage, hPSCs with depleted CENP-A arrest in G2/M and undergo apoptosis. Analysis of CENP-A dynamics following DNA damage in hPSCs reveals that 60 min after irradiation, CENP-A is found in multiple small nuclear foci that are mutually exclusive to gH2AX as well as CENP-C. Furthermore, following irradiation, hPSCs with depleted CENP-A mount a normal apoptotic response at 6 h; however at 24 h, apoptosis is significantly increased in CENP-A-depleted hPSCs relative to control. Taken together, our results indicate that hPSCs exhibit a unique mechanism for maintaining genomic integrity by possessing the flexibility to reduce the amount of CENP-A required to maintain a functional centromere under self-renewing conditions, and maintaining a reserve of CENP-A mRNA to rebuild the centromere following differentiation or DNA damage.
Genetics, 2006
The inactive X chromosome of female mammals displays several properties of heterochromatin includ... more The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of g-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.
Developmental Biology, 2006
homolog HLH-1 acts downstream of mls-2 to specify Mderived coelomocyte cell fates, whereas mls-2 ... more homolog HLH-1 acts downstream of mls-2 to specify Mderived coelomocyte cell fates, whereas mls-2 regulates cell proliferation through the G1 cell cycle regulator CYE-1. Thus, MLS-2 functions in a cell-type-specific manner to regulate both cell proliferation and fate specification. We have also found that the M lineage expression of mls-2 appears to be directly regulated by the Exd/Pbx homolog CEH-20. Eliminating CEH-20 or mutating a putative CEH-20 binding site in the mls-2 promoter leads to the specific loss of mls-2 in the M lineage. We are currently testing whether CEH-20 directly binds to the mls-2 promoter and whether CEH-20 functions together with the Abd-B class Hox proteins (EGL-5/PHP-3/NOB-1) and the Hth/Meis homolog UNC-62 to regulate mls-2 expression.
Biochemical and Biophysical Research Communications, 2005
13 Abstract 14 ATM and ATR are well documented for their roles in maintaining the integrity of ge... more 13 Abstract 14 ATM and ATR are well documented for their roles in maintaining the integrity of genomic DNA by responding to DNA damage and 15 preparing the cell for repair. Since ATM and ATR have been reported to exist in complexes with histone deacetylases, we asked whether 16 Atm and Atr might also uphold gene silencing by heterochromatin. We show that the Atm/Atr inhibitor 2-aminopurine causes the inac-17 tive X chromosome to accumulate abnormal chromatin and undergo unwanted gene reactivation. We provide evidence that this gene 18 expression from the inactive X chromosome is not a byproduct of the accumulation of DNA breaks. Individually inhibiting Atm and 19 Atr by either small interfering RNA or the expression of dominant-negative ATM and ATR constructs also compromised X-inactivation. 20 Atm and Atr, therefore, not only function in responding to DNA damage but perhaps also are involved in gene silencing via the main-21 tenance of heterochromatin. 22
Genetics, 2005
In female mammalian cells, the inactive X chromosome is replicated late in S phase while the acti... more In female mammalian cells, the inactive X chromosome is replicated late in S phase while the active X chromosome is replicated earlier. The replication times of the X chromosomes reflect a general trend in which late replication is associated with gene repression and earlier replication with transcriptional competence. The X-linked Xist gene is expressed exclusively from the inactive X chromosome where it is involved in the initiation and maintenance of X-inactivation. In contrast, no biological activity has been assigned to the Xist locus of the active X chromosome where the Xist gene is transcriptionally silenced. Here, we provide evidence that the element(s) at the nontranscribed Xist locus of the active X chromosome controls chromosomal replication timing in cis.
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Papers by Silvia V . Diaz-Perez